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Virshup, David Marc

Overview:

Wnt Signaling and Cancer Biology; Circadian Rhythms; Pediatric Hematology/Oncology

Positions:

Professor of Pediatrics

Pediatrics
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

M.D. 1981

M.D. — Johns Hopkins University

Pediatric Internship & Residency, Pediatrics

Johns Hopkins University

Hem/Onc Clinical Fellowship, Pediatric

Johns Hopkins University

News:

Publications:

Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors.

Porcupine is an O-acyltransferase that regulates Wnt secretion. Inhibiting porcupine may block the Wnt pathway which is often dysregulated in various cancers. Consequently porcupine inhibitors are thought to be promising oncology therapeutics. A high throughput screen against porcupine revealed several potent hits that were confirmed to be Wnt pathway inhibitors in secondary assays. We developed a pharmacophore model and used the putative bioactive conformation of a xanthine inhibitor for scaffold hopping. The resulting maleimide scaffold was optimized to subnanomolar potency while retaining good physical druglike properties. A preclinical development candidate was selected for which extensive in vitro and in vivo profiling is reported.

Authors
Ho, SY; Alam, J; Jeyaraj, DA; Wang, W; Lin, GR; Ang, SH; Tan, ESW; Lee, MA; Ke, Z; Madan, B; Virshup, DM; Ding, LJ; Manoharan, V; Chew, YS; Low, CB; Pendharkar, V; Sangthongpitag, K; Hill, J; Keller, TH; Poulsen, A
MLA Citation
Ho, SY, Alam, J, Jeyaraj, DA, Wang, W, Lin, GR, Ang, SH, Tan, ESW, Lee, MA, Ke, Z, Madan, B, Virshup, DM, Ding, LJ, Manoharan, V, Chew, YS, Low, CB, Pendharkar, V, Sangthongpitag, K, Hill, J, Keller, TH, and Poulsen, A. "Scaffold Hopping and Optimization of Maleimide Based Porcupine Inhibitors." Journal of Medicinal Chemistry 60.15 (August 2017): 6678-6692.
PMID
28671458
Source
epmc
Published In
Journal of Medicinal Chemistry
Volume
60
Issue
15
Publish Date
2017
Start Page
6678
End Page
6692
DOI
10.1021/acs.jmedchem.7b00662

Wnt proteins synergize to activate β-catenin signaling.

Wnt ligands are involved in diverse signaling pathways that are active during development, maintenance of tissue homeostasis and in various disease states. While signaling regulated by individual Wnts has been extensively studied, Wnts are rarely expressed alone, and the consequences of Wnt gene co-expression are not well understood. Here, we studied the effect of co-expression of Wnts on the β-catenin signaling pathway. While some Wnts are deemed 'non-canonical' due to their limited ability to activate β-catenin when expressed alone, unexpectedly, we find that multiple Wnt combinations can synergistically activate β-catenin signaling in multiple cell types. WNT1- and WNT7B-mediated synergistic Wnt signaling requires FZD5, FZD8 and LRP6, as well as the WNT7B co-receptors GPR124 (also known as ADGRA2) and RECK. Unexpectedly, this synergistic signaling occurs downstream of β-catenin stabilization, and is correlated with increased lysine acetylation of β-catenin. Wnt synergy provides a general mechanism to confer increased combinatorial control over this important regulatory pathway.

Authors
Alok, A; Lei, Z; Jagannathan, NS; Kaur, S; Harmston, N; Rozen, SG; Tucker-Kellogg, L; Tucker-Kellogg, L; Virshup, DM
MLA Citation
Alok, A, Lei, Z, Jagannathan, NS, Kaur, S, Harmston, N, Rozen, SG, Tucker-Kellogg, L, Tucker-Kellogg, L, and Virshup, DM. "Wnt proteins synergize to activate β-catenin signaling." Journal of cell science 130.9 (May 2017): 1532-1544.
PMID
28289266
Source
epmc
Published In
Journal of cell science
Volume
130
Issue
9
Publish Date
2017
Start Page
1532
End Page
1544
DOI
10.1242/jcs.198093

A Flick of the Tail Keeps the Circadian Clock in Line.

Circadian clocks signal and adapt to an ever-changing world by juggling a panoply of transcriptional and post-translational modifications. In this issue of Molecular Cell, Gustafson et al. (2017) report an additional requirement for accurate timekeeping, a cis/trans conformational flicker in the transcriptional activation domain of the core clock protein BMAL1.

Authors
Narasimamurthy, R; Virshup, DM
MLA Citation
Narasimamurthy, R, and Virshup, DM. "A Flick of the Tail Keeps the Circadian Clock in Line." Molecular cell 66.4 (May 2017): 437-438.
PMID
28525736
Source
epmc
Published In
Molecular Cell
Volume
66
Issue
4
Publish Date
2017
Start Page
437
End Page
438
DOI
10.1016/j.molcel.2017.05.008

Molecular Mechanisms Regulating Temperature Compensation of the Circadian Clock.

An approximately 24-h biological timekeeping mechanism called the circadian clock is present in virtually all light-sensitive organisms from cyanobacteria to humans. The clock system regulates our sleep-wake cycle, feeding-fasting, hormonal secretion, body temperature, and many other physiological functions. Signals from the master circadian oscillator entrain peripheral clocks using a variety of neural and hormonal signals. Even centrally controlled internal temperature fluctuations can entrain the peripheral circadian clocks. But, unlike other chemical reactions, the output of the clock system remains nearly constant with fluctuations in ambient temperature, a phenomenon known as temperature compensation. In this brief review, we focus on recent advances in our understanding of the posttranslational modifications, especially a phosphoswitch mechanism controlling the stability of PER2 and its implications for the regulation of temperature compensation.

Authors
Narasimamurthy, R; Virshup, DM
MLA Citation
Narasimamurthy, R, and Virshup, DM. "Molecular Mechanisms Regulating Temperature Compensation of the Circadian Clock." Frontiers in neurology 8 (January 2017): 161-. (Review)
PMID
28496429
Source
epmc
Published In
Frontiers in Neurology
Volume
8
Publish Date
2017
Start Page
161
DOI
10.3389/fneur.2017.00161

Site-specific phosphorylation of casein kinase 1 δ (CK1δ) regulates its activity towards the circadian regulator PER2.

Circadian rhythms are intrinsic ~24 hour cycles that regulate diverse aspects of physiology, and in turn are regulated by interactions with the external environment. Casein kinase 1 delta (CK1δ, CSNK1D) is a key regulator of the clock, phosphorylating both stabilizing and destabilizing sites on the PER2 protein, in a mechanism known as the phosphoswitch. CK1δ can itself be regulated by phosphorylation on its regulatory domain, but the specific sites involved, and the role this plays in control of circadian rhythms as well as other CK1-dependent processes is not well understood. Using a sensitized PER2::LUC reporter assay, we identified a specific phosphorylation site, T347, on CK1δ, that regulates CK1δ activity towards PER2. A mutant CK1δ T347A was more active in promoting PER2 degradation. This CK1δ regulatory site is phosphorylated in cells in trans by dinaciclib- and staurosporine-sensitive kinases, consistent with their potential regulation by cyclin dependent and other proline-directed kinases. The regulation of CK1δ by site-specific phosphorylation via the cell cycle and other signaling pathways provides a mechanism to couple external stimuli to regulation of CK1δ-dependent pathways including the circadian clock.

Authors
Eng, GWL; Edison, ; Virshup, DM
MLA Citation
Eng, GWL, Edison, , and Virshup, DM. "Site-specific phosphorylation of casein kinase 1 δ (CK1δ) regulates its activity towards the circadian regulator PER2." PloS one 12.5 (January 2017): e0177834-.
PMID
28545154
Source
epmc
Published In
PloS one
Volume
12
Issue
5
Publish Date
2017
Start Page
e0177834
DOI
10.1371/journal.pone.0177834

Wnt Signaling Promotes Breast Cancer by Blocking ITCH-Mediated Degradation of YAP/TAZ Transcriptional Coactivator WBP2.

Cross-talk between the Hippo and Wnt pathways has been implicated recently in breast cancer development, but key intersections have yet to be fully defined. Here we report that WBP2, a transcription coactivator that binds the Hippo pathway transcription factor YAP/TAZ, contributes to Wnt signaling and breast cancer pathogenesis. Clinically, overexpression of WBP2 in breast cancer specimens correlated with malignant progression and poor patient survival. In breast cancer cells, nuclear entry and interaction of WBP2 with β-catenin was stimulated by Wnt3A, thereby activating TCF-mediated transcription and driving malignant invasive character. Mechanistic investigations showed WBP2 levels were controlled by the E3 ligase ITCH, which bound and target WBP2 for ubiquitin-dependent proteasomal degradation. Accordingly, ITCH silencing could elevate WBP2 levels. Wnt signaling upregulated WBP2 by disrupting ITCH-WBP2 interactions via EGFR-mediated tyrosine phosphorylation of WBP2 and TAZ/YAP competitive binding. Conversely, ITCH-mediated downregulation of WBP2 inhibited TCF/β-catenin transcription, in vitro transformation, and in vivo tumorigenesis. We identified somatic mutations in ITCH, which impaired its ability to degrade WBP2 and to block its function in cancer, even while retaining binding capacity to WBP2. Thus, the Wnt pathway appeared to engage WBP2 primarily by affecting its protein stability. Our findings show how WBP2/ITCH signaling functions to link the intricate Wnt and Hippo signaling networks in breast cancer. Cancer Res; 76(21); 6278-89. ©2016 AACR.

Authors
Lim, SK; Lu, SY; Kang, S-A; Tan, HJ; Li, Z; Adrian Wee, ZN; Guan, JS; Reddy Chichili, VP; Sivaraman, J; Putti, T; Thike, AA; Tan, PH; Sudol, M; Virshup, DM; Chan, SW; Hong, W; Lim, YP
MLA Citation
Lim, SK, Lu, SY, Kang, S-A, Tan, HJ, Li, Z, Adrian Wee, ZN, Guan, JS, Reddy Chichili, VP, Sivaraman, J, Putti, T, Thike, AA, Tan, PH, Sudol, M, Virshup, DM, Chan, SW, Hong, W, and Lim, YP. "Wnt Signaling Promotes Breast Cancer by Blocking ITCH-Mediated Degradation of YAP/TAZ Transcriptional Coactivator WBP2." Cancer research 76.21 (November 2016): 6278-6289.
PMID
27578003
Source
epmc
Published In
Cancer Research
Volume
76
Issue
21
Publish Date
2016
Start Page
6278
End Page
6289
DOI
10.1158/0008-5472.can-15-3537

CK1δ: a pharmacologically tractable Achilles' heel of Wnt-driven cancers?

Aberrant Wnt signaling has been widely accepted to be a key driver of a subset of human cancers and a heavily scrutinized molecular pathway for the development of personalized medicine. In a recently published issue of Science Translational Medicine, Rosenberg and coworkers reported that the delta isoform of the CK1 family of serine/threonine kinases (CK1δ), an important mediator of intracellular Wnt signaling, is amplified and overexpressed in human breast tumors. They further demonstrated that pharmacological inhibition of CK1δ is efficacious for these cancers and implicate β-catenin signaling as a key target of CK1δ. In this perspective, we will discuss the salient features of this novel anti-cancer therapeutic approach and the challenges that lie ahead to translate it into a viable treatment option for cancer patients.

Authors
Cheong, JK; Virshup, DM
MLA Citation
Cheong, JK, and Virshup, DM. "CK1δ: a pharmacologically tractable Achilles' heel of Wnt-driven cancers?." Annals of translational medicine 4.21 (November 2016): 433-.
PMID
27942524
Source
epmc
Published In
Annals of translational medicine
Volume
4
Issue
21
Publish Date
2016
Start Page
433

Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.

In most human somatic cells, the lack of telomerase activity results in progressive telomere shortening during each cell division. Eventually, DNA damage responses triggered by critically short telomeres induce an irreversible cell cycle arrest termed replicative senescence. However, the cellular responses of human pluripotent stem cells to telomere uncapping remain unknown. We generated telomerase knockout human embryonic stem (ES) cells through gene targeting. Telomerase inactivation in ES cells results in progressive telomere shortening. Telomere DNA damage in ES cells and neural progenitor cells induces rapid apoptosis when telomeres are uncapped, in contrast to fibroblast cells that enter a state of replicative senescence. Significantly, telomerase inactivation limits the proliferation capacity of human ES cells without affecting their pluripotency. By targeting telomerase activity, we can functionally separate the two unique properties of human pluripotent stem cells, namely unlimited self-renewal and pluripotency. We show that the potential of ES cells to form teratomas in vivo is dictated by their telomere length. By controlling telomere length of ES cells through telomerase inactivation, we can inhibit teratoma formation and potentially improve the safety of cell therapies involving terminally differentiated cells as well as specific progenitor cells that do not require sustained cellular proliferation in vivo, and thus sustained telomerase activity. Stem Cells 2016;34:2471-2484.

Authors
Liu, CC; Ma, DL; Yan, T-D; Fan, X; Poon, Z; Poon, L-F; Goh, S-A; Rozen, SG; Hwang, WYK; Tergaonkar, V; Tan, P; Ghosh, S; Virshup, DM; Goh, ELK; Li, S
MLA Citation
Liu, CC, Ma, DL, Yan, T-D, Fan, X, Poon, Z, Poon, L-F, Goh, S-A, Rozen, SG, Hwang, WYK, Tergaonkar, V, Tan, P, Ghosh, S, Virshup, DM, Goh, ELK, and Li, S. "Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy." Stem cells (Dayton, Ohio) 34.10 (October 2016): 2471-2484.
PMID
27299710
Source
epmc
Published In
Stem Cells
Volume
34
Issue
10
Publish Date
2016
Start Page
2471
End Page
2484
DOI
10.1002/stem.2431

Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways.

The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways.

Authors
Somarelli, JA; Schaeffer, D; Marengo, MS; Bepler, T; Rouse, D; Ware, KE; Hish, AJ; Zhao, Y; Buckley, AF; Epstein, JI; Armstrong, AJ; Virshup, DM; Garcia-Blanco, MA
MLA Citation
Somarelli, JA, Schaeffer, D, Marengo, MS, Bepler, T, Rouse, D, Ware, KE, Hish, AJ, Zhao, Y, Buckley, AF, Epstein, JI, Armstrong, AJ, Virshup, DM, and Garcia-Blanco, MA. "Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways." Oncogene 35.33 (August 2016): 4302-4311.
Website
http://hdl.handle.net/10161/11682
PMID
26751776
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Volume
35
Issue
33
Publish Date
2016
Start Page
4302
End Page
4311
DOI
10.1038/onc.2015.497

Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization.

Protein phosphatase 2A (PP2A) accounts for the majority of total Ser/Thr phosphatase activities in most cell types and regulates many biological processes. PP2A holoenzymes contain a scaffold A subunit, a catalytic C subunit, and one of the regulatory/targeting B subunits. How the B subunit controls PP2A localization and substrate specificity, which is a crucial aspect of PP2A regulation, remains poorly understood. The kinetochore is a critical site for PP2A functioning, where PP2A orchestrates chromosome segregation through its interactions with BubR1. The PP2A-BubR1 interaction plays important roles in both spindle checkpoint silencing and stable microtubule-kinetochore attachment. Here we present the crystal structure of a PP2A B56-BubR1 complex, which demonstrates that a conserved BubR1 LxxIxE motif binds to the concave side of the B56 pseudo-HEAT repeats. The BubR1 motif binds to a groove formed between B56 HEAT repeats 3 and 4, which is quite distant from the B56 binding surface for PP2A catalytic C subunit and thus is unlikely to affect PP2A activity. In addition, the BubR1 binding site on B56 is far from the B56 binding site of shugoshin, another kinetochore PP2A-binding protein, and thus BubR1 and shugoshin can potentially interact with PP2A-B56 simultaneously. Our structural and biochemical analysis indicates that other proteins with the LxxIxE motif may also bind to the same PP2A B56 surface. Thus, our structure of the PP2A B56-BubR1 complex provides important insights into how the B56 subunit directs the recruitment of PP2A to specific targets.

Authors
Wang, J; Wang, Z; Yu, T; Yang, H; Virshup, DM; Kops, GJPL; Lee, SH; Zhou, W; Li, X; Xu, W; Rao, Z
MLA Citation
Wang, J, Wang, Z, Yu, T, Yang, H, Virshup, DM, Kops, GJPL, Lee, SH, Zhou, W, Li, X, Xu, W, and Rao, Z. "Crystal structure of a PP2A B56-BubR1 complex and its implications for PP2A substrate recruitment and localization." Protein & cell 7.7 (July 2016): 516-526.
PMID
27350047
Source
epmc
Published In
Protein & Cell
Volume
7
Issue
7
Publish Date
2016
Start Page
516
End Page
526
DOI
10.1007/s13238-016-0283-4

USP6 oncogene promotes Wnt signaling by deubiquitylating Frizzleds.

The Wnt signaling pathways play pivotal roles in carcinogenesis. Modulation of the cell-surface abundance of Wnt receptors is emerging as an important mechanism for regulating sensitivity to Wnt ligands. Endocytosis and degradation of the Wnt receptors Frizzled (Fzd) and lipoprotein-related protein 6 (LRP6) are regulated by the E3 ubiquitin ligases zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43), which are disrupted in cancer. In a genome-wide small interfering RNA screen, we identified the deubiquitylase ubiquitin-specific protease 6 (USP6) as a potent activator of Wnt signaling. USP6 enhances Wnt signaling by deubiquitylating Fzds, thereby increasing their cell-surface abundance. Chromosomal translocations in nodular fasciitis result in USP6 overexpression, leading to transcriptional activation of the Wnt/β-catenin pathway. Inhibition of Wnt signaling using Dickkopf-1 (DKK1) or a Porcupine (PORCN) inhibitor significantly decreased the growth of USP6-driven xenograft tumors, indicating that Wnt signaling is a key target of USP6 during tumorigenesis. Our study defines an additional route to ectopic Wnt pathway activation in human disease, and identifies a potential approach to modulate Wnt signaling for therapeutic benefit.

Authors
Madan, B; Walker, MP; Young, R; Quick, L; Orgel, KA; Ryan, M; Gupta, P; Henrich, IC; Ferrer, M; Marine, S; Roberts, BS; Arthur, WT; Berndt, JD; Oliveira, AM; Moon, RT; Virshup, DM; Chou, MM; Major, MB
MLA Citation
Madan, B, Walker, MP, Young, R, Quick, L, Orgel, KA, Ryan, M, Gupta, P, Henrich, IC, Ferrer, M, Marine, S, Roberts, BS, Arthur, WT, Berndt, JD, Oliveira, AM, Moon, RT, Virshup, DM, Chou, MM, and Major, MB. "USP6 oncogene promotes Wnt signaling by deubiquitylating Frizzleds." Proceedings of the National Academy of Sciences of the United States of America 113.21 (May 9, 2016): E2945-E2954.
PMID
27162353
Source
epmc
Published In
Proceedings of the National Academy of Sciences of USA
Volume
113
Issue
21
Publish Date
2016
Start Page
E2945
End Page
E2954
DOI
10.1073/pnas.1605691113

Experimental inhibition of porcupine-mediated Wnt O-acylation attenuates kidney fibrosis.

Activated Wnt signaling is critical in the pathogenesis of renal fibrosis, a final common pathway for most forms of chronic kidney disease. Therapeutic intervention by inhibition of individual Wnts or downstream Wnt/β-catenin signaling has been proposed, but these approaches do not interrupt the functions of all Wnts nor block non-canonical Wnt signaling pathways. Alternatively, an orally bioavailable small molecule, Wnt-C59, blocks the catalytic activity of the Wnt-acyl transferase porcupine, and thereby prevents secretion of all Wnt isoforms. We found that inhibiting porcupine dramatically attenuates kidney fibrosis in the murine unilateral ureteral obstruction model. Wnt-C59 treatment similarly blunts collagen mRNA expression in the obstructed kidney. Consistent with its actions to broadly arrest Wnt signaling, porcupine inhibition reduces expression of Wnt target genes and bolsters nuclear exclusion of β-catenin in the kidney following ureteral obstruction. Importantly, prevention of Wnt secretion by Wnt-C59 blunts expression of inflammatory cytokines in the obstructed kidney that otherwise provoke a positive feedback loop of Wnt expression in collagen-producing fibroblasts and epithelial cells. Thus, therapeutic targeting of porcupine abrogates kidney fibrosis not only by overcoming the redundancy of individual Wnt isoforms but also by preventing upstream cytokine-induced Wnt generation. These findings reveal a novel therapeutic maneuver to protect the kidney from fibrosis by interrupting a pathogenic crosstalk loop between locally generated inflammatory cytokines and the Wnt/β-catenin signaling pathway.

Authors
Madan, B; Patel, MB; Zhang, J; Bunte, RM; Rudemiller, NP; Griffiths, R; Virshup, DM; Crowley, SD
MLA Citation
Madan, B, Patel, MB, Zhang, J, Bunte, RM, Rudemiller, NP, Griffiths, R, Virshup, DM, and Crowley, SD. "Experimental inhibition of porcupine-mediated Wnt O-acylation attenuates kidney fibrosis." Kidney international 89.5 (May 2016): 1062-1074.
Website
http://hdl.handle.net/10161/11962
PMID
27083283
Source
epmc
Published In
Kidney international
Volume
89
Issue
5
Publish Date
2016
Start Page
1062
End Page
1074
DOI
10.1016/j.kint.2016.01.017

Keeping autophagy in cheCK1.

Mutant RAS-driven cancer cells cope with proliferative stress by increasing basal autophagy to maintain protein/organelle and energy homeostasis. We recently demonstrated that casein kinase 1 alpha (CK1α), a therapeutically tractable enzyme, is critical for fine-tuning the transcriptional regulation of mutant RAS-induced autophagy and the development of mutant RAS-driven cancers.

Authors
Cheong, JK; Virshup, DM
MLA Citation
Cheong, JK, and Virshup, DM. "Keeping autophagy in cheCK1." Molecular & Cellular Oncology 3.3 (May 2016): e1045117-.
PMID
27314070
Source
epmc
Published In
Molecular & cellular oncology
Volume
3
Issue
3
Publish Date
2016
Start Page
e1045117
DOI
10.1080/23723556.2015.1045117

Wnt addiction of genetically defined cancers reversed by PORCN inhibition.

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

Authors
Madan, B; Ke, Z; Harmston, N; Ho, SY; Frois, AO; Alam, J; Jeyaraj, DA; Pendharkar, V; Ghosh, K; Virshup, IH; Manoharan, V; Ong, EHQ; Sangthongpitag, K; Hill, J; Petretto, E; Keller, TH; Lee, MA; Matter, A; Virshup, DM
MLA Citation
Madan, B, Ke, Z, Harmston, N, Ho, SY, Frois, AO, Alam, J, Jeyaraj, DA, Pendharkar, V, Ghosh, K, Virshup, IH, Manoharan, V, Ong, EHQ, Sangthongpitag, K, Hill, J, Petretto, E, Keller, TH, Lee, MA, Matter, A, and Virshup, DM. "Wnt addiction of genetically defined cancers reversed by PORCN inhibition." Oncogene 35.17 (April 2016): 2197-2207.
PMID
26257057
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Volume
35
Issue
17
Publish Date
2016
Start Page
2197
End Page
2207
DOI
10.1038/onc.2015.280

Wnt inhibition enhances browning of mouse primary white adipocytes.

The global epidemic in obesity and metabolic syndrome requires novel approaches to tackle. White adipose tissue, traditionally seen as a passive energy-storage organ, can be induced to take on certain characteristics of brown fat in a process called browning. The "browned" white adipose tissue, or beige fat, is a potential anti-obesity target. Various signaling pathways can enhance browning. Wnt is a key regulator of adipocyte biology, but its role in browning has not been explored. In this study, we found that in primary mouse adipocytes derived from the inguinal depot, Wnt inhibition by both chemical and genetic methods significantly enhanced browning. The effect of Wnt inhibition on browning most likely targets the beige precursor cells in selected adipose depots.

Authors
Lo, KA; Ng, PY; Kabiri, Z; Virshup, D; Sun, L
MLA Citation
Lo, KA, Ng, PY, Kabiri, Z, Virshup, D, and Sun, L. "Wnt inhibition enhances browning of mouse primary white adipocytes." Adipocyte 5.2 (April 2016): 224-231.
PMID
27386162
Source
epmc
Published In
Adipocyte
Volume
5
Issue
2
Publish Date
2016
Start Page
224
End Page
231
DOI
10.1080/21623945.2016.1148834

NOTUM is a potential pharmacodynamic biomarker of Wnt pathway inhibition.

Activation of Wnt signaling due to Wnt overexpression or mutations of Wnt pathway components is associated with various cancers. Blocking Wnt secretion by inhibiting PORCN enzymatic activity has shown efficacy in a subset of cancers with elevated Wnt signaling. Predicting response to upstream Wnt inhibitors and monitoring response to therapeutics is challenging due to the paucity of well-defined biomarkers. In this study we identify Notum as a potential biomarker for Wnt driven cancers and show that coordinate regulation of NOTUM and AXIN2 expression may be a useful predictor of response to PORCN inhibitors. Most importantly, as NOTUM is a secreted protein and its levels in blood correlate with tumor growth, it has potential as a pharmacodynamic biomarker for PORCN and other Wnt pathway inhibitors.

Authors
Madan, B; Ke, Z; Lei, ZD; Oliver, FA; Oshima, M; Lee, MA; Rozen, S; Virshup, DM
MLA Citation
Madan, B, Ke, Z, Lei, ZD, Oliver, FA, Oshima, M, Lee, MA, Rozen, S, and Virshup, DM. "NOTUM is a potential pharmacodynamic biomarker of Wnt pathway inhibition." Oncotarget 7.11 (March 2016): 12386-12392.
PMID
26848981
Source
epmc
Published In
Oncotarget
Volume
7
Issue
11
Publish Date
2016
Start Page
12386
End Page
12392
DOI
10.18632/oncotarget.7157

Abstract P4-08-03: DEAD-box RNA helicase DP103 as a novel regulator of Wnt/β-catenin signaling pathway and promotes cancer stem cell-like behavior in triple negative breast cancers

Authors
Cai, W; Cheong, JK; Edison, E; Banerjee, A; Tan, TZ; Gaboury, L; Yousef, EM; Thiery, JP; Lobie, PE; Virshup, DM; Yap, CT; Kumar, AP
MLA Citation
Cai, W, Cheong, JK, Edison, E, Banerjee, A, Tan, TZ, Gaboury, L, Yousef, EM, Thiery, JP, Lobie, PE, Virshup, DM, Yap, CT, and Kumar, AP. "Abstract P4-08-03: DEAD-box RNA helicase DP103 as a novel regulator of Wnt/β-catenin signaling pathway and promotes cancer stem cell-like behavior in triple negative breast cancers." February 15, 2016.
Source
crossref
Published In
Cancer Research
Volume
76
Issue
4 Supplement
Publish Date
2016
Start Page
P4-08-03
End Page
P4-08-03
DOI
10.1158/1538-7445.SABCS15-P4-08-03

A Period2 Phosphoswitch Regulates and Temperature Compensates Circadian Period.

Period (PER) protein phosphorylation is a critical regulator of circadian period, yet an integrated understanding of the role and interaction between phosphorylation sites that can both increase and decrease PER2 stability remains elusive. Here, we propose a phosphoswitch model, where two competing phosphorylation sites determine whether PER2 has a fast or slow degradation rate. This mathematical model accurately reproduces the three-stage degradation kinetics of endogenous PER2. We predict and demonstrate that the phosphoswitch is intrinsically temperature sensitive, slowing down PER2 degradation as a result of faster reactions at higher temperatures. The phosphoswitch provides a biochemical mechanism for circadian temperature compensation of circadian period. This phosphoswitch additionally explains the phenotype of Familial Advanced Sleep Phase (FASP) and CK1ε(tau) genetic circadian rhythm disorders, metabolic control of PER2 stability, and how drugs that inhibit CK1 alter period. The phosphoswitch provides a general mechanism to integrate diverse stimuli to regulate circadian period.

Authors
Zhou, M; Kim, JK; Eng, GWL; Forger, DB; Virshup, DM
MLA Citation
Zhou, M, Kim, JK, Eng, GWL, Forger, DB, and Virshup, DM. "A Period2 Phosphoswitch Regulates and Temperature Compensates Circadian Period." Molecular cell 60.1 (October 2015): 77-88.
PMID
26431025
Source
epmc
Published In
Molecular Cell
Volume
60
Issue
1
Publish Date
2015
Start Page
77
End Page
88
DOI
10.1016/j.molcel.2015.08.022

Pyrvinium selectively targets blast phase-chronic myeloid leukemia through inhibition of mitochondrial respiration.

The use of BCR-ABL1 tyrosine kinase inhibitors (TKI) has led to excellent clinical responses in patients with chronic phase chronic myeloid leukemia (CML). However these inhibitors have been less effective as single agents in the terminal blast phase (BP). We show that pyrvinium, a FDA-approved anthelminthic drug, selectively targets BP-CML CD34+ progenitor cells. Pyrvinium is effective in inducing apoptosis, inhibiting colony formation and self-renewal capacity of CD34+ cells from TKI-resistant BP-CML patients, while cord blood CD34+ are largely unaffected. The effects of pyrvinium are further enhanced upon combination with dasatinib, a second generation BCR-ABL1 TKI. In a CML xenograft model pyrvinium significantly inhibits tumor growth as a single agent, with complete inhibition in combination with dasatinib. While pyrvinium has been shown to inhibit the Wnt/β-catenin signalling pathway via activation of casein kinase 1α , we find its activity in CML is not dependent on this pathway. Instead, we show that pyrvinium localizes to mitochondria and induces apoptosis by inhibiting mitochondrial respiration. Our study suggests that pyrvinium is a useful addition to the treatment armamentarium for BP-CML and that targeting mitochondrial respiration may be a potential therapeutic strategy in aggressive leukemia.

Authors
Xiang, W; Cheong, JK; Ang, SH; Teo, B; Xu, P; Asari, K; Sun, WT; Than, H; Bunte, RM; Virshup, DM; Chuah, C
MLA Citation
Xiang, W, Cheong, JK, Ang, SH, Teo, B, Xu, P, Asari, K, Sun, WT, Than, H, Bunte, RM, Virshup, DM, and Chuah, C. "Pyrvinium selectively targets blast phase-chronic myeloid leukemia through inhibition of mitochondrial respiration." Oncotarget 6.32 (October 2015): 33769-33780.
PMID
26378050
Source
epmc
Published In
Oncotarget
Volume
6
Issue
32
Publish Date
2015
Start Page
33769
End Page
33780
DOI
10.18632/oncotarget.5615

Abstract 4449: A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations

Authors
Ke, Z; Madan, B; Lim, SQY; Wang, S; Alam, J; Ho, SY; Jeyaraj, DA; Ghosh, K; Chew, YS; Ding, LJ; Monoharan, V; Pendharkar, V; Ong, E; Hill, J; Sangthongpitag, K; Keller, T; Lee, MA; Virshup, DM
MLA Citation
Ke, Z, Madan, B, Lim, SQY, Wang, S, Alam, J, Ho, SY, Jeyaraj, DA, Ghosh, K, Chew, YS, Ding, LJ, Monoharan, V, Pendharkar, V, Ong, E, Hill, J, Sangthongpitag, K, Keller, T, Lee, MA, and Virshup, DM. "Abstract 4449: A novel Porcupine inhibitor is effective in the treatment of cancers with RNF43 mutations." August 1, 2015.
Source
crossref
Published In
Cancer Research
Volume
75
Issue
15 Supplement
Publish Date
2015
Start Page
4449
End Page
4449
DOI
10.1158/1538-7445.AM2015-4449

Wnts are dispensable for differentiation and self-renewal of adult murine hematopoietic stem cells.

Wnt signaling controls early embryonic hematopoiesis and dysregulated β-catenin is implicated in leukemia. However, the role of Wnts and their source in adult hematopoiesis is still unclear, and is clinically important as upstream Wnt inhibitors enter clinical trials. We blocked Wnt secretion in hematopoietic lineages by targeting Porcn, a membrane-bound O-acyltransferase that is indispensable for the activity and secretion of all vertebrate Wnts. Surprisingly, deletion of Porcn in Rosa-CreER(T2)/Porcn(Del), MX1-Cre/Porcn(Del), and Vav-Cre/Porcn(Del) mice had no effects on proliferation, differentiation, or self-renewal of adult hematopoietic stem cells. Targeting Wnt secretion in the bone marrow niche by treatment with a PORCN inhibitor, C59, similarly had no effect on hematopoiesis. These results exclude a role for hematopoietic PORCN-dependent Wnts in adult hematopoiesis. Clinical use of upstream Wnt inhibitors is not likely to be limited by effects on hematopoiesis.

Authors
Kabiri, Z; Numata, A; Kawasaki, A; Edison, ; Tenen, DG; Virshup, DM
MLA Citation
Kabiri, Z, Numata, A, Kawasaki, A, Edison, , Tenen, DG, and Virshup, DM. "Wnts are dispensable for differentiation and self-renewal of adult murine hematopoietic stem cells." Blood 126.9 (August 2015): 1086-1094.
PMID
26089398
Source
epmc
Published In
Blood
Volume
126
Issue
9
Publish Date
2015
Start Page
1086
End Page
1094
DOI
10.1182/blood-2014-09-598540

Discovery and Optimization of a Porcupine Inhibitor.

Wnt proteins regulate various cellular functions and serve distinct roles in normal development throughout life. Wnt signaling is dysregulated in various diseases including cancers. Porcupine (PORCN) is a membrane-bound O-acyltransferase that palmitoleates the Wnts and hence is essential for their secretion and function. The inhibition of PORCN could serve as a therapeutic approach for the treatment of a number of Wnt-dependent cancers. Herein, we describe the identification of a Wnt secretion inhibitor from cellular high throughput screening. Classical SAR based cellular optimization provided us with a PORCN inhibitor with nanomolar activity and excellent bioavailability that demonstrated efficacy in a Wnt-driven murine tumor model. Finally, we also discovered that enantiomeric PORCN inhibitors show very different activity in our reporter assay, suggesting that such compounds may be useful for mode of action studies on the PORCN O-acyltransferase.

Authors
Duraiswamy, AJ; Lee, MA; Madan, B; Ang, SH; Tan, ESW; Cheong, WWV; Ke, Z; Pendharkar, V; Ding, LJ; Chew, YS; Manoharan, V; Sangthongpitag, K; Alam, J; Poulsen, A; Ho, SY; Virshup, DM; Keller, TH
MLA Citation
Duraiswamy, AJ, Lee, MA, Madan, B, Ang, SH, Tan, ESW, Cheong, WWV, Ke, Z, Pendharkar, V, Ding, LJ, Chew, YS, Manoharan, V, Sangthongpitag, K, Alam, J, Poulsen, A, Ho, SY, Virshup, DM, and Keller, TH. "Discovery and Optimization of a Porcupine Inhibitor." Journal of medicinal chemistry 58.15 (August 2015): 5889-5899.
PMID
26110200
Source
epmc
Published In
Journal of Medicinal Chemistry
Volume
58
Issue
15
Publish Date
2015
Start Page
5889
End Page
5899
DOI
10.1021/acs.jmedchem.5b00507

Targeting Wnts at the source--new mechanisms, new biomarkers, new drugs.

Wnt signaling is dysregulated in many cancers and is therefore an attractive therapeutic target. The focus of drug development has recently shifted away from downstream inhibitors of β-catenin. Active inhibitors of Wnt secretion and Wnt/receptor interactions have been developed that are now entering clinical trials. Such agents include inhibitors of Wnt secretion, as well as recombinant proteins that minimize Wnt-Frizzled interactions. These new therapies arrive together with the recent insight that cancer-specific upregulation of Wnt receptors at the cell surface regulates cellular sensitivity to Wnts. Loss-of-function mutations in RNF43 or ZNRF3 and gain-of-function chromosome translocations involving RSPO2 and RSPO3 are surprisingly common and markedly increase Wnt/β-catenin signaling in response to secreted Wnts. These mutations may be predictive biomarkers to select patients responsive to newly developed upstream Wnt inhibitors.

Authors
Madan, B; Virshup, DM
MLA Citation
Madan, B, and Virshup, DM. "Targeting Wnts at the source--new mechanisms, new biomarkers, new drugs." Molecular cancer therapeutics 14.5 (May 2015): 1087-1094. (Review)
PMID
25901018
Source
epmc
Published In
Molecular cancer therapeutics
Volume
14
Issue
5
Publish Date
2015
Start Page
1087
End Page
1094
DOI
10.1158/1535-7163.mct-14-1038

Casein kinase 1α-dependent feedback loop controls autophagy in RAS-driven cancers.

Activating mutations in the RAS oncogene are common in cancer but are difficult to therapeutically target. RAS activation promotes autophagy, a highly regulated catabolic process that metabolically buffers cells in response to diverse stresses. Here we report that casein kinase 1α (CK1α), a ubiquitously expressed serine/threonine kinase, is a key negative regulator of oncogenic RAS-induced autophagy. Depletion or pharmacologic inhibition of CK1α enhanced autophagic flux in oncogenic RAS-driven human fibroblasts and multiple cancer cell lines. FOXO3A, a master longevity mediator that transcriptionally regulates diverse autophagy genes, was a critical target of CK1α, as depletion of CK1α reduced levels of phosphorylated FOXO3A and increased expression of FOXO3A-responsive genes. Oncogenic RAS increased CK1α protein abundance via activation of the PI3K/AKT/mTOR pathway. In turn, elevated levels of CK1α increased phosphorylation of nuclear FOXO3A, thereby inhibiting transactivation of genes critical for RAS-induced autophagy. In both RAS-driven cancer cells and murine xenograft models, pharmacologic CK1α inactivation synergized with lysosomotropic agents to inhibit growth and promote tumor cell death. Together, our results identify a kinase feedback loop that influences RAS-dependent autophagy and suggest that targeting CK1α-regulated autophagy offers a potential therapeutic opportunity to treat oncogenic RAS-driven cancers.

Authors
Cheong, JK; Zhang, F; Chua, PJ; Bay, BH; Thorburn, A; Virshup, DM
MLA Citation
Cheong, JK, Zhang, F, Chua, PJ, Bay, BH, Thorburn, A, and Virshup, DM. "Casein kinase 1α-dependent feedback loop controls autophagy in RAS-driven cancers." The Journal of clinical investigation 125.4 (April 2015): 1401-1418.
PMID
25798617
Source
epmc
Published In
Journal of Clinical Investigation
Volume
125
Issue
4
Publish Date
2015
Start Page
1401
End Page
1418
DOI
10.1172/jci78018

TP53 intron 1 hotspot rearrangements are specific to sporadic osteosarcoma and can cause Li-Fraumeni syndrome.

Somatic mutations of TP53 are among the most common in cancer and germline mutations of TP53 (usually missense) can cause Li-Fraumeni syndrome (LFS). Recently, recurrent genomic rearrangements in intron 1 of TP53 have been described in osteosarcoma (OS), a highly malignant neoplasm of bone belonging to the spectrum of LFS tumors. Using whole-genome sequencing of OS, we found features of TP53 intron 1 rearrangements suggesting a unique mechanism correlated with transcription. Screening of 288 OS and 1,090 tumors of other types revealed evidence for TP53 rearrangements in 46 (16%) OS, while none were detected in other tumor types, indicating this rearrangement to be highly specific to OS. We revisited a four-generation LFS family where no TP53 mutation had been identified and found a 445 kb inversion spanning from the TP53 intron 1 towards the centromere. The inversion segregated with tumors in the LFS family. Cancers in this family had loss of heterozygosity, retaining the rearranged allele and resulting in TP53 expression loss. In conclusion, intron 1 rearrangements cause p53-driven malignancies by both germline and somatic mechanisms and provide an important mechanism of TP53 inactivation in LFS, which might in part explain the diagnostic gap of formerly classified "TP53 wild-type" LFS.

Authors
Ribi, S; Baumhoer, D; Lee, K; Edison, ; Teo, ASM; Madan, B; Zhang, K; Kohlmann, WK; Yao, F; Lee, WH; Hoi, Q; Cai, S; Woo, XY; Tan, P; Jundt, G; Smida, J; Nathrath, M; Sung, W-K; Schiffman, JD; Virshup, DM; Hillmer, AM
MLA Citation
Ribi, S, Baumhoer, D, Lee, K, Edison, , Teo, ASM, Madan, B, Zhang, K, Kohlmann, WK, Yao, F, Lee, WH, Hoi, Q, Cai, S, Woo, XY, Tan, P, Jundt, G, Smida, J, Nathrath, M, Sung, W-K, Schiffman, JD, Virshup, DM, and Hillmer, AM. "TP53 intron 1 hotspot rearrangements are specific to sporadic osteosarcoma and can cause Li-Fraumeni syndrome." Oncotarget 6.10 (April 2015): 7727-7740.
PMID
25762628
Source
epmc
Published In
Oncotarget
Volume
6
Issue
10
Publish Date
2015
Start Page
7727
End Page
7740

Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression.

The oncogenic role of WNT is well characterized. Wntless (WLS) (also known as GPR177, or Evi), a key modulator of WNT protein secretion, was recently found to be highly overexpressed in malignant astrocytomas. We hypothesized that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signaling such as ovarian, gastric, and breast cancers. Immunohistochemical analysis using a TMA platform revealed WLS overexpression in a subset of ovarian, gastric, and breast tumors; this overexpression was associated with poorer clinical outcomes in gastric cancer (P=0.025). In addition, a strong correlation was observed between WLS expression and human epidermal growth factor receptor 2 (HER2) overexpression. Indeed, 100% of HER2-positive intestinal gastric carcinomas, 100% of HER2-positive serous ovarian carcinomas, and 64% of HER2-positive breast carcinomas coexpressed WLS protein. Although HER2 protein expression or gene amplification is an established predictive biomarker for trastuzumab response in breast and gastric cancers, a significant proportion of HER2-positive tumors display resistance to trastuzumab, which may be in part explainable by a possible mechanistic link between WLS and HER2.

Authors
Stewart, J; James, J; McCluggage, GW; McQuaid, S; Arthur, K; Boyle, D; Mullan, P; McArt, D; Yan, B; Irwin, G; Harkin, DP; Zhengdeng, L; Ong, C-W; Yu, J; Virshup, DM; Salto-Tellez, M
MLA Citation
Stewart, J, James, J, McCluggage, GW, McQuaid, S, Arthur, K, Boyle, D, Mullan, P, McArt, D, Yan, B, Irwin, G, Harkin, DP, Zhengdeng, L, Ong, C-W, Yu, J, Virshup, DM, and Salto-Tellez, M. "Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression." Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 28.3 (March 2015): 428-436.
PMID
25258105
Source
epmc
Published In
Modern Pathology
Volume
28
Issue
3
Publish Date
2015
Start Page
428
End Page
436
DOI
10.1038/modpathol.2014.114

Moving upstream in the war on WNTs.

Cholangiocarcinoma is a relatively rare cancer of the biliary ducts that is highly refractory to treatment. The factors that drive cholangiocarcinoma are poorly understood, though chronic liver fluke infection is a risk factor for disease. In this issue of the JCI, Boulter and colleagues demonstrate that the WNT/β-catenin signaling pathway is upregulated in patients with sporadic cholangiocarcinoma. The authors determined that macrophages generate WNT ligands in cholangiocarcinomas and depletion or inhibition of this cell population in animal models of cholangiocarcinoma reduced tumor burden and proliferation. Moreover, pharmacological inhibition of WNT secretion or β-catenin activity was efficacious in animal models. Together the results of this study suggest that targeting WNT has potential as a therapeutic strategy for cholangiocarcinoma.

Authors
Virshup, DM
MLA Citation
Virshup, DM. "Moving upstream in the war on WNTs." The Journal of clinical investigation 125.3 (March 2015): 975-977.
PMID
25689251
Source
epmc
Published In
Journal of Clinical Investigation
Volume
125
Issue
3
Publish Date
2015
Start Page
975
End Page
977
DOI
10.1172/jci80819

Casein kinase 1 regulates Sprouty2 in FGF-ERK signaling

Sprouty2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase signaling, and is implicated as a tumor suppressor. SPRY2 inhibits FGF-RAS-ERK signaling by binding to growth factor receptor bound protein 2 (GRB2) during fibroblast growth factor receptor (FGFR) activation, disrupting the GRB2-SOS (son of sevenless) complex that transduces signals from FGFR to RAS. SPRY2 binding to GRB2 is modulated by phosphorylation but the key regulatory kinase(s) are not known. Prior studies identified the frequent presence of CK1 phosphorylation motifs on SPRY2. We therefore tested if CK1 has a role in SPRY2 phosphorylation and function. Loss of CK1 binding and inhibition of CK1 activity by two structurally distinct small molecules abrogated SPRY2 inhibition of FGF-ERK signaling, leading to decreased SPRY2 interaction with GRB2. Moreover, CK1 activity and binding are necessary for SPRY2 inhibition of FGF-stimulated neurite outgrowth in PC12 cells. Consistent with its proposed role as an inhibitor of FGF signaling, we find that CSNK1E transcript abundance negatively correlates with FGF1/FGF7 message in human gastric cancer samples. Modulation of CK1 activity may be therapeutically useful in the treatment of FGF/SPRY2-related diseases.

Authors
Yim, DGR; Ghosh, S; Guy, GR; Virshup, DM
MLA Citation
Yim, DGR, Ghosh, S, Guy, GR, and Virshup, DM. "Casein kinase 1 regulates Sprouty2 in FGF-ERK signaling." Oncogene 34.4 (January 22, 2015): 474-484.
Source
scopus
Published In
Oncogene: Including Oncogene Reviews
Volume
34
Issue
4
Publish Date
2015
Start Page
474
End Page
484
DOI
10.1038/onc.2013.564

Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression

© 2015 USCAP, Inc. All rights reserved. The oncogenic role of WNT is well characterized. Wntless (WLS) (also known as GPR177, or Evi), a key modulator of WNT protein secretion, was recently found to be highly overexpressed in malignant astrocytomas. We hypothesized that this molecule may be aberrantly expressed in other cancers known to possess aberrant WNT signaling such as ovarian, gastric, and breast cancers. Immunohistochemical analysis using a TMA platform revealed WLS overexpression in a subset of ovarian, gastric, and breast tumors; this overexpression was associated with poorer clinical outcomes in gastric cancer (P=0.025). In addition, a strong correlation was observed between WLS expression and human epidermal growth factor receptor 2 (HER2) overexpression. Indeed, 100% of HER2-positive intestinal gastric carcinomas, 100% of HER2-positive serous ovarian carcinomas, and 64% of HER2-positive breast carcinomas coexpressed WLS protein. Although HER2 protein expression or gene amplification is an established predictive biomarker for trastuzumab response in breast and gastric cancers, a significant proportion of HER2-positive tumors display resistance to trastuzumab, which may be in part explainable by a possible mechanistic link between WLS and HER2.

Authors
Stewart, J; James, J; McCluggage, GW; McQuaid, S; Arthur, K; Boyle, D; Mullan, P; McArt, D; Yan, B; Irwin, G; Harkin, DP; Zhengdeng, L; Ong, CW; Yu, J; Virshup, DM; Salto-Tellez, M
MLA Citation
Stewart, J, James, J, McCluggage, GW, McQuaid, S, Arthur, K, Boyle, D, Mullan, P, McArt, D, Yan, B, Irwin, G, Harkin, DP, Zhengdeng, L, Ong, CW, Yu, J, Virshup, DM, and Salto-Tellez, M. "Analysis of wntless (WLS) expression in gastric, ovarian, and breast cancers reveals a strong association with HER2 overexpression." Modern Pathology 28.3 (January 1, 2015): 428-436.
Source
scopus
Published In
Modern Pathology
Volume
28
Issue
3
Publish Date
2015
Start Page
428
End Page
436
DOI
10.1038/modpathol.2014.114

Casein kinase 1 regulates Sprouty2 in FGF-ERK signaling.

Sprouty2 (SPRY2) is a potent negative regulator of receptor tyrosine kinase signaling, and is implicated as a tumor suppressor. SPRY2 inhibits FGF-RAS-ERK signaling by binding to growth factor receptor bound protein 2 (GRB2) during fibroblast growth factor receptor (FGFR) activation, disrupting the GRB2-SOS (son of sevenless) complex that transduces signals from FGFR to RAS. SPRY2 binding to GRB2 is modulated by phosphorylation but the key regulatory kinase(s) are not known. Prior studies identified the frequent presence of CK1 phosphorylation motifs on SPRY2. We therefore tested if CK1 has a role in SPRY2 phosphorylation and function. Loss of CK1 binding and inhibition of CK1 activity by two structurally distinct small molecules abrogated SPRY2 inhibition of FGF-ERK signaling, leading to decreased SPRY2 interaction with GRB2. Moreover, CK1 activity and binding are necessary for SPRY2 inhibition of FGF-stimulated neurite outgrowth in PC12 cells. Consistent with its proposed role as an inhibitor of FGF signaling, we find that CSNK1E transcript abundance negatively correlates with FGF1/FGF7 message in human gastric cancer samples. Modulation of CK1 activity may be therapeutically useful in the treatment of FGF/SPRY2-related diseases.

Authors
Yim, DGR; Ghosh, S; Guy, GR; Virshup, DM
MLA Citation
Yim, DGR, Ghosh, S, Guy, GR, and Virshup, DM. "Casein kinase 1 regulates Sprouty2 in FGF-ERK signaling." Oncogene 34.4 (January 2015): 474-484.
PMID
24469046
Source
epmc
Published In
Oncogene: Including Oncogene Reviews
Volume
34
Issue
4
Publish Date
2015
Start Page
474
End Page
484
DOI
10.1038/onc.2013.564

B56-PP2A regulates motor dynamics for mitotic chromosome alignment.

Proper alignment of duplicated chromosomes at the metaphase plate involves both motor-driven chromosome movement and the functional and physical end-on connection (K-fiber formation) between the kinetochore and the plus-end of microtubules. The B56 family of protein phosphatase 2A (PP2A) regulatory subunits (B56-PP2A), through their interaction with the mitotic checkpoint protein BUBR1, are required for proper chromosome alignment, but the underlying mechanism(s) has remained elusive. Here, we show that B56-PP2A promotes chromosome alignment primarily by balancing chromosome movement towards the metaphase plate, rather than by directly establishing stable K-fibers. Notably, the poleward movement of chromosomes in cells depleted of the B56 family can be rescued by depletion of HSET (also known as kinesin-14 or KIFC1), a major minus-end-directed motor protein. Strikingly, K-fiber formation can be restored if chromosome movement to the metaphase plate is rescued in B56-depleted cells. Furthermore, the B56-BUBR1 interaction is required for promoting motor-driven chromosome movement towards the metaphase plate. Thus, we propose that B56-PP2A functions in mitotic chromosome alignment by balancing chromosome movement towards the metaphase plate, which is essential for the subsequent establishment of stable and functional kinetochore-microtubule attachments, and mitotic exit.

Authors
Xu, P; Virshup, DM; Lee, SH
MLA Citation
Xu, P, Virshup, DM, and Lee, SH. "B56-PP2A regulates motor dynamics for mitotic chromosome alignment." Journal of cell science 127.Pt 21 (November 2014): 4567-4573.
PMID
25179604
Source
epmc
Published In
Journal of cell science
Volume
127
Issue
Pt 21
Publish Date
2014
Start Page
4567
End Page
4573
DOI
10.1242/jcs.154609

Updating the Wnt pathways.

In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases.

Authors
Yu, J; Virshup, DM
MLA Citation
Yu, J, and Virshup, DM. "Updating the Wnt pathways." Bioscience reports 34.5 (October 17, 2014).
PMID
25208913
Source
epmc
Published In
Bioscience Reports
Volume
34
Issue
5
Publish Date
2014
DOI
10.1042/bsr20140119

Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56δ stabilizes its antiapoptotic activity.

Bcl-2 is frequently overexpressed in hematopoietic malignancies, and selective phosphorylation at ser70 enhances its antiapoptotic activity. Phospho-ser70 is dephosphorylated by specific heterotrimers of protein phosphatase 2A (PP2A). We report here that a mild pro-oxidant intracellular milieu induced by either pharmacological inhibition or genetic knockdown of superoxide dismutase 1 (SOD1) inhibits the functional holoenzyme assembly of PP2A and prevents Bcl-2 ser70 dephosphorylation. This redox-dependent regulation of Bcl-2 phosphorylation is due to nitrosative modification of B56δ, which we identify as the regulatory subunit mediating PP2A-dependent Bcl-2 dephosphorylation. Redox inhibition of PP2A results from peroxynitrite-mediated nitration of a conserved tyrosine residue within B56δ (B56δ(Y289)). Although nitrated B56δ(Y289) binds efficiently to ser70-phosphorylated Bcl-2, this specific modification inhibits the recruitment of the PP2A catalytic core (A and C subunits). Furthermore, inhibition of B56δ(Y289) nitration restores PP2A holoenzyme assembly, thereby permitting S70 dephosphorylation of Bcl-2 and inhibiting its antiapoptotic activity. More important, in primary cells derived from clinical lymphomas, Bcl-2 phosphorylation at S70 directly correlates with B56δ nitration and repression of SOD1, but inversely correlates with B56δ interaction with the PP2A-C catalytic subunit. These data underscore the role of a pro-oxidant milieu in chemoresistance of hematopoietic and other cancers via selective targeting of tumor suppressors such as PP2A.

Authors
Low, ICC; Loh, T; Huang, Y; Virshup, DM; Pervaiz, S
MLA Citation
Low, ICC, Loh, T, Huang, Y, Virshup, DM, and Pervaiz, S. "Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56δ stabilizes its antiapoptotic activity." Blood 124.14 (October 2014): 2223-2234.
PMID
25082878
Source
epmc
Published In
Blood
Volume
124
Issue
14
Publish Date
2014
Start Page
2223
End Page
2234
DOI
10.1182/blood-2014-03-563296

Phosphatase WIP1 regulates adult neurogenesis and WNT signaling during aging.

The number of newly formed neurons declines rapidly during aging, and this decrease in neurogenesis is associated with decreased function of neural stem/progenitor cells (NPCs). Here, we determined that a WIP1-dependent pathway regulates NPC differentiation and contributes to the age-associated decline of neurogenesis. Specifically, we found that WIP1 is expressed in NPCs of the mouse subventricular zone (SVZ) and aged animals with genetically enhanced WIP1 expression exhibited higher NPC numbers and neuronal differentiation compared with aged WT animals. Additionally, augmenting WIP1 expression in aged animals markedly improved neuron formation and rescued a functional defect in fine odor discrimination in aged mice. We identified the WNT signaling pathway inhibitor DKK3 as a key downstream target of WIP1 and found that expression of DKK3 in the SVZ is restricted to NPCs. Using murine reporter strains, we determined that DKK3 inhibits neuroblast formation by suppressing WNT signaling and Dkk3 deletion or pharmacological activation of the WNT pathway improved neuron formation and olfactory function in aged mice. We propose that WIP1 controls DKK3-dependent inhibition of neuronal differentiation during aging and suggest that regulating WIP1 levels could prevent certain aspects of functional decline of the aging brain.

Authors
Zhu, Y; Demidov, ON; Goh, AM; Virshup, DM; Lane, DP; Bulavin, DV
MLA Citation
Zhu, Y, Demidov, ON, Goh, AM, Virshup, DM, Lane, DP, and Bulavin, DV. "Phosphatase WIP1 regulates adult neurogenesis and WNT signaling during aging." The Journal of clinical investigation 124.7 (July 2014): 3263-3273.
PMID
24911145
Source
epmc
Published In
Journal of Clinical Investigation
Volume
124
Issue
7
Publish Date
2014
Start Page
3263
End Page
3273
DOI
10.1172/jci73015

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.

Wnt/β-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth cells as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (Porcn(Del)/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating that epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of Porcn(Del) organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable and that stromal production of Wnts can fully support normal murine intestinal homeostasis.

Authors
Kabiri, Z; Greicius, G; Madan, B; Biechele, S; Zhong, Z; Zaribafzadeh, H; Edison, ; Aliyev, J; Wu, Y; Bunte, R; Williams, BO; Rossant, J; Virshup, DM
MLA Citation
Kabiri, Z, Greicius, G, Madan, B, Biechele, S, Zhong, Z, Zaribafzadeh, H, Edison, , Aliyev, J, Wu, Y, Bunte, R, Williams, BO, Rossant, J, and Virshup, DM. "Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts." Development (Cambridge, England) 141.11 (June 2014): 2206-2215.
PMID
24821987
Source
epmc
Published In
Development (Cambridge)
Volume
141
Issue
11
Publish Date
2014
Start Page
2206
End Page
2215
DOI
10.1242/dev.104976

Disulfide bond requirements for active Wnt ligands.

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.

Authors
MacDonald, BT; Hien, A; Zhang, X; Iranloye, O; Virshup, DM; Waterman, ML; He, X
MLA Citation
MacDonald, BT, Hien, A, Zhang, X, Iranloye, O, Virshup, DM, Waterman, ML, and He, X. "Disulfide bond requirements for active Wnt ligands." The Journal of biological chemistry 289.26 (June 2014): 18122-18136.
PMID
24841207
Source
epmc
Published In
The Journal of biological chemistry
Volume
289
Issue
26
Publish Date
2014
Start Page
18122
End Page
18136
DOI
10.1074/jbc.m114.575027

WLS Retrograde transport to the endoplasmic reticulum during Wnt secretion

Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasizesd WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator A RF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle. © 2014 Elsevier Inc.

Authors
Yu, J; Chia, J; Canning, CA; Jones, CM; Bard, FA; Virshup, DM
MLA Citation
Yu, J, Chia, J, Canning, CA, Jones, CM, Bard, FA, and Virshup, DM. "WLS Retrograde transport to the endoplasmic reticulum during Wnt secretion." Developmental Cell 29.3 (May 12, 2014): 277-291.
Source
scopus
Published In
Developmental Cell
Volume
29
Issue
3
Publish Date
2014
Start Page
277
End Page
291
DOI
10.1016/j.devcel.2014.03.016

WLS retrograde transport to the endoplasmic reticulum during Wnt secretion.

Wnts are transported to the cell surface by the integral membrane protein WLS (also known as Wntless, Evi, and GPR177). Previous studies of WLS trafficking have emphasized WLS movement from the Golgi to the plasma membrane (PM) and then back to the Golgi via retromer-mediated endocytic recycling. We find that endogenous WLS binds Wnts in the endoplasmic reticulum (ER), cycles to the PM, and then returns to the ER through the Golgi. We identify an ER-targeting sequence at the carboxyl terminus of native WLS that is critical for ER retrograde recycling and contributes to Wnt secretory function. Golgi-to-ER recycling of WLS requires the COPI regulator ARF as well as ERGIC2, an ER-Golgi intermediate compartment protein that is also required for the retrograde trafficking of the KDEL receptor and certain toxins. ERGIC2 is required for efficient Wnt secretion. ER retrieval is an integral part of the WLS transport cycle.

Authors
Yu, J; Chia, J; Canning, CA; Jones, CM; Bard, FA; Virshup, DM
MLA Citation
Yu, J, Chia, J, Canning, CA, Jones, CM, Bard, FA, and Virshup, DM. "WLS retrograde transport to the endoplasmic reticulum during Wnt secretion." Developmental cell 29.3 (May 2014): 277-291.
PMID
24768165
Source
epmc
Published In
Developmental Cell
Volume
29
Issue
3
Publish Date
2014
Start Page
277
End Page
291
DOI
10.1016/j.devcel.2014.03.016

FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway.

Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell-cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell-cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1), profilin 2 (PFN2), protocadherin 9 (PCDH9), protocadherin α1 (PCDHA1), protocadherin β17 pseudogene (PCDHB17), protocadherin β3 (PCDHB3), sprouty homolog 1 (SPRY1) and protein tyrosine kinase 7 (PTK7)) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the Mes phenotype and cell migration via casein kinase 1ɛ-mediated non-canonical Wnt/PCP pathway.

Authors
Asad, M; Wong, MK; Tan, TZ; Choolani, M; Low, J; Mori, S; Virshup, D; Thiery, JP; Huang, RY-J
MLA Citation
Asad, M, Wong, MK, Tan, TZ, Choolani, M, Low, J, Mori, S, Virshup, D, Thiery, JP, and Huang, RY-J. "FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway." Cell death & disease 5 (January 2014): e1346-.
PMID
25032869
Source
epmc
Published In
Cell Death and Disease
Volume
5
Publish Date
2014
Start Page
e1346
DOI
10.1038/cddis.2014.302

Abstract C248: Novel PORCN inhibitors are safe and effective in the treatment of WNT-dependent cancers.

Authors
Madan, B; Ke, Z; Lim, SQY; Alam, J; Ho, SY; Jeyaraj, DA; Ghosh, K; Chew, YS; Aliyev, J; Ding, LJ; Pendharkar, V; Wang, S; Sangthongpitag, K; Keller, T; Lee, MA; Virshup, DM
MLA Citation
Madan, B, Ke, Z, Lim, SQY, Alam, J, Ho, SY, Jeyaraj, DA, Ghosh, K, Chew, YS, Aliyev, J, Ding, LJ, Pendharkar, V, Wang, S, Sangthongpitag, K, Keller, T, Lee, MA, and Virshup, DM. "Abstract C248: Novel PORCN inhibitors are safe and effective in the treatment of WNT-dependent cancers." Molecular Cancer Therapeutics 12.11_Supplement (November 1, 2013): C248-C248.
Source
crossref
Published In
Molecular cancer therapeutics
Volume
12
Issue
11_Supplement
Publish Date
2013
Start Page
C248
End Page
C248
DOI
10.1158/1535-7163.TARG-13-C248

Unwinding the Wnt action of casein kinase 1.

The casein kinase 1 (CK1) family, a major intracellular serine/threonine kinase, is implicated in multiple pathways; however, understanding its regulation has proven challenging. A recent study published in Science identifying allosteric activation of CK1 by the DEAD-box RNA helicase DDX3 expands our understanding of the control of this abundant kinase family.

Authors
Yim, DGR; Virshup, DM
MLA Citation
Yim, DGR, and Virshup, DM. "Unwinding the Wnt action of casein kinase 1." Cell Res 23.6 (June 2013): 737-738.
PMID
23567556
Source
pubmed
Published In
Cell Research
Volume
23
Issue
6
Publish Date
2013
Start Page
737
End Page
738
DOI
10.1038/cr.2013.51

BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression.

BUBR1 is a mitotic phosphoprotein essential for the maintenance of chromosome stability by promoting chromosome congression and proper kinetochore-microtubule (K-fiber) attachment, but the underlying mechanism(s) has remained elusive. Here we identify BUBR1 as a binding partner of the B56 family of Protein Phosphatase 2A regulatory subunits. The interaction between BUBR1 and the B56 family is required for chromosome congression, since point mutations in BUBR1 that block B56 binding abolish chromosome congression. The BUBR1:B56-PP2A complex opposes Aurora B kinase activity, since loss of the complex can be reverted by inhibiting Aurora B. Importantly, we show that the failure of BUBR1 to recruit B56-PP2A also contributes to the chromosome congression defects found in cells derived from patients with the Mosaic Variegated Aneuploidy (MVA) syndrome. Together, we propose that B56-PP2A is a key mediator of BUBR1's role in chromosome congression and functions by antagonizing Aurora B activity at the kinetochore for establishing stable kinetochore-microtubule attachment at the metaphase plate.

Authors
Xu, P; Raetz, EA; Kitagawa, M; Virshup, DM; Lee, SH
MLA Citation
Xu, P, Raetz, EA, Kitagawa, M, Virshup, DM, and Lee, SH. "BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression. (Published online)" Biol Open 2.5 (May 15, 2013): 479-486.
Website
http://hdl.handle.net/10161/11681
PMID
23789096
Source
pubmed
Published In
Biology Open
Volume
2
Issue
5
Publish Date
2013
Start Page
479
End Page
486
DOI
10.1242/bio.20134051

A strong correlation between expression of Wntless and of human epidermal growth factor receptor 2 in gastric, ovarian, and breast cancers suggests a novel-signalling pathway involving NFκB and STAT3

Authors
Stewart, J; James, J; McCluggage, G; McQuaid, S; Boyle, D; Arthur, K; Mullan, P; Kidson, C; McCabe, N; Kennedy, R; McArt, D; Carson, A; Yan, B; Zhengdeng, L; Tan, P; Virshup, D; Salto-Tellez, M
MLA Citation
Stewart, J, James, J, McCluggage, G, McQuaid, S, Boyle, D, Arthur, K, Mullan, P, Kidson, C, McCabe, N, Kennedy, R, McArt, D, Carson, A, Yan, B, Zhengdeng, L, Tan, P, Virshup, D, and Salto-Tellez, M. "A strong correlation between expression of Wntless and of human epidermal growth factor receptor 2 in gastric, ovarian, and breast cancers suggests a novel-signalling pathway involving NFκB and STAT3." The Lancet 381 (February 2013): S106-S106.
Source
crossref
Published In
The Lancet
Volume
381
Publish Date
2013
Start Page
S106
End Page
S106
DOI
10.1016/S0140-6736(13)60546-2

Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer.

Porcupine (PORCN) is a membrane bound O-acyltransferase that is required for Wnt palmitoylation, secretion, and biologic activity. All evaluable human Wnts require PORCN for their activity, suggesting that inhibition of PORCN could be an effective treatment for cancers dependent on excess Wnt activity. In this study, we evaluated the PORCN inhibitor Wnt-C59 (C59), to determine its activity and toxicity in cultured cells and mice. C59 inhibits PORCN activity in vitro at nanomolar concentrations, as assessed by inhibition of Wnt palmitoylation, Wnt interaction with the carrier protein Wntless/WLS, Wnt secretion, and Wnt activation of β-catenin reporter activity. In mice, C59 displayed good bioavailability, as once daily oral administration was sufficient to maintain blood concentrations well above the IC(50). C59 blocked progression of mammary tumors in MMTV-WNT1 transgenic mice while downregulating Wnt/β-catenin target genes. Surprisingly, mice exhibit no apparent toxicity, such that at a therapeutically effective dose there were no pathologic changes in the gut or other tissues. These results offer preclinical proof-of-concept that inhibiting mammalian Wnts can be achieved by targeting PORCN with small-molecule inhibitors such as C59, and that this is a safe and feasible strategy in vivo.

Authors
Proffitt, KD; Madan, B; Ke, Z; Pendharkar, V; Ding, L; Lee, MA; Hannoush, RN; Virshup, DM
MLA Citation
Proffitt, KD, Madan, B, Ke, Z, Pendharkar, V, Ding, L, Lee, MA, Hannoush, RN, and Virshup, DM. "Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer." Cancer Res 73.2 (January 15, 2013): 502-507.
PMID
23188502
Source
pubmed
Published In
Cancer Research
Volume
73
Issue
2
Publish Date
2013
Start Page
502
End Page
507
DOI
10.1158/0008-5472.CAN-12-2258

Unwinding the Wnt action of casein kinase 1

The casein kinase 1 (CK1) family, a major intracellular serine/threonine kinase, is implicated in multiple pathways; however, understanding its regulation has proven challenging. A recent study published in Science identifying allosteric activation of CK1 by the DEAD-box RNA helicase DDX3 expands our understanding of the control of this abundant kinase family. © 2013 IBCB, SIBS, CAS All rights reserved.

Authors
Yim, DGR; Virshup, DM
MLA Citation
Yim, DGR, and Virshup, DM. "Unwinding the Wnt action of casein kinase 1." Cell Research 23.6 (2013): 737-738.
Source
scival
Published In
Cell Research
Volume
23
Issue
6
Publish Date
2013
Start Page
737
End Page
738
DOI
10.1038/cr.2013.51

Identification of Small Molecule Inhibitors of Wnt Secretion

Authors
Ke, Z; Lim, S; Wang, S; Fulwood, J; Proffitt, K; Madan, B; Choong, M; Flotow, H; Virshup, D; Lee, M
MLA Citation
Ke, Z, Lim, S, Wang, S, Fulwood, J, Proffitt, K, Madan, B, Choong, M, Flotow, H, Virshup, D, and Lee, M. "Identification of Small Molecule Inhibitors of Wnt Secretion." November 2012.
Source
wos-lite
Published In
European Journal of Cancer
Volume
48
Publish Date
2012
Start Page
52
End Page
53

Precise regulation of porcupine activity is required for physiological Wnt signaling.

Gradients of diverse Wnt proteins regulate development, renewal, and differentiation. Porcupine (PORCN) is a membrane-bound O-acyltransferase that is required for post-translational modification of all Wnts to enable their transport, secretion, and activity. Mutations in PORCN are associated with focal dermal hypoplasia (FDH), whereas gene deletion causes embryonic lethality in mice. To study the protein in more detail, zinc finger nucleases were used to edit the PORCN genomic locus, establishing two HT1080 fibrosarcoma clones null for PORCN activity that facilitate the study of PORCN structure and function. We establish that PORCN is a key non-redundant node for the regulation of global Wnt signaling because PORCN null cells are completely incapable of autocrine Wnt signaling. The strength of Wnt signaling is exquisitely sensitive to PORCN expression, with a dynamic range of at least 3 orders of magnitude, suggesting that PORCN activity is a key modulator of all Wnt ligand activity. Consistent with this, we find that multiple FDH-associated mutants have only subtle alterations in enzyme activity yet are associated with a severe FDH phenotype. These studies support an essential regulatory role of PORCN in shaping Wnt signaling gradients.

Authors
Proffitt, KD; Virshup, DM
MLA Citation
Proffitt, KD, and Virshup, DM. "Precise regulation of porcupine activity is required for physiological Wnt signaling." J Biol Chem 287.41 (October 5, 2012): 34167-34178.
PMID
22888000
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
41
Publish Date
2012
Start Page
34167
End Page
34178
DOI
10.1074/jbc.M112.381970

A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities.

Wnt ligands are secreted morphogens that control multiple developmental processes during embryogenesis and adult homeostasis. A diverse set of receptors and signals have been linked to individual Wnts, but the lack of tools for comparative analysis has limited the ability to determine which of these signals are general for the entire Wnt family, and which define subsets of differently acting ligands. We have created a versatile Gateway library of clones for all 19 human Wnts. An analysis comparing epitope-tagged and untagged versions of each ligand shows that despite their similar expression at the mRNA level, Wnts exhibit considerable variation in stability, processing and secretion. At least 14 out of the 19 Wnts activate β-catenin-dependent signaling, an activity that is cell type-dependent and tracks with the stabilization of β-catenin and LRP6 phosphorylation. We find that the core Wnt modification and secretion proteins Porcupine (PORCN) and Wntless (WLS) are essential for all Wnts to signal through β-catenin-dependent and independent pathways. This comprehensive toolkit provides critical tools and new insights into human Wnt gene expression and function.

Authors
Najdi, R; Proffitt, K; Sprowl, S; Kaur, S; Yu, J; Covey, TM; Virshup, DM; Waterman, ML
MLA Citation
Najdi, R, Proffitt, K, Sprowl, S, Kaur, S, Yu, J, Covey, TM, Virshup, DM, and Waterman, ML. "A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities." Differentiation 84.2 (September 2012): 203-213.
PMID
22784633
Source
pubmed
Published In
Differentiation
Volume
84
Issue
2
Publish Date
2012
Start Page
203
End Page
213
DOI
10.1016/j.diff.2012.06.004

Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer.

Using a screen for Wnt/β-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe(2+) with a dissociation constant of ∼10(-19) M, with much weaker binding to Fe(3+) and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific β-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on β-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse β-catenin-independent cell lines. HQBA is a promising specific intracellular Fe(2+) chelator with activity against spontaneous mouse mammary cancers.

Authors
Coombs, GS; Schmitt, AA; Canning, CA; Alok, A; Low, ICC; Banerjee, N; Kaur, S; Utomo, V; Jones, CM; Pervaiz, S; Toone, EJ; Virshup, DM
MLA Citation
Coombs, GS, Schmitt, AA, Canning, CA, Alok, A, Low, ICC, Banerjee, N, Kaur, S, Utomo, V, Jones, CM, Pervaiz, S, Toone, EJ, and Virshup, DM. "Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer." Oncogene 31.2 (January 12, 2012): 213-225.
PMID
21666721
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
31
Issue
2
Publish Date
2012
Start Page
213
End Page
225
DOI
10.1038/onc.2011.228

PORCN moonlights in a Wnt-independent pathway that regulates cancer cell proliferation.

Porcupine (PORCN) is a membrane-bound O-acyl transferase that is required for the palmitoylation of Wnt proteins, and that is essential in diverse Wnt pathways for Wnt-Wntless (WLS) binding, Wnt secretion, and Wnt signaling activity. We tested if PORCN was required for the proliferation of transformed cells. Knockdown of PORCN by multiple independent siRNAs results in a cell growth defect in a subset of epithelial cancer cell lines. The growth defect is transformation-dependent in human mammary epithelial (HMEC) cells. Additionally, inducible PORCN knockdown by two independent shRNAs markedly reduces the growth of established MDA-MB-231 cancers in orthotopic xenografts in immunodeficient mice. Unexpectedly, the proliferation defect resulting from loss of PORCN occurs in a Wnt-independent manner, as it is rescued by re-expression of catalytically inactive PORCN, and is not seen after RNAi-mediated knockdown of the Wnt carrier protein WLS, nor after treatment with the PORCN inhibitor IWP. Consistent with a role in a Wnt-independent pathway, knockdown of PORCN regulates a distinct set of genes that are not altered by other inhibitors of Wnt signaling. PORCN protein thus appears to moonlight in a novel signaling pathway that is rate-limiting for cancer cell growth and tumorigenesis independent of its enzymatic function in Wnt biosynthesis and secretion.

Authors
Covey, TM; Kaur, S; Tan Ong, T; Proffitt, KD; Wu, Y; Tan, P; Virshup, DM
MLA Citation
Covey, TM, Kaur, S, Tan Ong, T, Proffitt, KD, Wu, Y, Tan, P, and Virshup, DM. "PORCN moonlights in a Wnt-independent pathway that regulates cancer cell proliferation." PLoS One 7.4 (2012): e34532-.
PMID
22509316
Source
pubmed
Published In
PloS one
Volume
7
Issue
4
Publish Date
2012
Start Page
e34532
DOI
10.1371/journal.pone.0034532

A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures.

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.

Authors
Ooi, CH; Oh, HK; Wang, HZ; Tan, ALK; Wu, J; Lee, M; Rha, SY; Chung, HC; Virshup, DM; Tan, P
MLA Citation
Ooi, CH, Oh, HK, Wang, HZ, Tan, ALK, Wu, J, Lee, M, Rha, SY, Chung, HC, Virshup, DM, and Tan, P. "A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures." PLoS Genet 7.12 (December 2011): e1002415-.
PMID
22194702
Source
pubmed
Published In
PLoS genetics
Volume
7
Issue
12
Publish Date
2011
Start Page
e1002415
DOI
10.1371/journal.pgen.1002415

IC261 induces cell cycle arrest and apoptosis of human cancer cells via CK1δ/ɛ and Wnt/β-catenin independent inhibition of mitotic spindle formation.

Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/β-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/β-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/β-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.

Authors
Cheong, JK; Nguyen, TH; Wang, H; Tan, P; Voorhoeve, PM; Lee, SH; Virshup, DM
MLA Citation
Cheong, JK, Nguyen, TH, Wang, H, Tan, P, Voorhoeve, PM, Lee, SH, and Virshup, DM. "IC261 induces cell cycle arrest and apoptosis of human cancer cells via CK1δ/ɛ and Wnt/β-catenin independent inhibition of mitotic spindle formation." Oncogene 30.22 (June 2, 2011): 2558-2569.
PMID
21258417
Source
pubmed
Published In
Oncogene: Including Oncogene Reviews
Volume
30
Issue
22
Publish Date
2011
Start Page
2558
End Page
2569
DOI
10.1038/onc.2010.627

Casein kinase 1: Complexity in the family.

The CK1 family of serine/threonine kinases regulates diverse cellular processes, through binding to and phosphorylation a myriad of protein substrates. CK1 prefers substrates primed by prior phosphorylation, and works closely with other kinases in the Wnt pathway. CK1 is itself regulated by posttranslational modification, including autophosphorylation. We provide a brief overview of the fundamentals of CK1 biology with an emphasis on scaffold binding and kinase regulation in Wnt signaling and circadian rhythms.

Authors
Cheong, JK; Virshup, DM
MLA Citation
Cheong, JK, and Virshup, DM. "Casein kinase 1: Complexity in the family." Int J Biochem Cell Biol 43.4 (April 2011): 465-469. (Review)
PMID
21145983
Source
pubmed
Published In
The International Journal of Biochemistry and Cell Biology
Volume
43
Issue
4
Publish Date
2011
Start Page
465
End Page
469
DOI
10.1016/j.biocel.2010.12.004

Cell biology. Enforcing the Greatwall in mitosis.

Authors
Virshup, DM; Kaldis, P
MLA Citation
Virshup, DM, and Kaldis, P. "Cell biology. Enforcing the Greatwall in mitosis." Science 330.6011 (December 17, 2010): 1638-1639.
PMID
21164006
Source
pubmed
Published In
Science
Volume
330
Issue
6011
Publish Date
2010
Start Page
1638
End Page
1639
DOI
10.1126/science.1199898

Alkylation of the tumor suppressor PTEN activates Akt and β-catenin signaling: a mechanism linking inflammation and oxidative stress with cancer.

PTEN, a phosphoinositide-3-phosphatase, serves dual roles as a tumor suppressor and regulator of cellular anabolic/catabolic metabolism. Adaptation of a redox-sensitive cysteinyl thiol in PTEN for signal transduction by hydrogen peroxide may have superimposed a vulnerability to other mediators of oxidative stress and inflammation, especially reactive carbonyl species, which are commonly occurring by-products of arachidonic acid peroxidation. Using MCF7 and HEK-293 cells, we report that several reactive aldehydes and ketones, e.g. electrophilic α,β-enals (acrolein, 4-hydroxy-2-nonenal) and α,β-enones (prostaglandin A(2), Δ12-prostaglandin J(2) and 15-deoxy-Δ-12,14-prostaglandin J(2)) covalently modify and inactivate cellular PTEN, with ensuing activation of PKB/Akt kinase; phosphorylation of Akt substrates; increased cell proliferation; and increased nuclear β-catenin signaling. Alkylation of PTEN by α,β-enals/enones and interference with its restraint of cellular PKB/Akt signaling may accentuate hyperplastic and neoplastic disorders associated with chronic inflammation, oxidative stress, or aging.

Authors
Covey, TM; Edes, K; Coombs, GS; Virshup, DM; Fitzpatrick, FA
MLA Citation
Covey, TM, Edes, K, Coombs, GS, Virshup, DM, and Fitzpatrick, FA. "Alkylation of the tumor suppressor PTEN activates Akt and β-catenin signaling: a mechanism linking inflammation and oxidative stress with cancer. (Published online)" PLoS One 5.10 (October 21, 2010): e13545-.
PMID
20975834
Source
pubmed
Published In
PloS one
Volume
5
Issue
10
Publish Date
2010
Start Page
e13545
DOI
10.1371/journal.pone.0013545

WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification.

Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/β-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding β-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

Authors
Coombs, GS; Yu, J; Canning, CA; Veltri, CA; Covey, TM; Cheong, JK; Utomo, V; Banerjee, N; Zhang, ZH; Jadulco, RC; Concepcion, GP; Bugni, TS; Harper, MK; Mihalek, I; Jones, CM; Ireland, CM; Virshup, DM
MLA Citation
Coombs, GS, Yu, J, Canning, CA, Veltri, CA, Covey, TM, Cheong, JK, Utomo, V, Banerjee, N, Zhang, ZH, Jadulco, RC, Concepcion, GP, Bugni, TS, Harper, MK, Mihalek, I, Jones, CM, Ireland, CM, and Virshup, DM. "WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification." J Cell Sci 123.Pt 19 (October 1, 2010): 3357-3367.
PMID
20826466
Source
pubmed
Published In
Journal of cell science
Volume
123
Issue
Pt 19
Publish Date
2010
Start Page
3357
End Page
3367
DOI
10.1242/jcs.072132

Carteriosulfonic acids A-C, GSK-3beta inhibitors from a Carteriospongia sp.

Modulators of Wnt signaling have therapeutic potential in a number of human diseases. A fractionated library from marine invertebrates was screened in a luciferase assay designed to identify modulators of Wnt signaling. A fraction from a Carteriospongia sp. sponge activated Wnt signaling and was subsequently shown to inhibit GSK-3beta, which inhibits Wnt signaling through phosphorylation of beta-catenin. Three novel natural products, carteriosulfonic acids A (1), B (2), and C (3), were identified as active constituents. The carteriosulfonic acids contain unprecedented 4,6,7,9-tetrahydroxylated decanoic acid subunits. Their structures were elucidated through analysis of NMR data and a detailed analysis of pseudo MS(3) spectra.

Authors
McCulloch, MWB; Bugni, TS; Concepcion, GP; Coombs, GS; Harper, MK; Kaur, S; Mangalindan, GC; Mutizwa, MM; Veltri, CA; Virshup, DM; Ireland, CM
MLA Citation
McCulloch, MWB, Bugni, TS, Concepcion, GP, Coombs, GS, Harper, MK, Kaur, S, Mangalindan, GC, Mutizwa, MM, Veltri, CA, Virshup, DM, and Ireland, CM. "Carteriosulfonic acids A-C, GSK-3beta inhibitors from a Carteriospongia sp." J Nat Prod 72.9 (September 2009): 1651-1656.
PMID
19778090
Source
pubmed
Published In
Journal of Natural Products
Volume
72
Issue
9
Publish Date
2009
Start Page
1651
End Page
1656
DOI
10.1021/np900336f

08-P022 Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells

Authors
Wang, C; Chang, K; Somers, G; Virshup, D; Ti Ang, B; Tang, C; Yu, F; Wang, H
MLA Citation
Wang, C, Chang, K, Somers, G, Virshup, D, Ti Ang, B, Tang, C, Yu, F, and Wang, H. "08-P022 Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells." August 2009.
Source
crossref
Published In
Mechanisms of Development
Volume
126
Publish Date
2009
Start Page
S150
End Page
S150
DOI
10.1016/j.mod.2009.06.329

Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells.

Drosophila larval brain neural stem cells, also known as neuroblasts, divide asymmetrically to generate a self-renewing neuroblast and a ganglion mother cell (GMC) that divides terminally to produce two differentiated neurons or glia. Failure of asymmetric cell division can result in hyperproliferation of neuroblasts, a phenotype resembling brain tumors. Here we have identified Drosophila Protein phosphatase 2A (PP2A) as a brain tumor-suppressor that can inhibit self-renewal of neuroblasts. Supernumerary larval brain neuroblasts are generated at the expense of differentiated neurons in PP2A mutants. Neuroblast overgrowth was observed in both dorsomedial (DM)/posterior Asense-negative (PAN) neuroblast lineages and non-DM neuroblast lineages. The PP2A heterotrimeric complex, composed of the catalytic subunit (Mts), scaffold subunit (PP2A-29B) and a B-regulatory subunit (Tws), is required for the asymmetric cell division of neuroblasts. The PP2A complex regulates asymmetric localization of Numb, Pon and Atypical protein kinase C, as well as proper mitotic spindle orientation. Interestingly, PP2A and Polo kinase enhance Numb and Pon phosphorylation. PP2A, like Polo, acts to prevent excess neuroblast self-renewal primarily by regulating asymmetric localization and activation of Numb. Reduction of PP2A function in larval brains or S2 cells causes a marked decrease in Polo transcript and protein abundance. Overexpression of Polo or Numb significantly suppresses neuroblast overgrowth in PP2A mutants, suggesting that PP2A inhibits excess neuroblast self-renewal in the Polo/Numb pathway.

Authors
Wang, C; Chang, KC; Somers, G; Virshup, D; Ang, BT; Tang, C; Yu, F; Wang, H
MLA Citation
Wang, C, Chang, KC, Somers, G, Virshup, D, Ang, BT, Tang, C, Yu, F, and Wang, H. "Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells." Development 136.13 (July 2009): 2287-2296.
PMID
19502489
Source
pubmed
Published In
Development (Cambridge)
Volume
136
Issue
13
Publish Date
2009
Start Page
2287
End Page
2296
DOI
10.1242/dev.035758

Keeping the beat in the rising heat.

Circadian clocks use temperature compensation to keep accurate time over a range of temperatures, thus allowing reliable timekeeping under diverse environmental conditions. Mehra et al. (2009) and Baker et al. (2009) now show that phosphorylation-regulated protein degradation plays a key role in circadian temperature compensation.

Authors
Virshup, DM; Forger, DB
MLA Citation
Virshup, DM, and Forger, DB. "Keeping the beat in the rising heat." Cell 137.4 (May 15, 2009): 602-604.
PMID
19450508
Source
pubmed
Published In
Cell
Volume
137
Issue
4
Publish Date
2009
Start Page
602
End Page
604
DOI
10.1016/j.cell.2009.04.051

Psammaplin A as a general activator of cell-based signaling assays via HDAC inhibition and studies on some bromotyrosine derivatives.

The Wnt signaling pathway regulates cell growth and development in metazoans, and is therefore of interest for drug discovery. By screening a library of 5808 pre-fractionated marine extracts in a cell-based Wnt signaling assay, several signaling activators and inhibitors were observed. LCMS-based fractionation rapidly identified an active compound from Pseudoceratina purpurea as psammaplin A, a known HDAC inhibitor. Other HDAC inhibitors similarly activated signaling in this assay, indicating HDAC inhibitors will be identified through many cell-based reporter assays. In a large scale analysis of P. purpurea, three previously undescribed bromotyrosine based natural products were identified; the structure of one of these was confirmed by synthesis. Additionally, three other derivatives of psammaplin A were prepared: a mixed disulfide and two sulfinate esters. Finally, evidence to support a structural reassignment of psammaplin I from a sulfone to the isomeric sulfinate ester is presented.

Authors
McCulloch, MWB; Coombs, GS; Banerjee, N; Bugni, TS; Cannon, KM; Harper, MK; Veltri, CA; Virshup, DM; Ireland, CM
MLA Citation
McCulloch, MWB, Coombs, GS, Banerjee, N, Bugni, TS, Cannon, KM, Harper, MK, Veltri, CA, Virshup, DM, and Ireland, CM. "Psammaplin A as a general activator of cell-based signaling assays via HDAC inhibition and studies on some bromotyrosine derivatives." Bioorg Med Chem 17.6 (March 15, 2009): 2189-2198.
PMID
19022675
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
17
Issue
6
Publish Date
2009
Start Page
2189
End Page
2198
DOI
10.1016/j.bmc.2008.10.077

From promiscuity to precision: protein phosphatases get a makeover.

The control of biological events requires strict regulation using complex protein phosphorylation and dephosphorylation strategies. The bulk of serine-threonine dephosphorylations are catalyzed by a handful of phosphatase catalytic subunits, giving rise to the misconception that these phosphatases are promiscuous and unregulated enzymes in vivo. The reality is much more nuanced: PP1 and PP2A, the most abundant serine-threonine phosphatases, are, in fact, families of hundreds of protein serine/threonine phosphatases, assembled from a few catalytic subunits in combination with a highly diverse array of regulators. As recent publications illustrate, these regulatory subunits confer specificity, selectivity, localization, and regulation on these important enzymes.

Authors
Virshup, DM; Shenolikar, S
MLA Citation
Virshup, DM, and Shenolikar, S. "From promiscuity to precision: protein phosphatases get a makeover." Mol Cell 33.5 (March 13, 2009): 537-545. (Review)
PMID
19285938
Source
pubmed
Published In
Molecular Cell
Volume
33
Issue
5
Publish Date
2009
Start Page
537
End Page
545
DOI
10.1016/j.molcel.2009.02.015

Erratum: Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells (Development (2009) vol. 136 (2287-2296))

Authors
Wang, C; Chang, KC; Somers, G; Virshup, D; Ang, BT; Tang, C; Yu, F; Wang, H
MLA Citation
Wang, C, Chang, KC, Somers, G, Virshup, D, Ang, BT, Tang, C, Yu, F, and Wang, H. "Erratum: Protein phosphatase 2A regulates self-renewal of Drosophila neural stem cells (Development (2009) vol. 136 (2287-2296))." Development 136.17 (2009): 3031--.
Source
scival
Published In
Development (Cambridge)
Volume
136
Issue
17
Publish Date
2009
Start Page
3031-
DOI
10.1242/dev.042432

Wnt signaling in development, disease and translational medicine.

Wnt signaling regulates a multitude of critical processes in development and tissue homeostasis. The wingless (wg) gene product was first identified in Drosophila in 1973. Subsequently, the proto-oncogene INT-1 was identified in mice in 1984 when its activation by mouse mammary tumor virus' proviral insertion was found to induce tumor formation. The discovery in 1987 that wg and INT-1 are orthologues contributed to an appreciation of the intimate connection between oncogenic and developmental processes. Diverse diseases including cancer, diabetes, osteoporosis and psychiatric disorders may be amenable to treatment via modulation of Wnt-mediated signaling pathways. There are a number of attractive targets that are the object of ongoing drug development studies aiming to capitalize on these opportunities. In this review, we present a historical overview of key events in this field that have elucidated the known signaling cascades associated with Wnt ligands and shaped our understanding of the roles of these cascades in physiological and pathological processes.

Authors
Coombs, GS; Covey, TM; Virshup, DM
MLA Citation
Coombs, GS, Covey, TM, and Virshup, DM. "Wnt signaling in development, disease and translational medicine." Curr Drug Targets 9.7 (July 2008): 513-531. (Review)
PMID
18673238
Source
pubmed
Published In
Current Drug Targets
Volume
9
Issue
7
Publish Date
2008
Start Page
513
End Page
531

Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins.

The intrinsic period of circadian clocks is their defining adaptive property. To identify the biochemical mechanisms whereby casein kinase1 (CK1) determines circadian period in mammals, we created mouse null and tau mutants of Ck1 epsilon. Circadian period lengthened in CK1epsilon-/-, whereas CK1epsilon(tau/tau) shortened circadian period of behavior in vivo and suprachiasmatic nucleus firing rates in vitro, by accelerating PERIOD-dependent molecular feedback loops. CK1epsilon(tau/tau) also accelerated molecular oscillations in peripheral tissues, revealing its global role in circadian pacemaking. CK1epsilon(tau) acted by promoting degradation of both nuclear and cytoplasmic PERIOD, but not CRYPTOCHROME, proteins. Together, these whole-animal and biochemical studies explain how tau, as a gain-of-function mutation, acts at a specific circadian phase to promote degradation of PERIOD proteins and thereby accelerate the mammalian clockwork in brain and periphery.

Authors
Meng, Q-J; Logunova, L; Maywood, ES; Gallego, M; Lebiecki, J; Brown, TM; Sládek, M; Semikhodskii, AS; Glossop, NRJ; Piggins, HD; Chesham, JE; Bechtold, DA; Yoo, S-H; Takahashi, JS; Virshup, DM; Boot-Handford, RP; Hastings, MH; Loudon, ASI
MLA Citation
Meng, Q-J, Logunova, L, Maywood, ES, Gallego, M, Lebiecki, J, Brown, TM, Sládek, M, Semikhodskii, AS, Glossop, NRJ, Piggins, HD, Chesham, JE, Bechtold, DA, Yoo, S-H, Takahashi, JS, Virshup, DM, Boot-Handford, RP, Hastings, MH, and Loudon, ASI. "Setting clock speed in mammals: the CK1 epsilon tau mutation in mice accelerates circadian pacemakers by selectively destabilizing PERIOD proteins." Neuron 58.1 (April 10, 2008): 78-88.
PMID
18400165
Source
pubmed
Published In
Neuron
Volume
58
Issue
1
Publish Date
2008
Start Page
78
End Page
88
DOI
10.1016/j.neuron.2008.01.019

Control of mitotic exit by PP2A regulation of Cdc25C and Cdk1.

Inactivation of maturation-promoting factor [(MPF) Cdk1/Cyclin B] is a key event in the exit from mitosis. Although degradation of Cyclin B is important for MPF inactivation, recent studies indicate that Cdk1 phosphorylation and inactivation occur before Cyclin B degradation and, therefore, also may be important steps in the exit from mitosis. Cdk1 activity is controlled by the Cdc25C phosphatase, which is turned on at the G(2)/M transition to catalyze Cdk1 activation. PP2A:B56delta is a negative regulator of Cdc25C during interphase. We show here that PP2A:B56delta also regulates Cdc25C at mitosis. Failure of PP2A:B56delta to dephosphorylate Cdc25C at mitosis results in prolonged hyperphosphorylation and activation of Cdc25C, causing persistent dephosphorylation and, hence, activation of Cdk1. This constitutive activation of Cdc25C and Cdk1 leads to a delayed exit from mitosis. Consistent with Cdk1 as a major biological target of B56delta, stable knockdown and germ-line mouse KO of B56delta leads to compensatory transcriptional up-regulation of Wee1 kinase to oppose the Cdc25C activity and permit cell survival. These observations place PP2A:B56delta as a key upstream regulator of Cdk1 activity upon exit from mitosis.

Authors
Forester, CM; Maddox, J; Louis, JV; Goris, J; Virshup, DM
MLA Citation
Forester, CM, Maddox, J, Louis, JV, Goris, J, and Virshup, DM. "Control of mitotic exit by PP2A regulation of Cdc25C and Cdk1." Proc Natl Acad Sci U S A 104.50 (December 11, 2007): 19867-19872.
PMID
18056802
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
104
Issue
50
Publish Date
2007
Start Page
19867
End Page
19872
DOI
10.1073/pnas.0709879104

After hours keeps clock researchers CRYing Overtime.

Three recent reports, including one in this issue of Cell, reveal that the circadian regulator CRY is targeted for degradation by the F box E3 ubiquitin ligase FBXL3 (Siepka et al., 2007; Busino et al., 2007; Godinho et al., 2007). These studies confirm the importance of targeted protein degradation as a key design feature of the mammalian circadian clock.

Authors
Virshup, DM; Forger, DB
MLA Citation
Virshup, DM, and Forger, DB. "After hours keeps clock researchers CRYing Overtime." Cell 129.5 (June 1, 2007): 857-859.
PMID
17540165
Source
pubmed
Published In
Cell
Volume
129
Issue
5
Publish Date
2007
Start Page
857
End Page
859
DOI
10.1016/j.cell.2007.05.015

Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex.

The Wnt/beta-catenin signaling pathway is critical in both cellular proliferation and organismal development. However, how the beta-catenin degradation complex is inhibited upon Wnt activation remains unclear. Using a directed RNAi screen we find that protein phosphatase 1 (PP1), a ubiquitous serine/threonine phosphatase, is a novel potent positive physiologic regulator of the Wnt/beta-catenin signaling pathway. PP1 expression synergistically activates, and inhibition of PP1 inhibits, Wnt/beta-catenin signaling in Drosophila and mammalian cells as well as in Xenopus embryos. The data suggest that PP1 controls Wnt signaling through interaction with, and regulated dephosphorylation of, axin. Inhibition of PP1 leads to enhanced phosphorylation of specific sites on axin by casein kinase I. Axin phosphorylation markedly enhances the binding of glycogen synthase kinase 3, leading to a more active beta-catenin destruction complex. Wnt-regulated changes in axin phosphorylation, mediated by PP1, may therefore determine beta-catenin transcriptional activity. Specific inhibition of PP1 in this pathway may offer therapeutic approaches to disorders with increased beta-catenin signaling.

Authors
Luo, W; Peterson, A; Garcia, BA; Coombs, G; Kofahl, B; Heinrich, R; Shabanowitz, J; Hunt, DF; Yost, HJ; Virshup, DM
MLA Citation
Luo, W, Peterson, A, Garcia, BA, Coombs, G, Kofahl, B, Heinrich, R, Shabanowitz, J, Hunt, DF, Yost, HJ, and Virshup, DM. "Protein phosphatase 1 regulates assembly and function of the beta-catenin degradation complex." EMBO J 26.6 (March 21, 2007): 1511-1521.
PMID
17318175
Source
pubmed
Published In
EMBO Journal
Volume
26
Issue
6
Publish Date
2007
Start Page
1511
End Page
1521
DOI
10.1038/sj.emboj.7601607

Disease-associated casein kinase I delta mutation may promote adenomatous polyps formation via a Wnt/beta-catenin independent mechanism.

The Wnt signaling pathway is critical for embryonic development and is dysregulated in multiple cancers. Two closely related isoforms of casein kinase I (CKIdelta and epsilon) are positive regulators of this pathway. We speculated that mutations in the autoinhibitory domain of CKIdelta/epsilon might upregulate CKIdelta/epsilon activity and hence Wnt signaling and increase the risk of adenomatous polyps and colon cancer. Exons encoding the CKIepsilon and CKIdelta regulatory domains were sequenced from DNA obtained from individuals with adenomatous polyps and a family history of colon cancer unaffected by familial adenomatous polyposis or hereditary nonpolyposis colorectal cancer (HNPCC). A CKIdelta missense mutation, changing a highly conserved residue, Arg324, to His (R324H), was found in an individual with large and multiple polyps diagnosed at a relatively young age. Two findings indicate that this mutation is biologically active. First, ectopic ventral expression of CKIdelta(R324H) in Xenopus embryos results in secondary axis formation with an additional distinctive phenotype (altered morphological movements) similar to that seen with unregulated CKIepsilon. Second, CKIdelta(R324H) is more potent than wildtype CKIdelta in transformation of RKO colon cancer cells. Although the R324H mutation does not significantly change CKIdelta kinase activity in an in vitro kinase assay or Wnt/beta-catenin signal transduction as assessed by a beta-catenin reporter assay, it alters morphogenetic movements via a beta-catenin-independent mechanism in early Xenopus development. This novel human CKIdelta mutation may alter the physiological role and enhance the transforming ability of CKIdelta through a Wnt/beta-catenin independent mechanism and thereby influence colonic adenoma development.

Authors
Tsai, I-C; Woolf, M; Neklason, DW; Branford, WW; Yost, HJ; Burt, RW; Virshup, DM
MLA Citation
Tsai, I-C, Woolf, M, Neklason, DW, Branford, WW, Yost, HJ, Burt, RW, and Virshup, DM. "Disease-associated casein kinase I delta mutation may promote adenomatous polyps formation via a Wnt/beta-catenin independent mechanism." Int J Cancer 120.5 (March 1, 2007): 1005-1012.
PMID
17131344
Source
pubmed
Published In
International Journal of Cancer
Volume
120
Issue
5
Publish Date
2007
Start Page
1005
End Page
1012
DOI
10.1002/ijc.22368

A Wnt-CKIvarepsilon-Rap1 pathway regulates gastrulation by modulating SIPA1L1, a Rap GTPase activating protein.

Noncanonical Wnt signals control morphogenetic movements during vertebrate gastrulation. Casein kinase I epsilon (CKIvarepsilon) is a Wnt-regulated kinase that regulates Wnt/beta-catenin signaling and has a beta-catenin-independent role(s) in morphogenesis that is poorly understood. Here we report the identification of a CKIvarepsilon binding partner, SIPA1L1/E6TP1, a GAP (GTPase activating protein) of the Rap small GTPase family. We show that CKIvarepsilon phosphorylates SIPA1L1 to reduce its stability and thereby increase Rap1 activation. Wnt-8, which activates CKIvarepsilon, enhances the CKIvarepsilon-dependent phosphorylation and degradation of SIPA1L1. In early Xenopus or zebrafish development, inactivation of the Rap1 pathway results in abnormal gastrulation and a shortened anterior-posterior axis. Although CKIvarepsilon also transduces Wnt/beta-catenin signaling, inhibition of Rap1 does not alter beta-catenin-regulated gene expression. Our data demonstrate a role for CKIvarepsilon in noncanonical Wnt signaling and indicate that Wnt regulates morphogenesis in part through CKIvarepsilon-mediated control of Rap1 signaling.

Authors
Tsai, I-C; Amack, JD; Gao, Z-H; Band, V; Yost, HJ; Virshup, DM
MLA Citation
Tsai, I-C, Amack, JD, Gao, Z-H, Band, V, Yost, HJ, and Virshup, DM. "A Wnt-CKIvarepsilon-Rap1 pathway regulates gastrulation by modulating SIPA1L1, a Rap GTPase activating protein." Dev Cell 12.3 (March 2007): 335-347.
PMID
17336901
Source
pubmed
Published In
Developmental Cell
Volume
12
Issue
3
Publish Date
2007
Start Page
335
End Page
347
DOI
10.1016/j.devcel.2007.02.009

Post-translational modifications regulate the ticking of the circadian clock.

Getting a good night's sleep is on everyone's to-do list. So is, no doubt, staying awake during late afternoon seminars. Our internal clocks control these and many more workings of the body, and disruptions of the circadian clocks predispose individuals to depression, obesity and cancer. Mutations in kinases and phosphatases in hamsters, flies, fungi and humans highlight how our timepieces are regulated and provide clues as to how we might be able to manipulate them.

Authors
Gallego, M; Virshup, DM
MLA Citation
Gallego, M, and Virshup, DM. "Post-translational modifications regulate the ticking of the circadian clock." Nat Rev Mol Cell Biol 8.2 (February 2007): 139-148. (Review)
PMID
17245414
Source
pubmed
Published In
Nature Reviews Molecular Cell Biology
Volume
8
Issue
2
Publish Date
2007
Start Page
139
End Page
148
DOI
10.1038/nrm2106

Differential expression of the B'beta regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

Authors
Saraf, A; Virshup, DM; Strack, S
MLA Citation
Saraf, A, Virshup, DM, and Strack, S. "Differential expression of the B'beta regulatory subunit of protein phosphatase 2A modulates tyrosine hydroxylase phosphorylation and catecholamine synthesis." J Biol Chem 282.1 (January 5, 2007): 573-580.
PMID
17085438
Source
pubmed
Published In
The Journal of biological chemistry
Volume
282
Issue
1
Publish Date
2007
Start Page
573
End Page
580
DOI
10.1074/jbc.M607407200

Beyond intuitive modeling: Combining biophysical models with innovative experiments to move the circadian clock field forward

Two major approaches have been used to model circadian clocks. Qualitative modeling, used prior to the recent wealth of detailed molecular knowledge, makes general predictions but cannot provide detailed mechanistic insights. The more recent biophysical approach, on the other hand, incorporates the biochemical events that drive the clock and can make detailed and testable molecular predictions. These predictions are being tested using new experimental techniques that measure reaction kinetics and the behavior of individual cells. A joint modeling and experimental approach has recently been used to understand how mutations affecting phosphorylation can lead to a short circadian period in tau mutant hamsters and in humans with familial advanced sleep phase syndrome (FASPS). Another recent study has revealed novel single-cell phenotypes of clock gene mutations, demanding revision of current biophysical models yet validating certain model predictions that were previously overlooked. A new paradigm for clock research is emerging in which modeling inspires new experimental efforts, experimental data inspire new modeling efforts, and joint modeling/experimental studies lead to a deeper understanding of mammalian circadian rhythms. © 2007 Sage Publications.

Authors
Forger, D; Gonze, D; Virshup, D; Welsh, DK
MLA Citation
Forger, D, Gonze, D, Virshup, D, and Welsh, DK. "Beyond intuitive modeling: Combining biophysical models with innovative experiments to move the circadian clock field forward." Journal of Biological Rhythms 22.3 (2007): 200-210.
PMID
17517910
Source
scival
Published In
Journal of Biological Rhythms
Volume
22
Issue
3
Publish Date
2007
Start Page
200
End Page
210
DOI
10.1177/0748730407301823

Reversible protein phosphorylation regulates circadian rhythms.

Protein phosphorylation regulates the period of the circadian clock within mammalian cells. Circadian rhythms are an approximately 24-hour cycle that regulates key biological processes. Daily fluctuations of wakefulness, stress hormones, lipid metabolism, immune function, and the cell division cycle are controlled by the molecular clocks that function throughout our bodies. Mutations in regulatory components of the clock can shorten or lengthen the timing of the rhythms and have significant physiological consequences. The clock is formed by a negative feedback loop of transcription, translation, and inhibition of transcription. The precision of clock timing is controlled by protein kinases and phosphatases. Casein kinase Iepsilon is a protein kinase that regulates the circadian clock by periodic phosphorylation of the proteins PER1 and PER2, controlling their stability and localization. The role of phosphorylation in regulating PER function in the clock has been explored in detail. Quantitative modeling has proven to be very useful in making important predictions about how changes in phosphorylation alter the clock's behavior. Quantitative data from biological studies can be used to refine the quantitative model and make additional testable predictions. A detailed understanding of how reversible protein phosphorylation regulates circadian rhythms and a detailed quantitative model that makes clear, testable, and accurate predictions about the clock and how we may manipulate it can have important benefits for human health. Pharmacological manipulation of rhythms could mitigate stress from jet lag, shift work, and perhaps even seasonal affective disorder.

Authors
Virshup, DM; Eide, EJ; Forger, DB; Gallego, M; Harnish, EV
MLA Citation
Virshup, DM, Eide, EJ, Forger, DB, Gallego, M, and Harnish, EV. "Reversible protein phosphorylation regulates circadian rhythms." Cold Spring Harb Symp Quant Biol 72 (2007): 413-420. (Review)
PMID
18419299
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
72
Publish Date
2007
Start Page
413
End Page
420
DOI
10.1101/sqb.2007.72.048

Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis.

DNA-responsive checkpoints prevent cell-cycle progression following DNA damage or replication inhibition. The mitotic activator Cdc25 is suppressed by checkpoints through inhibitory phosphorylation at Ser287 (Xenopus numbering) and docking of 14-3-3. Ser287 phosphorylation is a major locus of G2/M checkpoint control, although several checkpoint-independent kinases can phosphorylate this site. We reported previously that mitotic entry requires 14-3-3 removal and Ser287 dephosphorylation. We show here that DNA-responsive checkpoints also activate PP2A/B56delta phosphatase complexes to dephosphorylate Cdc25 at a site distinct from Ser287 (T138), the phosphorylation of which is required for 14-3-3 release. However, phosphorylation of T138 is not sufficient for 14-3-3 release from Cdc25. Our data suggest that creation of a 14-3-3 "sink," consisting of phosphorylated 14-3-3 binding intermediate filament proteins, including keratins, coupled with reduced Cdc25-14-3-3 affinity, contribute to Cdc25 activation. These observations identify PP2A/B56delta as a central checkpoint effector and suggest a mechanism for controlling 14-3-3 interactions to promote mitosis.

Authors
Margolis, SS; Perry, JA; Forester, CM; Nutt, LK; Guo, Y; Jardim, MJ; Thomenius, MJ; Freel, CD; Darbandi, R; Ahn, J-H; Arroyo, JD; Wang, X-F; Shenolikar, S; Nairn, AC; Dunphy, WG; Hahn, WC; Virshup, DM; Kornbluth, S
MLA Citation
Margolis, SS, Perry, JA, Forester, CM, Nutt, LK, Guo, Y, Jardim, MJ, Thomenius, MJ, Freel, CD, Darbandi, R, Ahn, J-H, Arroyo, JD, Wang, X-F, Shenolikar, S, Nairn, AC, Dunphy, WG, Hahn, WC, Virshup, DM, and Kornbluth, S. "Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis." Cell 127.4 (November 17, 2006): 759-773.
PMID
17110335
Source
pubmed
Published In
Cell
Volume
127
Issue
4
Publish Date
2006
Start Page
759
End Page
773
DOI
10.1016/j.cell.2006.10.035

Protein phosphatase 1 regulates the stability of the circadian protein PER2.

The circadian clock is regulated by a transcription/translation negative feedback loop. A key negative regulator of circadian rhythm in mammals is the PER2 (mammalian PERIOD 2) protein. Its daily degradation at the end of the night accompanies de-repression of transcription. CKI (casein kinase I ) has been identified as the kinase that phosphorylates PER2, targeting it for ubiquitin-mediated proteasomal degradation. We now report that PER2 degradation is also negatively regulated by PP1 (protein phosphatase 1)-mediated dephosphorylation. In Xenopus egg extract, PP1 inhibition by Inhibitor-2 accelerated mPER2 degradation. Co-immunoprecipitation experiments showed that PER2 bound to PP1c in transfected HEK-293 cells. PP1 immunoprecipitated from HEK-293 cells, mouse liver and mouse brain, dephosphorylated CKI-phosphorylated PER2, showing that PER2 is a substrate for mammalian endogenous PP1. Moreover, over-expression of the dominant negative form of PP1c, the D95N mutant, accelerated ubiquitin and proteasome-mediated degradation of PER2, and shortened the PER2 half-life in HEK-293 cells. Over-expression of the PP1 inhibitors, protein phosphatase 1 holoenzyme inhibitor-1 and Inhibitor-2, confirmed these results. Thus PP1 regulates PER2 stability and is therefore a candidate to regulate mammalian circadian rhythms.

Authors
Gallego, M; Kang, H; Virshup, DM
MLA Citation
Gallego, M, Kang, H, and Virshup, DM. "Protein phosphatase 1 regulates the stability of the circadian protein PER2." Biochem J 399.1 (October 1, 2006): 169-175.
PMID
16813562
Source
pubmed
Published In
The Biochemical journal
Volume
399
Issue
1
Publish Date
2006
Start Page
169
End Page
175
DOI
10.1042/BJ20060678

Site-specific casein kinase 1epsilon-dependent phosphorylation of Dishevelled modulates beta-catenin signaling.

Careful regulation of the Wnt-Beta-catenin signaling pathway is critical to many aspects of development and cancer. Casein kinase Iepsilon is a Wnt-activated positive regulator of this pathway. Members of the Dishevelled family have been identified as key substrates of casein kinase I (CKI). However, the specific sites phosphorylated in vivo by CKI and their relative importance in the physiologic regulation of these proteins in the canonical Wnt-beta-catenin signaling pathway remain unclear. To address this question, recombinant mouse Dishevelled (mDvl-1) was phosphorylated by CKIin vitro and phosphorylation sites were identified by MS. CKI phosphorylation of mDvl-1 at two highly conserved residues, serines 139 and 142, was observed by MS and confirmed by phosphopeptide mapping of in vivo phosphorylated protein. Phosphorylation of these sites is dependent on casein kinase I epsilon activity in vivo. Phenotypic analysis of mutant mDvl-1 indicates that phosphorylation of these sites stimulates the Dvl-activated beta-catenin-dependent Wnt signaling pathway in both cell culture and in Xenopus development. Casein kinase I epsilon is a Wnt-regulated kinase, and regulated phosphorylation of Dvl allows fine tuning of the Wnt-beta-catenin signaling pathway.

Authors
Klimowski, LK; Garcia, BA; Shabanowitz, J; Hunt, DF; Virshup, DM
MLA Citation
Klimowski, LK, Garcia, BA, Shabanowitz, J, Hunt, DF, and Virshup, DM. "Site-specific casein kinase 1epsilon-dependent phosphorylation of Dishevelled modulates beta-catenin signaling." FEBS J 273.20 (October 2006): 4594-4602.
PMID
16965538
Source
pubmed
Published In
FEBS Journal
Volume
273
Issue
20
Publish Date
2006
Start Page
4594
End Page
4602
DOI
10.1111/j.1742-4658.2006.05462.x

An opposite role for tau in circadian rhythms revealed by mathematical modeling.

Biological clocks with a period of approximately 24 h (circadian) exist in most organisms and time a variety of functions, including sleep-wake cycles, hormone release, bioluminescence, and core body temperature fluctuations. Much of our understanding of the clock mechanism comes from the identification of specific mutations that affect circadian behavior. A widely studied mutation in casein kinase I (CKI), the CKIepsilon(tau) mutant, has been shown to cause a loss of kinase function in vitro, but it has been difficult to reconcile this loss of function with the current model of circadian clock function. Here we show that mathematical modeling predicts the opposite, that the kinase mutant CKIepsilon(tau) increases kinase activity, and we verify this prediction experimentally. CKIepsilon(tau) is a highly specific gain-of-function mutation that increases the in vivo phosphorylation and degradation of the circadian regulators PER1 and PER2. These findings experimentally validate a mathematical modeling approach to a complex biological function, clarify the role of CKI in the clock, and demonstrate that a specific mutation can be both a gain and a loss of function depending on the substrate.

Authors
Gallego, M; Eide, EJ; Woolf, MF; Virshup, DM; Forger, DB
MLA Citation
Gallego, M, Eide, EJ, Woolf, MF, Virshup, DM, and Forger, DB. "An opposite role for tau in circadian rhythms revealed by mathematical modeling." Proc Natl Acad Sci U S A 103.28 (July 11, 2006): 10618-10623.
PMID
16818876
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
103
Issue
28
Publish Date
2006
Start Page
10618
End Page
10623
DOI
10.1073/pnas.0604511103

Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation.

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.

Authors
Swiatek, W; Kang, H; Garcia, BA; Shabanowitz, J; Coombs, GS; Hunt, DF; Virshup, DM
MLA Citation
Swiatek, W, Kang, H, Garcia, BA, Shabanowitz, J, Coombs, GS, Hunt, DF, and Virshup, DM. "Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation." J Biol Chem 281.18 (May 5, 2006): 12233-12241.
PMID
16513652
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
18
Publish Date
2006
Start Page
12233
End Page
12241
DOI
10.1074/jbc.M510580200

Protein serine/threonine phosphatases: life, death, and sleeping.

Protein serine/threonine phosphatases control key biological pathways including early embryonic development, cell proliferation, cell death, circadian rhythm and cancer. Recent studies have provided important insights into how several of the many phosphatase regulators, through their interaction with a conserved phosphatase catalytic subunit, control the activity of critical substrates in these diverse pathways. Recent co-crystal structures provided a major insight into how the diverse protein serine/threonine regulators rein in the otherwise promiscuous catalytic subunits.

Authors
Gallego, M; Virshup, DM
MLA Citation
Gallego, M, and Virshup, DM. "Protein serine/threonine phosphatases: life, death, and sleeping." Curr Opin Cell Biol 17.2 (April 2005): 197-202. (Review)
PMID
15780597
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
17
Issue
2
Publish Date
2005
Start Page
197
End Page
202
DOI
10.1016/j.ceb.2005.01.002

Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

Authors
Eide, EJ; Woolf, MF; Kang, H; Woolf, P; Hurst, W; Camacho, F; Vielhaber, EL; Giovanni, A; Virshup, DM
MLA Citation
Eide, EJ, Woolf, MF, Kang, H, Woolf, P, Hurst, W, Camacho, F, Vielhaber, EL, Giovanni, A, and Virshup, DM. "Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation." Mol Cell Biol 25.7 (April 2005): 2795-2807.
PMID
15767683
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
25
Issue
7
Publish Date
2005
Start Page
2795
End Page
2807
DOI
10.1128/MCB.25.7.2795-2807.2005

Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities.

Autosomal dominant mutations in the gene encoding the basic helix-loop-helix transcription factor Twist1 are associated with limb and craniofacial defects in humans with Saethre-Chotzen syndrome. The molecular mechanism underlying these phenotypes is poorly understood. We show that ectopic expression of the related basic helix-loop-helix factor Hand2 phenocopies Twist1 loss of function in the limb and that the two factors have a gene dosage-dependent antagonistic interaction. Dimerization partner choice by Twist1 and Hand2 can be modulated by protein kinase A- and protein phosphatase 2A-regulated phosphorylation of conserved helix I residues. Notably, multiple Twist1 mutations associated with Saethre-Chotzen syndrome alter protein kinase A-mediated phosphorylation of Twist1, suggesting that misregulation of Twist1 dimerization through either stoichiometric or post-translational mechanisms underlies phenotypes of individuals with Saethre-Chotzen syndrome.

Authors
Firulli, BA; Krawchuk, D; Centonze, VE; Vargesson, N; Virshup, DM; Conway, SJ; Cserjesi, P; Laufer, E; Firulli, AB
MLA Citation
Firulli, BA, Krawchuk, D, Centonze, VE, Vargesson, N, Virshup, DM, Conway, SJ, Cserjesi, P, Laufer, E, and Firulli, AB. "Altered Twist1 and Hand2 dimerization is associated with Saethre-Chotzen syndrome and limb abnormalities." Nat Genet 37.4 (April 2005): 373-381.
PMID
15735646
Source
pubmed
Published In
Nature Genetics
Volume
37
Issue
4
Publish Date
2005
Start Page
373
End Page
381
DOI
10.1038/ng1525

Casein kinase I in the mammalian circadian clock.

The circadian clock is characterized by daily fluctuations in gene expression, protein abundance, and posttranslational modification of regulatory proteins. The Drosophila PERIOD (dPER) protein is phosphorylated by the serine?threonine protein kinase, DOUBLETIME (DBT). Similarly, the murine PERIOD proteins, mPER1 and mPER2, are phosphorylated by casein kinase I epsilon (CKI), the mammalian homolog of DBT. CKIepsilon also phosphorylates and partially activates the transcription factor BMAL1. Given the variety of potential targets for CKIepsilon and other cellular kinases, the precise role of phosphorylation is likely to be a complex one. Biochemical analysis of these and other circadian regulatory proteins has proven to be a fruitful approach in determining how they function within the context of the molecular clockworks.

Authors
Eide, EJ; Kang, H; Crapo, S; Gallego, M; Virshup, DM
MLA Citation
Eide, EJ, Kang, H, Crapo, S, Gallego, M, and Virshup, DM. "Casein kinase I in the mammalian circadian clock." Methods Enzymol 393 (2005): 408-418.
PMID
15817302
Source
pubmed
Published In
Methods in Enzymology
Volume
393
Publish Date
2005
Start Page
408
End Page
418
DOI
10.1016/S0076-6879(05)93019-X

Hand2 and Twist1 interact antagonistically in the vertebrate limb.

Authors
Laufer, E; Firulli, B; Krawchuk, D; Centonze, V; Virshup, D; Conway, S; Cserjesi, P; Firulli, A
MLA Citation
Laufer, E, Firulli, B, Krawchuk, D, Centonze, V, Virshup, D, Conway, S, Cserjesi, P, and Firulli, A. "Hand2 and Twist1 interact antagonistically in the vertebrate limb." July 15, 2004.
Source
wos-lite
Published In
Developmental Biology
Volume
271
Issue
2
Publish Date
2004
Start Page
590
End Page
590

A disease-associated casein kinase 1 delta mutation alters its physiological roles and may promote adenomatous polyps via wnt/beta-catenin independent mechanism

Authors
Tsai, IC; Woolf, M; Neklason, D; Burt, R; Virshup, DM
MLA Citation
Tsai, IC, Woolf, M, Neklason, D, Burt, R, and Virshup, DM. "A disease-associated casein kinase 1 delta mutation alters its physiological roles and may promote adenomatous polyps via wnt/beta-catenin independent mechanism." May 14, 2004.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
18
Issue
8
Publish Date
2004
Start Page
C299
End Page
C299

A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1.

In resting cells, the NFAT1 transcription factor is kept inactive in the cytoplasm by phosphorylation on multiple serine residues. These phosphorylated residues are primarily contained within two types of serine-rich motifs, the SRR-1 and SP motifs, which are conserved within the NFAT family. Several different kinases have been proposed to regulate NFAT, but no single candidate displays the specificity required to fully phosphorylate both types of motifs; thus, the identity of the kinase that regulates NFAT activity remains unclear. Here we show that the NFAT1 serine motifs are regulated by distinct kinases that must coordinate to control NFAT1 activation. CK1 phosphorylates only the SRR-1 motif, the primary region required for NFAT1 nuclear import. CK1 exists with NFAT1 in a high-molecular-weight complex in resting T cells but dissociates upon activation. GSK3 does not phosphorylate the SRR-1 region but can target the NFAT1 SP-2 motif, and it synergizes with CK1 to regulate NFAT1 nuclear export. We identify a conserved docking site for CK1 in NFAT proteins and show that mutation of this site disrupts NFAT1-CK1 interaction and causes constitutive nuclear localization of NFAT1. The CK1 docking motif is present in proteins of the Wnt, Hedgehog, and circadian-rhythm pathways, which also integrate the activities of CK1 and GSK3.

Authors
Okamura, H; Garcia-Rodriguez, C; Martinson, H; Qin, J; Virshup, DM; Rao, A
MLA Citation
Okamura, H, Garcia-Rodriguez, C, Martinson, H, Qin, J, Virshup, DM, and Rao, A. "A conserved docking motif for CK1 binding controls the nuclear localization of NFAT1." Mol Cell Biol 24.10 (May 2004): 4184-4195.
PMID
15121840
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
24
Issue
10
Publish Date
2004
Start Page
4184
End Page
4195

Regulation of casein kinase I epsilon activity by Wnt signaling.

The Wnt/beta-catenin signaling pathway is important in both development and cancer. Casein kinase Iepsilon (CKIepsilon) is a positive regulator of the canonical Wnt pathway. CKIepsilon itself can be regulated in vitro by inhibitory autophosphorylation, and recent data suggest that in vivo kinase activity can be regulated by extracellular stimuli. We show here that the phosphorylation state and kinase activity of CKIepsilon are directly regulated by Wnt signaling. Coexpression of XWnt-8 or addition of soluble Wnt-3a ligand led to a significant and rapid increase in the activity of endogenous CKIepsilon. The increase in CKIepsilon activity is the result of decreased inhibitory autophosphorylation because it is abolished by preincubation of immunoprecipitated kinase with ATP. Furthermore, mutation of CKIepsilon inhibitory autophosphorylation sites creates a kinase termed CKIepsilon(MM2) that is significantly more active than CKIepsilon and is not activated further upon Wnt stimulation. Autoinhibition of CKIepsilon is biologically relevant because CKIepsilon(MM2) is more effective than CKIepsilon at activating transcription from a Lef1-dependent promoter. Finally, CKIepsilon(MM2) expression in Xenopus embryos induces both axis duplication and additional developmental abnormalities. The data suggest that Wnt signaling activates CKIepsilon by causing transient dephosphorylation of critical inhibitory sites present in the carboxyl-terminal domain of the kinase. Activation of the Wnt pathway may therefore stimulate a cellular phosphatase to dephosphorylate and activate CKIepsilon

Authors
Swiatek, W; Tsai, I-C; Klimowski, L; Pepler, A; Barnette, J; Yost, HJ; Virshup, DM
MLA Citation
Swiatek, W, Tsai, I-C, Klimowski, L, Pepler, A, Barnette, J, Yost, HJ, and Virshup, DM. "Regulation of casein kinase I epsilon activity by Wnt signaling." J Biol Chem 279.13 (March 26, 2004): 13011-13017.
PMID
14722104
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
13
Publish Date
2004
Start Page
13011
End Page
13017
DOI
10.1074/jbc.M304682200

Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.

Authors
Firulli, BA; Virshup, DM; Firulli, AB
MLA Citation
Firulli, BA, Virshup, DM, and Firulli, AB. "Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines." Biol Proced Online 6 (2004): 16-22.
PMID
15103396
Source
pubmed
Published In
Biological Procedures Online
Volume
6
Publish Date
2004
Start Page
16
End Page
22
DOI
10.1251/bpo69

PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation.

The bHLH factors HAND1 and HAND2 are required for heart, vascular, neuronal, limb, and extraembryonic development. Unlike most bHLH proteins, HAND factors exhibit promiscuous dimerization properties. We report that phosphorylation/dephosphorylation via PKA, PKC, and a specific heterotrimeric protein phosphatase 2A (PP2A) modulates HAND function. The PP2A targeting-subunit B56delta specifically interacts with HAND1 and -2, but not other bHLH proteins. PKA and PKC phosphorylate HAND proteins in vivo, and only B56delta-containing PP2A complexes reduce levels of HAND1 phosphorylation. During RCHOI trophoblast stem cell differentiation, B56delta expression is downregulated and HAND1 phosphorylation increases. Mutations in phosphorylated residues result in altered HAND1 dimerization and biological function. Taken together, these results suggest that site-specific phosphorylation regulates HAND factor functional specificity.

Authors
Firulli, BA; Howard, MJ; McDaid, JR; McIlreavey, L; Dionne, KM; Centonze, VE; Cserjesi, P; Virshup, DM; Firulli, AB
MLA Citation
Firulli, BA, Howard, MJ, McDaid, JR, McIlreavey, L, Dionne, KM, Centonze, VE, Cserjesi, P, Virshup, DM, and Firulli, AB. "PKA, PKC, and the protein phosphatase 2A influence HAND factor function: a mechanism for tissue-specific transcriptional regulation." Mol Cell 12.5 (November 2003): 1225-1237.
PMID
14636580
Source
pubmed
Published In
Molecular Cell
Volume
12
Issue
5
Publish Date
2003
Start Page
1225
End Page
1237

Mechanism of regulation of casein kinase I activity by group I metabotropic glutamate receptors.

Previously, we reported that (S)-3,5-dihydroxypenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRs), stimulates CK1 and Cdk5 kinase activities in neostriatal neurons, leading to enhanced phosphorylation, respectively, of Ser-137 and Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa). We have now investigated the signaling pathway that leads from mGluRs to casein kinase 1 (CK1) activation. In mouse neostriatal slices, the effect of DHPG on phosphorylation of Ser-137 or Thr-75 of DARPP-32 was blocked by the phospholipase Cbeta inhibitor, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), and the calcineurin inhibitor cyclosporin A. In neuroblastoma N2a cells, the effect of DHPG on the activity of transfected HA-tagged CK1(epsilon) was blocked by BAPTA/AM and cyclosporin A. In neostriatal slices, the effect of DHPG on Cdk5 activity was also abolished by BAPTA/AM and cyclosporin A, presumably through blocking activation of CK1. Metabolic labeling studies and phosphopeptide mapping revealed that a set of C-terminal sites in HA-CK1epsilon were transiently dephosphorylated in N2a cells upon treatment with DHPG, and this was blocked by cyclosporin A. A mutant CK1epsilon with a nonphosphorylatable C-terminal domain was not activated by DHPG. Together, these studies suggest that DHPG activates CK1(epsilon) via Ca(2+)-dependent stimulation of calcineurin and subsequent dephosphorylation of inhibitory C-terminal autophosphorylation sites.

Authors
Liu, F; Virshup, DM; Nairn, AC; Greengard, P
MLA Citation
Liu, F, Virshup, DM, Nairn, AC, and Greengard, P. "Mechanism of regulation of casein kinase I activity by group I metabotropic glutamate receptors." J Biol Chem 277.47 (November 22, 2002): 45393-45399.
PMID
12223474
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
47
Publish Date
2002
Start Page
45393
End Page
45399
DOI
10.1074/jbc.M204499200

Regulation of BRCA1 phosphorylation by interaction with protein phosphatase 1alpha.

Numerous reports have revealed that the tumor suppressor BRCA1 may play an important role in DNA damage repair. BRCA1 is expressed and phosphorylated during cell cycle progression and after DNA damage. BRCA1 is hypophosphorylated in G0-G1 and probably during mitosis as well. Kinases known to phosphorylate BRCA1 include cyclin-dependent kinase 2, as well as ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related kinase (ATR), which function in G2 checkpoint control. However, protein phosphatases responsible for dephosphorylation of BRCA1 had yet to be identified. hCds1, which acts downstream of ATM, also phosphorylates a BRCA1 fragment containing amino acids 759-1064 [BRCA1 fragment 4 (BF4)]. We have used a GST-BF4 protein phosphorylated by hCds1 [glutathione S-transferase (GST)-BF4-P] as a substrate to identify potential phosphatases responsible for BRCA1 dephosphorylation. Data presented here show that both recombinant protein phosphatase 1 alpha (PP1alpha) catalytic subunit and endogenous PP1alpha dephosphorylate GST-BF4-P. Inhibitor 2 abolishes this activity. Overexpression of PP1alpha partially inhibits hyperphosphorylation of BRCA1 after ionizing radiation, indicating that PP1alpha dephosphorylates BRCA1 in vivo. BRCA1 and PP1alpha reciprocally coimmunoprecipitate, and a glutathione S-transferase pull-down assay shows that PP1alpha catalytic subunit associates directly with the BF4 region of BRCA1. In addition, BRCA1 inhibits PP1alpha activity. Therefore, BRCA1 is both a substrate and a regulator of PP1alpha. The interaction between BRCA1 and PP1alpha thus may play a role in DNA damage repair and cell cycle progression.

Authors
Liu, Y; Virshup, DM; White, RL; Hsu, L-C
MLA Citation
Liu, Y, Virshup, DM, White, RL, and Hsu, L-C. "Regulation of BRCA1 phosphorylation by interaction with protein phosphatase 1alpha." Cancer Res 62.22 (November 15, 2002): 6357-6361.
PMID
12438214
Source
pubmed
Published In
Cancer Research
Volume
62
Issue
22
Publish Date
2002
Start Page
6357
End Page
6361

The nucleophosmin-anaplastic lymphoma kinase fusion protein induces c-Myc expression in pediatric anaplastic large cell lymphomas.

The majority of pediatric anaplastic large cell lymphomas (ALCLs) carry the t(2;5)(p23;q35) chromosomal translocation that juxtaposes the dimerization domain of nucleophosmin with anaplastic lymphoma kinase (ALK). The nucleophosmin-ALK fusion induces constitutive, ligand-independent activation of the ALK tyrosine kinase leading to aberrant activation of cellular signaling pathways. To study the early consequences of ectopic ALK activation, a GyrB-ALK fusion was constructed that allowed regulated dimerization with the addition of coumermycin. Expression of the fusion protein caused a coumermycin-dependent increase in cellular tyrosine phosphorylation and c-Myc immunoreactivity, which was paralleled by a rise in c-myc RNA. To assess the clinical relevance of this observation, c-Myc expression was determined in pediatric ALK-positive and -negative lymphomas. Co-expression of c-Myc and ALK was seen in tumor cells in 15 of 15 (100%) ALK-positive ALCL samples, whereas no expression of either ALK or c-Myc was seen in six of six cases of ALK-negative T-cell lymphoma. C-Myc may be a downstream target of ALK signaling and its expression a defining characteristic of ALK-positive ALCLs.

Authors
Raetz, EA; Perkins, SL; Carlson, MA; Schooler, KP; Carroll, WL; Virshup, DM
MLA Citation
Raetz, EA, Perkins, SL, Carlson, MA, Schooler, KP, Carroll, WL, and Virshup, DM. "The nucleophosmin-anaplastic lymphoma kinase fusion protein induces c-Myc expression in pediatric anaplastic large cell lymphomas." Am J Pathol 161.3 (September 2002): 875-883.
PMID
12213716
Source
pubmed
Published In
The American journal of pathology
Volume
161
Issue
3
Publish Date
2002
Start Page
875
End Page
883
DOI
10.1016/S0002-9440(10)64248-4

B56-associated protein phosphatase 2A is required for survival and protects from apoptosis in Drosophila melanogaster.

Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. B56:PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase DRICE: B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.

Authors
Li, X; Scuderi, A; Letsou, A; Virshup, DM
MLA Citation
Li, X, Scuderi, A, Letsou, A, and Virshup, DM. "B56-associated protein phosphatase 2A is required for survival and protects from apoptosis in Drosophila melanogaster." Mol Cell Biol 22.11 (June 2002): 3674-3684.
PMID
11997504
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
22
Issue
11
Publish Date
2002
Start Page
3674
End Page
3684

The circadian regulatory proteins BMAL1 and cryptochromes are substrates of casein kinase Iepsilon.

The serine/threonine protein kinase casein kinase I epsilon (CKIepsilon) is a key regulator of metazoan circadian rhythm. Genetic and biochemical data suggest that CKIepsilon binds to and phosphorylates the PERIOD proteins. However, the PERIOD proteins interact with a variety of circadian regulators, suggesting the possibility that CKIepsilon may interact with and phosphorylate additional clock components as well. We find that CRY1 and BMAL1 are phosphoproteins in cultured cells. Mammalian PERIOD proteins act as a scaffold with distinct domains that simultaneously bind CKIepsilon and mCRY1 and mCRY2 (mCRY). mCRY is phosphorylated by CKIepsilon only when both proteins are bound to mammalian PERIOD proteins. BMAL1 is also a substrate for CKIepsilon in vitro, and CKIepsilon kinase activity positively regulates BMAL1-dependent transcription from circadian promoters in reporter assays. We conclude that CKIepsilon phosphorylates multiple circadian substrates and may exert its effects on circadian rhythm in part by a direct effect on BMAL1-dependent transcription.

Authors
Eide, EJ; Vielhaber, EL; Hinz, WA; Virshup, DM
MLA Citation
Eide, EJ, Vielhaber, EL, Hinz, WA, and Virshup, DM. "The circadian regulatory proteins BMAL1 and cryptochromes are substrates of casein kinase Iepsilon." J Biol Chem 277.19 (May 10, 2002): 17248-17254.
PMID
11875063
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
19
Publish Date
2002
Start Page
17248
End Page
17254
DOI
10.1074/jbc.M111466200

Sequential multisite phosphorylation by casein kinase I epsilon (CKI epsilon)

Authors
Toh, KL; Thulin, C; Fu, YH; Ptacek, LJ; Virshup, DM
MLA Citation
Toh, KL, Thulin, C, Fu, YH, Ptacek, LJ, and Virshup, DM. "Sequential multisite phosphorylation by casein kinase I epsilon (CKI epsilon)." FASEB JOURNAL 16.5 (March 22, 2002): A917-A917.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
16
Issue
5
Publish Date
2002
Start Page
A917
End Page
A917

Lineage-specific trisomy 21 in a neonate with resolving transient myeloproliferative syndrome.

The cellular events that lead to transient myeloproliferative syndrome (TMS) in patients with trisomy 21 mosaicism confined to the hematopoietic system are poorly understood. The authors attempt to define the event that led to the development of TMS in a single patient with clonal trisomy 21. A phenotypically normal neonate with clonal trisomy 21 is described. At the time when his TMS was resolving, fluorescent in situ hybridization analysis was performed on cell populations sorted by flow cytometry to determine what cell populations contained trisomic cells. Trisomy 21 was found in cells of the erythrocytic and monocytic lineages, but not in the stem cells, progenitor compartment, megakaryocytes, lymphocytes, or neutrophils. These results support the hypothesis that, in this neonate, trisomy 21 occurred in a multipotent hematopoietic progenitor, and a subsequent event led to the appearance of the blast population.

Authors
Slayton, WB; Spangrude, GJ; Chen, Z; Greene, WF; Virshup, D
MLA Citation
Slayton, WB, Spangrude, GJ, Chen, Z, Greene, WF, and Virshup, D. "Lineage-specific trisomy 21 in a neonate with resolving transient myeloproliferative syndrome." J Pediatr Hematol Oncol 24.3 (March 2002): 224-226.
PMID
11990311
Source
pubmed
Published In
Journal of Pediatric Hematology/Oncology
Volume
24
Issue
3
Publish Date
2002
Start Page
224
End Page
226

Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex.

Wnt signaling plays a key role in cell proliferation and development. Recently, casein kinase I (CKI) and protein phosphatase 2A (PP2A) have emerged as positive and negative regulators of the Wnt pathway, respectively. However, it is not clear how these two enzymes with opposing functions regulate Wnt signaling. Here we show that both CKI delta and CKI epsilon interacted directly with Dvl-1, and that CKI phosphorylated multiple components of the Wnt-regulated beta-catenin degradation complex in vitro, including Dvl-1, adenomatous polyposis coli (APC), axin, and beta-catenin. Comparison of peptide maps from in vivo and in vitro phosphorylated beta-catenin and axin suggests that CKI phosphorylates these proteins in vivo as well. CKI abrogated beta-catenin degradation in Xenopus egg extracts. Notably, CKI decreased, whereas inhibition of CKI increased, the association of PP2A with the beta-catenin degradation complex in vitro. Additionally, inhibition of CKI in vivo stabilized the beta-catenin degradation complex, suggesting that CKI actively destabilizes the complex in vivo. The ability of CKI to induce secondary body axes in Xenopus embryos was reduced by the B56 regulatory subunit of PP2A, and kinase-dead CKI epsilon acted synergistically with B56 in inhibiting Wnt signaling. The data suggest that CKI phosphorylates and destabilizes the beta-catenin degradation complex, likely through the dissociation of PP2A, providing a mechanism by which CKI stabilizes beta-catenin and propagates the Wnt signal.

Authors
Gao, Z-H; Seeling, JM; Hill, V; Yochum, A; Virshup, DM
MLA Citation
Gao, Z-H, Seeling, JM, Hill, V, Yochum, A, and Virshup, DM. "Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex." Proc Natl Acad Sci U S A 99.3 (February 5, 2002): 1182-1187.
PMID
11818547
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
99
Issue
3
Publish Date
2002
Start Page
1182
End Page
1187
DOI
10.1073/pnas.032468199

Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A.

Protein phosphatase 2A (PP2A) is an abundant heterotrimeric serine/threonine phosphatase containing highly conserved structural (A) and catalytic (C) subunits. Its diverse functions in the cell are determined by its association with a highly variable regulatory and targeting B subunit. At least three distinct gene families encoding B subunits are known: B/B55/CDC55, B'/B56/RTS1 and B"/PR72/130. No homology has been identified among the B families, and little is known about how these B subunits interact with the PP2A A and C subunits. In vitro expression of a series of B56alpha fragments identified two distinct domains that bound independently to the A subunit. Sequence alignment of these A subunit binding domains (ASBD) identified conserved residues in B/B55 and PR72 family members. The alignment successfully predicted domains in B55 and PR72 subunits that similarly bound to the PP2A A subunit. These results suggest that these B subunits share a common core structure and mode of interaction with the PP2A holoenzyme.

Authors
Li, X; Virshup, DM
MLA Citation
Li, X, and Virshup, DM. "Two conserved domains in regulatory B subunits mediate binding to the A subunit of protein phosphatase 2A." Eur J Biochem 269.2 (January 2002): 546-552.
PMID
11856313
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
269
Issue
2
Publish Date
2002
Start Page
546
End Page
552

Nuclear export of mammalian PERIOD proteins.

The timing of mammalian circadian rhythm is determined by interlocking negative and positive transcriptional feedback loops that govern the cyclic expression of both clock regulators and output genes. In mammals, nuclear localization of the circadian regulators PER1-3 is controlled by multiple mechanisms, including multimerization with PER and CRY proteins. In addition, nuclear entry of mammalian PER1 (mPER1) can be regulated by a phosphorylation-dependent masking of its nuclear localization signal. Here we present evidence suggesting that nuclear localization of PER proteins is a dynamic process determined by both nuclear import and previously unrecognized nuclear export pathways. Examination of the subcellular localization of a series of truncated mPER1 proteins demonstrated that cytoplasmic localization is mediated by an 11-amino acid region with homology to leucine-rich nuclear export signals (NESs). Similar sequences were identified in mPER2 and mPER3 as well as in several insect PER proteins. The putative NESs from mPER1 and mPER2 were able to direct cytoplasmic accumulation when fused to a heterologous protein. Mutations in conserved NES residues and the nuclear export inhibitor leptomycin B each blocked the function of the NES. Full-length mPER1 was also exported from microinjected Xenopus laevis oocyte nuclei in an NES-dependent manner. The presence of a functional NES in mPER1 and mPER2 as well as related sequences in a variety of other PER proteins suggests that nuclear export may be a conserved and important feature of circadian regulators.

Authors
Vielhaber, EL; Duricka, D; Ullman, KS; Virshup, DM
MLA Citation
Vielhaber, EL, Duricka, D, Ullman, KS, and Virshup, DM. "Nuclear export of mammalian PERIOD proteins." J Biol Chem 276.49 (December 7, 2001): 45921-45927.
PMID
11591712
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
49
Publish Date
2001
Start Page
45921
End Page
45927
DOI
10.1074/jbc.M107726200

An hPer2 phosphorylation site mutation in familial Advanced Sleep-Phase Syndrome.

Authors
Fu, Y; Jones, CR; Toh, K; Virshup, D; Ptacek, LJ
MLA Citation
Fu, Y, Jones, CR, Toh, K, Virshup, D, and Ptacek, LJ. "An hPer2 phosphorylation site mutation in familial Advanced Sleep-Phase Syndrome." October 2001.
Source
wos-lite
Published In
The American Journal of Human Genetics
Volume
69
Issue
4
Publish Date
2001
Start Page
597
End Page
597

Protein phosphatase 2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus.

Wnt signaling increases beta-catenin abundance and transcription of Wnt-responsive genes. Our previous work suggested that the B56 regulatory subunit of protein phosphatase 2A (PP2A) inhibits Wnt signaling. Okadaic acid (a phosphatase inhibitor) increases, while B56 expression reduces, beta-catenin abundance; B56 also reduces transcription of Wnt-responsive genes. Okadaic acid is a tumor promoter, and the structural A subunit of PP2A is mutated in multiple cancers. Taken together, the evidence suggests that PP2A is a tumor suppressor. However, other studies suggest that PP2A activates Wnt signaling. We now show that the B56, A and catalytic C subunits of PP2A each have ventralizing activity in Xenopus embryos. B56 was epistatically positioned downstream of GSK3beta and axin but upstream of beta-catenin, and axin co-immunoprecipitated B56, A and C subunits, suggesting that PP2A:B56 is in the beta-catenin degradation complex. PP2A appears to be essential for beta-catenin degradation, since beta-catenin degradation was reconstituted in phosphatase-depleted Xenopus egg extracts by PP2A, but not PP1. These results support the hypothesis that PP2A:B56 directly inhibits Wnt signaling and plays a role in development and carcinogenesis.

Authors
Li, X; Yost, HJ; Virshup, DM; Seeling, JM
MLA Citation
Li, X, Yost, HJ, Virshup, DM, and Seeling, JM. "Protein phosphatase 2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus." EMBO J 20.15 (August 1, 2001): 4122-4131.
PMID
11483515
Source
pubmed
Published In
EMBO Journal
Volume
20
Issue
15
Publish Date
2001
Start Page
4122
End Page
4131
DOI
10.1093/emboj/20.15.4122

PP2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus.

Authors
Seeling, JM; Li, X; Yost, HJ; Virshup, DM
MLA Citation
Seeling, JM, Li, X, Yost, HJ, and Virshup, DM. "PP2A and its B56 regulatory subunit inhibit Wnt signaling in Xenopus." DEVELOPMENTAL BIOLOGY 235.1 (July 1, 2001): 254-254.
Source
wos-lite
Published In
Developmental Biology
Volume
235
Issue
1
Publish Date
2001
Start Page
254
End Page
254

Casein kinase I: another cog in the circadian clockworks.

Multiple components of the circadian central clock are phosphoproteins, and it has become increasingly clear that posttranslational modification is an important regulator of circadian rhythm in diverse organisms, from dinoflagellates to humans. Genetic studies in Drosophila have identified double-time (dbt), a serine/threonine protein kinase that is highly homologous to human casein kinase I epsilon (CKIepsilon), as the first kinase linked to behavioral rhythms. Identification of a missense mutation in CKIepsilon as the tau mutation in the Syrian hamster places CKIepsilon within the core clock machinery in mammals. Most recently, identification of a phosphorylation site mutant of hPER2 in a family with an inherited circadian rhythm abnormality strongly suggests that PER2 is a physiologically relevant substrate of CKI. Phosphorylation may regulate multiple properties of clock proteins, including stability and intracellular localization.

Authors
Eide, EJ; Virshup, DM
MLA Citation
Eide, EJ, and Virshup, DM. "Casein kinase I: another cog in the circadian clockworks." Chronobiol Int 18.3 (May 2001): 389-398. (Review)
PMID
11475410
Source
pubmed
Published In
Chronobiology International: the journal of biological and medical rhythm research
Volume
18
Issue
3
Publish Date
2001
Start Page
389
End Page
398

An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.

Familial advanced sleep phase syndrome (FASPS) is an autosomal dominant circadian rhythm variant; affected individuals are "morning larks" with a 4-hour advance of the sleep, temperature, and melatonin rhythms. Here we report localization of the FASPS gene near the telomere of chromosome 2q. A strong candidate gene (hPer2), a human homolog of the period gene in Drosophila, maps to the same locus. Affected individuals have a serine to glycine mutation within the casein kinase Iepsilon (CKIepsilon) binding region of hPER2, which causes hypophosphorylation by CKIepsilon in vitro. Thus, a variant in human sleep behavior can be attributed to a missense mutation in a clock component, hPER2, which alters the circadian period.

Authors
Toh, KL; Jones, CR; He, Y; Eide, EJ; Hinz, WA; Virshup, DM; Ptácek, LJ; Fu, YH
MLA Citation
Toh, KL, Jones, CR, He, Y, Eide, EJ, Hinz, WA, Virshup, DM, Ptácek, LJ, and Fu, YH. "An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome." Science 291.5506 (February 9, 2001): 1040-1043.
PMID
11232563
Source
pubmed
Published In
Science
Volume
291
Issue
5506
Publish Date
2001
Start Page
1040
End Page
1043

Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2.

Casein kinase Iepsilon (CKIepsilon), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951-955]. A mutation in CKIepsilon causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Idelta (hCKIdelta), the closest homologue to hCKIepsilon, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIdelta and hCKIepsilon phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIdelta protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIdelta may play a role similar to CKIepsilon, suggesting that it may also be involved in regulating circadian rhythmicity by post-translation modification of mammalian clock proteins hPER1 and 2.

Authors
Camacho, F; Cilio, M; Guo, Y; Virshup, DM; Patel, K; Khorkova, O; Styren, S; Morse, B; Yao, Z; Keesler, GA
MLA Citation
Camacho, F, Cilio, M, Guo, Y, Virshup, DM, Patel, K, Khorkova, O, Styren, S, Morse, B, Yao, Z, and Keesler, GA. "Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2." FEBS Lett 489.2-3 (February 2, 2001): 159-165.
PMID
11165242
Source
pubmed
Published In
FEBS Letters
Volume
489
Issue
2-3
Publish Date
2001
Start Page
159
End Page
165

Casein kinase I: from obscurity to center stage.

The casein kinase I (CKI) family of protein kinases is a group of highly related, ubiquitously expressed serine/threonine kinases found in all eukaryotic organisms from protozoa to man. Recent advances in diverse fields, including developmental biology and chronobiology, have elucidated roles for CKI in regulating critical processes such as Wnt signaling, circadian rhythm, nuclear import, and Alzheimer's disease progression.

Authors
Vielhaber, E; Virshup, DM
MLA Citation
Vielhaber, E, and Virshup, DM. "Casein kinase I: from obscurity to center stage." IUBMB Life 51.2 (February 2001): 73-78. (Review)
PMID
11463166
Source
pubmed
Published In
IUBMB Life
Volume
51
Issue
2
Publish Date
2001
Start Page
73
End Page
78
DOI
10.1080/15216540117461

Pediatric ALK-positive lymphomas coexpress c-Myc.

Authors
Raetz, EA; Perkins, SL; Carlson, MA; Schooler, KP; Virshup, DM
MLA Citation
Raetz, EA, Perkins, SL, Carlson, MA, Schooler, KP, and Virshup, DM. "Pediatric ALK-positive lymphomas coexpress c-Myc." BLOOD 96.11 (November 16, 2000): 128A-128A.
Source
wos-lite
Published In
Blood
Volume
96
Issue
11
Publish Date
2000
Start Page
128A
End Page
128A

Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon.

The molecular oscillator that keeps circadian time is generated by a negative feedback loop. Nuclear entry of circadian regulatory proteins that inhibit transcription from E-box-containing promoters appears to be a critical component of this loop in both Drosophila and mammals. The Drosophila double-time gene product, a casein kinase I epsilon (CKIepsilon) homolog, has been reported to interact with dPER and regulate circadian cycle length. We find that mammalian CKIepsilon binds to and phosphorylates the murine circadian regulator mPER1. Unlike both dPER and mPER2, mPER1 expressed alone in HEK 293 cells is predominantly a nuclear protein. Two distinct mechanisms appear to retard mPER1 nuclear entry. First, coexpression of mPER2 leads to mPER1-mPER2 heterodimer formation and cytoplasmic colocalization. Second, coexpression of CKIepsilon leads to masking of the mPER1 nuclear localization signal and phosphorylation-dependent cytoplasmic retention of both proteins. CKIepsilon may regulate mammalian circadian rhythm by controlling the rate at which mPER1 enters the nucleus.

Authors
Vielhaber, E; Eide, E; Rivers, A; Gao, ZH; Virshup, DM
MLA Citation
Vielhaber, E, Eide, E, Rivers, A, Gao, ZH, and Virshup, DM. "Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I epsilon." Mol Cell Biol 20.13 (July 2000): 4888-4899.
PMID
10848614
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
13
Publish Date
2000
Start Page
4888
End Page
4899

Phosphorylation and destabilization of human period I clock protein by human casein kinase I epsilon.

Period (PER), a central component of the circadian clock in Drosophila, undergoes daily oscillation in abundance and phosphorylation state. Here we report that human casein kinase I epsilon (hCKI epsilon) can phosphorylate human PER I (hPER I). Purified recombinant hCKI epsilon (but not a kinase negative mutant of hCKI epsilon, hCKI epsilon-K38R) phosphorylated hPER I in vitro. When co-transfected with wild-type hCKI epsilon, in 293T cells, hPER I showed a significant increase in phosphorylation as evidenced by a shift in molecular mass. Furthermore, phosphorylation of hPER I by hCKI epsilon caused a decrease in protein stability in hPER I. Whereas phosphorylated hPER I had a half-life of approximately 12 h, unphosphorylated hPER I remained stable in the cell for > 24 h. hPER I protein could also be co-immunoprecipitated with transfected hCKI epsilon as well as endogenous hCKI epsilon, indicating physical association between hPER I and hCKI epsilon proteins in vivo.

Authors
Keesler, GA; Camacho, F; Guo, Y; Virshup, D; Mondadori, C; Yao, Z
MLA Citation
Keesler, GA, Camacho, F, Guo, Y, Virshup, D, Mondadori, C, and Yao, Z. "Phosphorylation and destabilization of human period I clock protein by human casein kinase I epsilon." Neuroreport 11.5 (April 7, 2000): 951-955.
PMID
10790862
Source
pubmed
Published In
NeuroReport
Volume
11
Issue
5
Publish Date
2000
Start Page
951
End Page
955

Protein phosphatase 2A: a panoply of enzymes.

Protein phosphatase 2A describes an extended family of intracellular protein serine/threonine phosphatases sharing a common catalytic subunit that regulates a variety of processes by means of diverse regulatory subunits. During the past year, studies have shown that protein phosphatase 2A influences events ranging from the initiation of DNA replication to vertebrate axis formation to apoptosis.

Authors
Virshup, DM
MLA Citation
Virshup, DM. "Protein phosphatase 2A: a panoply of enzymes." Curr Opin Cell Biol 12.2 (April 2000): 180-185. (Review)
PMID
10712915
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
12
Issue
2
Publish Date
2000
Start Page
180
End Page
185

Identification of casein kinase I substrates by in vitro expression cloning screening.

Casein kinase I (CKI) is a widely expressed protein kinase family implicated in diverse processes including membrane trafficking, DNA repair, and circadian rhythm. Despite the large number of CKI genes, few biologically relevant substrates have been identified. As an approach to better defining the spectrum of CKI substrates, we extended a recently described in vitro expression cloning (IVEC) strategy. Polypeptides pools were screened for kinase-dependent electrophoretic mobility shifts. Ten putative CKI substrates were isolated from an initial sample of 3000 random cDNA clones. Candidate substrates include proteins involved in RNA metabolism (a putative RNA helicase, the nucleolar protein hNOP56, and hnRNP A1, and ribosomal proteins L4, L8, and L13), as well as keratin 17, a necdin-related protein, and the calcium-binding proteins desmoglein 2 and annexin II. The same pools were also screened with active ERK2, and four substrates identified: aldolase, NSD-like protein, uracil-DNA glycosylase, and HHR23A. IVEC is an effective method to identify novel protein kinase substrates.

Authors
Gao, ZH; Metherall, J; Virshup, DM
MLA Citation
Gao, ZH, Metherall, J, and Virshup, DM. "Identification of casein kinase I substrates by in vitro expression cloning screening." Biochem Biophys Res Commun 268.2 (February 16, 2000): 562-566.
PMID
10679243
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
268
Issue
2
Publish Date
2000
Start Page
562
End Page
566
DOI
10.1006/bbrc.2000.2168

Residual trisomic cells in a patient with resolving transient myeloproliferative disorder and trisomy 21 mosaicism.

Authors
Slayton, WB; Spangrude, GJ; Chen, Z; Virshup, D
MLA Citation
Slayton, WB, Spangrude, GJ, Chen, Z, and Virshup, D. "Residual trisomic cells in a patient with resolving transient myeloproliferative disorder and trisomy 21 mosaicism." November 15, 1999.
Source
wos-lite
Published In
Blood
Volume
94
Issue
10
Publish Date
1999
Start Page
291B
End Page
291B

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.

Authors
Gietzen, KF; Virshup, DM
MLA Citation
Gietzen, KF, and Virshup, DM. "Identification of inhibitory autophosphorylation sites in casein kinase I epsilon." J Biol Chem 274.45 (November 5, 1999): 32063-32070.
PMID
10542239
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
45
Publish Date
1999
Start Page
32063
End Page
32070

Lymphoblastic lymphoma and excessive toxicity from chemotherapy: an unusual presentation for Fanconi anemia.

Authors
Goldsby, RE; Perkins, SL; Virshup, DM; Brothman, AR; Bruggers, CS
MLA Citation
Goldsby, RE, Perkins, SL, Virshup, DM, Brothman, AR, and Bruggers, CS. "Lymphoblastic lymphoma and excessive toxicity from chemotherapy: an unusual presentation for Fanconi anemia." J Pediatr Hematol Oncol 21.3 (May 1999): 240-243.
PMID
10363859
Source
pubmed
Published In
Journal of Pediatric Hematology/Oncology
Volume
21
Issue
3
Publish Date
1999
Start Page
240
End Page
243

Expression of multiple PP2A serine/threonine phosphatase B56 (B ') subunit isoforms in human cardiac and vascular smooth muscle myocytes.

Authors
Honeggar, M; Krall, J; Virshup, DM; Movsesian, MA
MLA Citation
Honeggar, M, Krall, J, Virshup, DM, and Movsesian, MA. "Expression of multiple PP2A serine/threonine phosphatase B56 (B ') subunit isoforms in human cardiac and vascular smooth muscle myocytes." JOURNAL OF INVESTIGATIVE MEDICINE 47.4 (April 1999): 194A-194A.
Source
wos-lite
Published In
Journal of Investigative Medicine
Volume
47
Issue
4
Publish Date
1999
Start Page
194A
End Page
194A

Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A.

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.

Authors
Seeling, JM; Miller, JR; Gil, R; Moon, RT; White, R; Virshup, DM
MLA Citation
Seeling, JM, Miller, JR, Gil, R, Moon, RT, White, R, and Virshup, DM. "Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A." Science 283.5410 (March 26, 1999): 2089-2091.
PMID
10092233
Source
pubmed
Published In
Science
Volume
283
Issue
5410
Publish Date
1999
Start Page
2089
End Page
2091

Expression of multiple PP2A serine/threonine phosphatase B56 (B ') subunit isoforms in human cardiac and vascular smooth muscle myocytes.

Authors
Honeggar, M; Krall, J; Virshup, DM; Movsesian, MA
MLA Citation
Honeggar, M, Krall, J, Virshup, DM, and Movsesian, MA. "Expression of multiple PP2A serine/threonine phosphatase B56 (B ') subunit isoforms in human cardiac and vascular smooth muscle myocytes." FASEB JOURNAL 13.4 (March 12, 1999): A468-A468.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
13
Issue
4
Publish Date
1999
Start Page
A468
End Page
A468

Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle.

Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K. F., Rivers, A., and Virshup, D. M. (1998) J. Biol. Chem. 273, 1357-1364). However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases. Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation. Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo.

Authors
Rivers, A; Gietzen, KF; Vielhaber, E; Virshup, DM
MLA Citation
Rivers, A, Gietzen, KF, Vielhaber, E, and Virshup, DM. "Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle." J Biol Chem 273.26 (June 26, 1998): 15980-15984.
PMID
9632646
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
26
Publish Date
1998
Start Page
15980
End Page
15984

Linkage of familial Wilms' tumor predisposition to chromosome 19 and a two-locus model for the etiology of familial tumors.

Familial predisposition to Wilms' tumor (WT), a childhood kidney tumor, is inherited as an autosomal dominant trait. For most WT families studied, the 11p13 gene WT1 and genomic regions implicated in tumorigenesis in a subset of tumors can be ruled out as the site of the familial predisposition gene. Following a genome-wide genetic linkage scan, we have obtained strong evidence (log of the odds ratio = 4.0) in five families for an inherited WT predisposition gene (FWT2) at 19q13.3-q13.4. In addition, we observed loss of heterozygosity at 19q in tumors from individuals from two families in which 19q can be ruled out as the site of the inherited predisposing mutation. From these data, we hypothesize that alterations at two distinct loci are critical rate-limiting steps in the etiology of familial WTs.

Authors
McDonald, JM; Douglass, EC; Fisher, R; Geiser, CF; Krill, CE; Strong, LC; Virshup, D; Huff, V
MLA Citation
McDonald, JM, Douglass, EC, Fisher, R, Geiser, CF, Krill, CE, Strong, LC, Virshup, D, and Huff, V. "Linkage of familial Wilms' tumor predisposition to chromosome 19 and a two-locus model for the etiology of familial tumors." Cancer Res 58.7 (April 1, 1998): 1387-1390.
PMID
9537236
Source
pubmed
Published In
Cancer Research
Volume
58
Issue
7
Publish Date
1998
Start Page
1387
End Page
1390

Autoinhibition of casein kinase I epsilon (CKI epsilon) is relieved by protein phosphatases and limited proteolysis.

Casein kinase I epsilon (CKI epsilon) is a member of the CKI gene family, members of which are involved in the control of SV40 DNA replication, DNA repair, and cell metabolism. The mechanisms that regulate CKI epsilon activity and substrate specificity are not well understood. We report that CKI epsilon, which contains a highly phosphorylated 123-amino acid carboxyl-terminal extension not present in CKI alpha, is substantially less active than CKI alpha in phosphorylating a number of substrates including SV40 large T antigen and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKI epsilon have been identified. First, limited tryptic digestion of CKI epsilon produces a protease-resistant amino-terminal 39-kDa core kinase with several-fold enhanced activity. Second, phosphatase treatment of CKI epsilon activates CKI epsilon 5-20-fold toward T antigen. Similar treatment of a truncated form of CKI epsilon produced only a 2-fold activation. Notably, this activation was transient; reautophosphorylation led to a rapid down-regulation of the kinase within 5 min. Phosphatase treatment also activated CKI epsilon toward the novel substrates I kappa B alpha and Ets-1. These mechanisms may serve to regulate CKI epsilon and related forms of CKI in the cell, perhaps in response to DNA damage.

Authors
Cegielska, A; Gietzen, KF; Rivers, A; Virshup, DM
MLA Citation
Cegielska, A, Gietzen, KF, Rivers, A, and Virshup, DM. "Autoinhibition of casein kinase I epsilon (CKI epsilon) is relieved by protein phosphatases and limited proteolysis." J Biol Chem 273.3 (January 16, 1998): 1357-1364.
PMID
9430669
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
3
Publish Date
1998
Start Page
1357
End Page
1364

Identifying protein phosphatase 2A interacting proteins using the yeast two-hybrid method.

Authors
McCright, B; Virshup, DM
MLA Citation
McCright, B, and Virshup, DM. "Identifying protein phosphatase 2A interacting proteins using the yeast two-hybrid method." Methods Mol Biol 93 (1998): 263-277.
PMID
9664544
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
93
Publish Date
1998
Start Page
263
End Page
277
DOI
10.1385/0-89603-468-2:263

Identification of Casein Kinase I epsilon (CKI epsilon) substrates using a membrane based kinase assay

Authors
Vielhaber, E; Virshup, D
MLA Citation
Vielhaber, E, and Virshup, D. "Identification of Casein Kinase I epsilon (CKI epsilon) substrates using a membrane based kinase assay." July 31, 1997.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1359
End Page
A1359

Regulation of casein kinase I epsilon

Authors
Virshup, D; Gietzen, K; Rivers, A
MLA Citation
Virshup, D, Gietzen, K, and Rivers, A. "Regulation of casein kinase I epsilon." July 31, 1997.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1359
End Page
A1359

Determinants of casein kinase I epsilon (CKI epsilon) autoinhibition

Authors
Gietzen, KJ; Virshup, DM
MLA Citation
Gietzen, KJ, and Virshup, DM. "Determinants of casein kinase I epsilon (CKI epsilon) autoinhibition." FASEB JOURNAL 11.9 (July 31, 1997): A1356-A1356.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1356
End Page
A1356

Determinants of casein kinase i epsilon (ckir) autoinhibition

Casein kinase I (CKI) is a rnonomeric, serine/threonine protein kinase that is known to participate in aspects of I")NA metabolism including repair and replication. Many isoforrns of CKI are known to exist in a number of organisms, and splice variants add additional diversity to the family. The functional differences between the various isofornis and splice variant are just beginning to be understood. For example, CKIo is not able to phosphorylat.e various protein substrates as effectively as CKIo. This lack of catalytic activity is the result of autophosphorylation events resulting in inhibition of the CKIa en/ynie. Inhibition is relieved in vitro by either dophosphorylation of CKIc with any of a number of cellular phosphatases or removal of the portion of the en/yme carboxy-terrninal to the kinase domain by cloning or partial proteolysis. The project goal was to identify the autophosphorylated residues responsible for inhibition of CKIe. Techniques included phospho-amino acid analysis. HPLC and peptide sequencing to identify phosphorylated peptides. Putative phosphory lated residues on such peptides will be changed using site-directed mutagenesis and the resulting changes in the activity of the enzyme observed. Although the majority of CKIf autophosphorylation occurs on the carboxy terminal tail of the kinase, autophosphorylation sites in the amino-terminus, in the kinase domain, appear to mediate inhibiton via an intramolecular mechanism.

Authors
Gietzen, KJ; Virshup, DM
MLA Citation
Gietzen, KJ, and Virshup, DM. "Determinants of casein kinase i epsilon (ckir) autoinhibition." FASEB Journal 11.9 (1997): A1355-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1355

Regulation of casein kinase i

Casein kinase I ( (CKIf) is a member of (he CKI gene family, members of which are involved in the control of SV40 DNA replication. DNA repair, and cell metabolism. The mechanisms that regulate CKIf activity and substrate specificity are not well understood. We report that, recomhinant CKIf, which contains a highly phosphorylated 123 ammo arid carboxy-terminal extension not present is CKIo, is substantially less active than CKIa in phosphorylating a number of substrates, including SV40 largo T antigen, and is unable to inhibit the initiation of SV40 DNA replication. Two mechanisms for the activation of CKIf have been identified. One, limited proteolysis of OKI products -A protease-resistant amino-terminal 39 kDa core kinase with enhanced activity. Two, phosphatase treatment of CKIf activated the kinase five- to twenty-fold towards T antigen. Similar treatment of CKIa and a truncated form of CKIf produced only a two-fold activation. Notably, this activation was transient; re-autophosphorylation lead to a rapid down-regulation of the kinase within five minutes. These results suggest that autoinhibition by the carboxy-terminal domain is a general mechanism for CKI family regulation. Phosphatase treatment also activated CKIe. towards the novel substrates I/tBa and Kts-1. Intra-molecular autophosphorylation of CKit also rapidly inactivates the kinase. However, in all cells and tissue examined CKIf is found in the dephosphorylated, active state. CKIf is actively maintained in this active state by a futile cycle of phosphorylation, as treatment of cells with phosphatase inhibitors leads to rapid kinase phosphorylation and inactivation. Physiologic stimuli that lead to CKIf inactivation are currently being investigated.

Authors
Virshup, D; Oietzen, K; Rivers, A; Utah, U
MLA Citation
Virshup, D, Oietzen, K, Rivers, A, and Utah, U. "Regulation of casein kinase i." FASEB Journal 11.9 (1997): A1358-.
Source
scival
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
11
Issue
9
Publish Date
1997
Start Page
A1358

The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm.

Protein phosphatase 2A is a heterotrimeric protein serine/threonine phosphatase consisting of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit. The B subunits determine the substrate specificity of the enzyme. There have been three families of cellular B subunits identified to date: B55, B56 (B'), and PR72/130. We have now cloned five genes encoding human B56 isoforms. Polypeptides encoded by all but one splice variant (B56gamma1) are phosphoproteins, as shown by mobility shift after treatment with alkaline phosphatase and metabolic labeling with [32P]phosphate. All labeled isoforms contain solely phosphoserine. Indirect immunofluorescence microscopy demonstrates distinct patterns of intracellular targeting by different B56 isoforms. Specifically, B56alpha, B56beta, and B56epsilon complexed with the protein phosphatase 2A A and C subunits localize to the cytoplasm, whereas B56delta, B56gamma1, and B56gamma3 are concentrated in the nucleus. Two isoforms (B56beta and B56delta) are highly expressed in adult brain; here we show that mRNA for these isoforms increases severalfold when neuroblastoma cell lines are induced to differentiate by retinoic acid treatment. These studies demonstrate an increasing diversity of regulatory mechanisms to control the activity of this key intracellular protein phosphatase and suggest distinct functions for isoforms targeted to different intracellular locations.

Authors
McCright, B; Rivers, AM; Audlin, S; Virshup, DM
MLA Citation
McCright, B, Rivers, AM, Audlin, S, and Virshup, DM. "The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm." J Biol Chem 271.36 (September 6, 1996): 22081-22089.
PMID
8703017
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
36
Publish Date
1996
Start Page
22081
End Page
22089

Assignment of human protein phosphatase 2A regulatory subunit genes b56alpha, b56beta, b56gamma, b56delta, and b56epsilon (PPP2R5A-PPP2R5E), highly expressed in muscle and brain, to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12.

The activity of the major intracellular protein phosphatase, protein phosphatase 2A (PP2A), is determined by the nature of the associated regulatory subunit. A new family of human PP2A regulatory subunits has recently been identified. Three of these subunits, B56beta, B56delta, and B56epsilon, are most highly expressed in brain, while the B56alpha and B56gamma isoforms are highly expressed in cardiac and skeletal muscle. Genes PPP2R5A-PPP2R5E encoding the phosphatase regulatory proteins B56alpha, B56beta, B56gamma, B56delta, and B56epsilon have now been mapped by fluorescence in situ hybridization to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12, respectively.

Authors
McCright, B; Brothman, AR; Virshup, DM
MLA Citation
McCright, B, Brothman, AR, and Virshup, DM. "Assignment of human protein phosphatase 2A regulatory subunit genes b56alpha, b56beta, b56gamma, b56delta, and b56epsilon (PPP2R5A-PPP2R5E), highly expressed in muscle and brain, to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12." Genomics 36.1 (August 15, 1996): 168-170.
PMID
8812429
Source
pubmed
Published In
Genomics
Volume
36
Issue
1
Publish Date
1996
Start Page
168
End Page
170

Comparing effects on temperature of early versus late leukodepletion in platelet transfusions.

Authors
Goldsby, RE; Carlson, D; Swalberg, ED; Virshup, D
MLA Citation
Goldsby, RE, Carlson, D, Swalberg, ED, and Virshup, D. "Comparing effects on temperature of early versus late leukodepletion in platelet transfusions." November 15, 1995.
Source
wos-lite
Published In
Blood
Volume
86
Issue
10
Publish Date
1995
Start Page
1397
End Page
1397

Identification of a new family of protein phosphatase 2A regulatory subunits.

Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. The ability of this widely distributed heterotrimeric enzyme to act on a diverse array of substrates is largely controlled by the nature of its regulatory B subunit. Only two gene families encoding endogenous B subunits have been cloned to date, although the existence of several additional regulatory subunits is likely. We have identified by two-hybrid interaction a new human gene family encoding PP2A B subunits. This family, denoted B56, contains three distinct genes, one of which is differentially spliced. B56 polypeptides co-immunoprecipitate with PP2A A and C subunits and with an okadaic acid-inhibitable, heparin-stimulated phosphatase activity. The three B56 family members are 70% identical to each other but share no obvious homology with previously identified B subunits. These phosphatase regulators are differentially expressed, with B56 alpha and B56 gamma highly expressed in heart and skeletal muscle and B56 beta highly expressed in brain. The identification of this novel phosphatase regulator gene family will facilitate future studies on the control of protein dephosphorylation and the role of PP2A in cellular function.

Authors
McCright, B; Virshup, DM
MLA Citation
McCright, B, and Virshup, DM. "Identification of a new family of protein phosphatase 2A regulatory subunits." J Biol Chem 270.44 (November 3, 1995): 26123-26128.
PMID
7592815
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
44
Publish Date
1995
Start Page
26123
End Page
26128

Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family.

The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.

Authors
Fish, KJ; Cegielska, A; Getman, ME; Landes, GM; Virshup, DM
MLA Citation
Fish, KJ, Cegielska, A, Getman, ME, Landes, GM, and Virshup, DM. "Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family." J Biol Chem 270.25 (June 23, 1995): 14875-14883.
PMID
7797465
Source
pubmed
Published In
The Journal of biological chemistry
Volume
270
Issue
25
Publish Date
1995
Start Page
14875
End Page
14883

Occurrence of Henoch-Schönlein purpura in a child with Wilms' tumor.

Authors
Sivak, LE; Virshup, DM
MLA Citation
Sivak, LE, and Virshup, DM. "Occurrence of Henoch-Schönlein purpura in a child with Wilms' tumor." Med Pediatr Oncol 24.3 (March 1995): 213-214.
PMID
7838045
Source
pubmed
Published In
Pediatric Blood and Cancer
Volume
24
Issue
3
Publish Date
1995
Start Page
213
End Page
214

Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication.

The ability of simian virus 40 (SV40) large T antigen to catalyze the initiation of viral DNA replication is regulated by its phosphorylation state. Previous studies have identified the free catalytic subunit of protein phosphatase 2A (PP2Ac) as the cellular phosphatase which can remove inhibitory phosphoryl groups from serines 120 and 123. The catalytic C subunit exists in the cell complexed with a 65-kDa A subunit and one of several B subunits. To determine if any of the holoenzymes could activate T antigen, we tested the ability of the heterodimeric AC and two heterotrimeric ABC forms to stimulate T-antigen function in unwinding the origin of SV40 DNA replication. Only free catalytic subunit C and the heterotrimeric form with a 72-kDa B subunit (PP2A-T72) could stimulate T-antigen-dependent origin unwinding. Both the dimeric form (PP2A-D) and the heterotrimer with a 55-kDa B subunit (PP2A-T55) actively inhibited T-antigen function. We found that PP2A-T72 activated T antigen by dephosphorylating serines 120 and 123, while PP2A-D and PP2A-T55 inactivated T antigen by dephosphorylating the p34cdc2 target site, threonine 124. Thus, alterations in the subunit composition of PP2A holoenzymes have significant functional consequences for the initiation of in vitro SV40 DNA replication. The regulatory B subunits of PP2A may play a role in regulating SV40 DNA replication in infected cells as well.

Authors
Cegielska, A; Shaffer, S; Derua, R; Goris, J; Virshup, DM
MLA Citation
Cegielska, A, Shaffer, S, Derua, R, Goris, J, and Virshup, DM. "Different oligomeric forms of protein phosphatase 2A activate and inhibit simian virus 40 DNA replication." Mol Cell Biol 14.7 (July 1994): 4616-4623.
PMID
8007966
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
14
Issue
7
Publish Date
1994
Start Page
4616
End Page
4623

Enhancing the psychological health of medical students: the student well-being committee

Authors
COOMBS, RH; VIRSHUP, BB
MLA Citation
COOMBS, RH, and VIRSHUP, BB. "Enhancing the psychological health of medical students: the student well-being committee." Medical Education 28.1 (January 1994): 47-54.
Source
crossref
Published In
Medical Education
Volume
28
Issue
1
Publish Date
1994
Start Page
47
End Page
54
DOI
10.1111/j.1365-2923.1994.tb02684.x

T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123.

Simian virus 40 (SV40) DNA replication begins after two large T-antigen hexamers assemble on the viral minimal origin of replication and locally unwind the template DNA. The activity of T antigen in this reaction is regulated by its phosphorylation state. A form of casein kinase I purified from HeLa nuclear extracts (T-antigen kinase) phosphorylates T antigen on physiologic sites and inhibits its activity in the unwinding reaction (A. Cegielska and D. M. Virshup, Mol. Cell. Biol. 13:1202-1211, 1993). Using a series of mutant T antigens expressed by recombinant baculoviruses in Sf9 cells, we find that the origin unwinding activities of both TS677-->A and TS677,679-->A are inhibited by the T-antigen kinase, as is wild-type T antigen. In contrast, mutants TS120-->A and TS123,679-->A are resistant to inhibition by the kinase. Thus, phosphorylation of serines 120 and 123 is necessary for inhibition of T-antigen activity. Previous studies of casein kinase I substrate specificity have suggested that acidic residues or a phosphorylated amino acid amino terminal to the target residue are required to create a casein kinase I recognition site. However, we find that the T-antigen kinase can add more than 3 mol of Pi per mol to full-length bacterially produced T antigen and that it inhibits the unwinding activity of p34cdc2-activated bacterially produced T antigen. Since no prior phosphorylation is present in this bacterially produced T antigen, and no acidic residues are present immediately amino terminal to serines 120 and 123, other structural elements of T antigen must contribute to the recognition signals for T-antigen kinase. In support of this conclusion, we find that while T-antigen kinase phosphorylates amino-terminal residues in bacterially produced full-length T antigen, it cannot phosphorylate bacterially produced truncated T antigen containing amino acids 1 to 259, a 17-kDa amino-terminal tryptic fragment of T antigen, nor can it phosphorylate denatured T antigen. These findings strongly suggest that the carboxy-terminal domain of T antigen is an important modifier of the recognition signals for phosphorylation of the critical amino-terminal sites by the T-antigen kinase. This conclusion is consistent with previous studies suggesting close apposition of amino- and carboxy-terminal domains of T antigen in the native protein. The three-dimensional conformation of the substrate appears to make a significant contribution to T-antigen kinase substrate specificity.

Authors
Cegielska, A; Moarefi, I; Fanning, E; Virshup, DM
MLA Citation
Cegielska, A, Moarefi, I, Fanning, E, and Virshup, DM. "T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123." J Virol 68.1 (January 1994): 269-275.
PMID
8254738
Source
pubmed
Published In
Journal of virology
Volume
68
Issue
1
Publish Date
1994
Start Page
269
End Page
275

Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I.

The initiation of simian virus 40 (SV40) DNA replication is regulated by the phosphorylation state of the viral initiator protein, large T antigen. We describe the purification from HeLa cell nuclei of a 35-kDa serine/threonine protein kinase that phosphorylates T antigen at sites that are phosphorylated in vivo and thereby inhibits its ability to initiate SV40 DNA replication. The inhibition of both origin unwinding and DNA replication by the kinase is reversed by protein phosphatase 2A. As determined by molecular weight, substrate specificity, autophosphorylation, immunoreactivity, and limited sequence analysis, this kinase appears to be identical to casein kinase I, a ubiquitous serine/threonine protein kinase that is closely related to a yeast kinase involved in DNA metabolism. The HeLa cell phosphorylation cycle that controls the initiation of SV40 DNA replication may also play a role in cellular DNA metabolism.

Authors
Cegielska, A; Virshup, DM
MLA Citation
Cegielska, A, and Virshup, DM. "Control of simian virus 40 DNA replication by the HeLa cell nuclear kinase casein kinase I." Mol Cell Biol 13.2 (February 1993): 1202-1211.
PMID
8380893
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
13
Issue
2
Publish Date
1993
Start Page
1202
End Page
1211

CONTROL OF SV40 DNA-REPLICATION BY THE RECIPROCAL ACTION OF CASEIN KINASE-I AND PROTEIN PHOSPHATASE 2AC

Authors
VIRSHUP, DM; CEGIELSKA, A; FISH, K; SHAFFER, S; HILLAM, D
MLA Citation
VIRSHUP, DM, CEGIELSKA, A, FISH, K, SHAFFER, S, and HILLAM, D. "CONTROL OF SV40 DNA-REPLICATION BY THE RECIPROCAL ACTION OF CASEIN KINASE-I AND PROTEIN PHOSPHATASE 2AC." JOURNAL OF CELLULAR BIOCHEMISTRY (January 9, 1993): 305-305.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1993
Start Page
305
End Page
305

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.

Authors
Virshup, DM; Russo, AA; Kelly, TJ
MLA Citation
Virshup, DM, Russo, AA, and Kelly, TJ. "Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A." Mol Cell Biol 12.11 (November 1992): 4883-4895.
PMID
1328866
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
12
Issue
11
Publish Date
1992
Start Page
4883
End Page
4895

Focal encephalitis with enterovirus infections.

We report on four pediatric patients with Enterovirus infections who were admitted to the hospital with signs or symptoms of acute, focal encephalitis. All four experienced focal seizures. Each had a cerebrospinal fluid pleocytosis at the initial lumbar puncture. In all four patients the diagnosis of herpes simplex encephalitis was entertained. Each child improved spontaneously within a few days of admission to the hospital, and only one had residual neurologic abnormalities at the time of discharge. A brief review of these cases, and three additional cases from the literature, indicate that the enteroviruses, particularly the group A Coxsackieviruses, are rare causes of acute focal encephalitis in children and adolescents.

Authors
Modlin, JF; Dagan, R; Berlin, LE; Virshup, DM; Yolken, RH; Menegus, M
MLA Citation
Modlin, JF, Dagan, R, Berlin, LE, Virshup, DM, Yolken, RH, and Menegus, M. "Focal encephalitis with enterovirus infections." Pediatrics 88.4 (October 1991): 841-845.
PMID
1896296
Source
pubmed
Published In
Pediatrics
Volume
88
Issue
4
Publish Date
1991
Start Page
841
End Page
845

Protein phosphatase 2A dephosphorylates simian virus 40 large T antigen specifically at residues involved in regulation of DNA-binding activity.

Treatment of purified simian virus 40 large T antigen (LT) with protein phosphatase 2A stimulates LT-dependent DNA unwinding and replication (D. M. Virshup, M. G. Kauffman, and T. J. Kelly, EMBO J. 8: 3891-3898, 1989). The specificity of the catalytic subunit of protein phosphatase 2A toward LT was investigated by two-dimensional peptide mapping. Increasing amounts of phosphatase sequentially removed the phosphates from serine residues 120, 123, 677, and perhaps 679, residues which have been implicated in regulating the DNA-binding activity of LT.

Authors
Scheidtmann, KH; Virshup, DM; Kelly, TJ
MLA Citation
Scheidtmann, KH, Virshup, DM, and Kelly, TJ. "Protein phosphatase 2A dephosphorylates simian virus 40 large T antigen specifically at residues involved in regulation of DNA-binding activity." J Virol 65.4 (April 1991): 2098-2101.
PMID
1848320
Source
pubmed
Published In
Journal of virology
Volume
65
Issue
4
Publish Date
1991
Start Page
2098
End Page
2101

INITIATION OF SV40 DNA-REPLICATION - MECHANISM AND CONTROL

Authors
ERDILE, LF; COLLINS, KL; RUSSO, A; SIMANCEK, P; SMALL, D; UMBRICHT, C; VIRSHUP, D; CHENG, L; RANDALL, S; WEINBERG, D; MOAREFI, I; FANNING, E; KELLY, T
MLA Citation
ERDILE, LF, COLLINS, KL, RUSSO, A, SIMANCEK, P, SMALL, D, UMBRICHT, C, VIRSHUP, D, CHENG, L, RANDALL, S, WEINBERG, D, MOAREFI, I, FANNING, E, and KELLY, T. "INITIATION OF SV40 DNA-REPLICATION - MECHANISM AND CONTROL." COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY 56 (1991): 303-313.
Source
wos-lite
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
56
Publish Date
1991
Start Page
303
End Page
313

Initiation of SV40 DNA replication: mechanism and control.

Authors
Erdile, LF; Collins, KL; Russo, A; Simancek, P; Small, D; Umbricht, C; Virshup, D; Cheng, L; Randall, S; Weinberg, D
MLA Citation
Erdile, LF, Collins, KL, Russo, A, Simancek, P, Small, D, Umbricht, C, Virshup, D, Cheng, L, Randall, S, and Weinberg, D. "Initiation of SV40 DNA replication: mechanism and control." Cold Spring Harb Symp Quant Biol 56 (1991): 303-313. (Review)
PMID
1668084
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
56
Publish Date
1991
Start Page
303
End Page
313

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.

Authors
Weinberg, DH; Collins, KL; Simancek, P; Russo, A; Wold, MS; Virshup, DM; Kelly, TJ
MLA Citation
Weinberg, DH, Collins, KL, Simancek, P, Russo, A, Wold, MS, Virshup, DM, and Kelly, TJ. "Reconstitution of simian virus 40 DNA replication with purified proteins." Proc Natl Acad Sci U S A 87.22 (November 1990): 8692-8696.
PMID
2174160
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
87
Issue
22
Publish Date
1990
Start Page
8692
End Page
8696

DNA replication.

Authors
Virshup, DM
MLA Citation
Virshup, DM. "DNA replication." Curr Opin Cell Biol 2.3 (June 1990): 453-460. (Review)
PMID
2165792
Source
pubmed
Published In
Current Opinion in Cell Biology
Volume
2
Issue
3
Publish Date
1990
Start Page
453
End Page
460

CELLULAR PROTEINS INVOLVED IN SV40 DNA-REPLICATION INVITRO

Authors
VIRSHUP, DM; WOLD, MS; WEINBERG, DH; KAUFFMAN, MG; KELLY, TJ
MLA Citation
VIRSHUP, DM, WOLD, MS, WEINBERG, DH, KAUFFMAN, MG, and KELLY, TJ. "CELLULAR PROTEINS INVOLVED IN SV40 DNA-REPLICATION INVITRO." 1990.
Source
wos-lite
Published In
MOLECULAR MECHANISMS IN DNA REPLICATION AND RECOMBINATION
Volume
127
Publish Date
1990
Start Page
303
End Page
314

Hb Catonsville (glutamic acid inserted between Pro-37(C2)alpha and Thr-38(C3)alpha). Nonallelic gene conversion in the globin system?

Hb Catonsville is an unstable variant in which glutamic acid is inserted into the alpha-globin chain between Pro-37(C2) and Thr-38(C3). The peptide sequence data are consistent with the DNA sequence of the polymerase chain reaction-amplified fragment of the variant globin gene, which shows the insertion of the triplet codon--GAA--into the mutant alpha-globin gene. In the normal alpha-globin gene cluster the codon for glutamic acid is GAG rather than GAA. Thus, there are two features unique to Hb Catonsville, one the insertion of a single residue into the interior of the alpha-globin chain, and two the presence of the alternate codon for glutamic acid. The experimental evidence suggests that Hb Catonsville may be an example of nonhomologous nonallelic gene conversion, an observation not previously reported in this gene family. The mutation occurs in the critical alpha 1 beta 2 interface of the hemoglobin tetramer and leads to a variant with high oxygen affinity, a reduced cooperativity, and Bohr effect.

Authors
Moo-Penn, WF; Swan, DC; Hine, TK; Baine, RM; Jue, DL; Benson, JM; Johnson, MH; Virshup, DM; Zinkham, WH
MLA Citation
Moo-Penn, WF, Swan, DC, Hine, TK, Baine, RM, Jue, DL, Benson, JM, Johnson, MH, Virshup, DM, and Zinkham, WH. "Hb Catonsville (glutamic acid inserted between Pro-37(C2)alpha and Thr-38(C3)alpha). Nonallelic gene conversion in the globin system?." J Biol Chem 264.36 (December 25, 1989): 21454-21457.
PMID
2574721
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
36
Publish Date
1989
Start Page
21454
End Page
21457

Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A.

We have made use of the cell-free SV40 DNA replication system to identify and characterize cellular proteins required for efficient DNA synthesis. One such protein, replication protein C (RP-C), was shown to be involved with SV40 large T antigen in the early stages of viral DNA replication in vitro. We demonstrate here that RP-C is identical to the catalytic subunit of cellular protein phosphatase 2A (PP2Ac). The purified protein dephosphorylates specific phosphoamino acid residues in T antigen, consistent with the hypothesis that SV40 DNA replication is regulated by modulating the phosphorylation state of the viral initiator protein. We also show that purified RP-C/PP2Ac preferentially stimulates SV40 DNA replication in extracts from early G1 phase cells. This finding suggests that the activity of a cellular factor that influences the net phosphorylation state of T antigen is cell cycle dependent.

Authors
Virshup, DM; Kauffman, MG; Kelly, TJ
MLA Citation
Virshup, DM, Kauffman, MG, and Kelly, TJ. "Activation of SV40 DNA replication in vitro by cellular protein phosphatase 2A." EMBO J 8.12 (December 1, 1989): 3891-3898.
PMID
2555176
Source
pubmed
Published In
EMBO Journal
Volume
8
Issue
12
Publish Date
1989
Start Page
3891
End Page
3898

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.

Authors
Virshup, DM; Kelly, TJ
MLA Citation
Virshup, DM, and Kelly, TJ. "Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro." Proc Natl Acad Sci U S A 86.10 (May 1989): 3584-3588.
PMID
2542940
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
10
Publish Date
1989
Start Page
3584
End Page
3588

Identification of cellular proteins required for simian virus 40 DNA replication.

Study of the proteins involved in DNA replication of a model system such as SV40 is a first step in understanding eukaryotic chromosomal replication. Using a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication, we conducted a series of systematic fractionation-reconstitution experiments for the purpose of identifying and characterizing the cellular proteins involved in SV40 DNA replication. In addition to the one viral-encoded replication protein, T antigen, we have identified and begun to characterize at least six cellular components from a HeLa cytoplasmic extract that are absolutely required for SV40 DNA replication in vitro. These include: (i) two partially purified fractions, CF IC and CF IIA, and (ii) four proteins that have been purified to near homogeneity, replication protein-A, proliferating cell nuclear antigen, DNA polymerase alpha-primase complex, and topoisomerase (I and II). Replication protein-A is a multi-subunit protein that has single-stranded DNA binding activity and is required for a T antigen-dependent, origin-dependent unwinding reaction which may be an important early step in initiation of replication. Fraction CF IC can stimulate this unwinding reaction, suggesting that it also may function during initiation. Proliferating cell nuclear antigen, DNA polymerase alpha-primase, and CF IIA all appear to be involved in elongation of nascent chains.

Authors
Wold, MS; Weinberg, DH; Virshup, DM; Li, JJ; Kelly, TJ
MLA Citation
Wold, MS, Weinberg, DH, Virshup, DM, Li, JJ, and Kelly, TJ. "Identification of cellular proteins required for simian virus 40 DNA replication." J Biol Chem 264.5 (February 15, 1989): 2801-2809.
PMID
2536723
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
5
Publish Date
1989
Start Page
2801
End Page
2809

Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes.

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.

Authors
Virshup, DM; Bennett, V
MLA Citation
Virshup, DM, and Bennett, V. "Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes." J Cell Biol 106.1 (January 1988): 39-50.
PMID
2892842
Source
pubmed
Published In
The Journal of Cell Biology
Volume
106
Issue
1
Publish Date
1988
Start Page
39
End Page
50

Cutaneous manifestations of Corynebacterium group JK sepsis.

A 14-year-old boy developed group JK corynebacteria sepsis and a generalized erythematous macular and papular skin eruption following chemotherapy for relapse of acute lymphocytic leukemia. Lesional skin biopsy demonstrated effacement of eccrine glands by numerous pleomorphic gram-positive bacilli, morphologically consistent with Corynebacterium and confirmed by culture. This is the first known report documenting the generalized skin manifestations and histopathologic features associated with Corynebacterium sepsis.

Authors
Jerdan, MS; Shapiro, RS; Smith, NB; Virshup, DM; Hood, AF
MLA Citation
Jerdan, MS, Shapiro, RS, Smith, NB, Virshup, DM, and Hood, AF. "Cutaneous manifestations of Corynebacterium group JK sepsis." J Am Acad Dermatol 16.2 Pt 2 (February 1987): 444-447.
PMID
3469229
Source
pubmed
Published In
Journal of The American Academy of Dermatology
Volume
16
Issue
2 Pt 2
Publish Date
1987
Start Page
444
End Page
447

CUTANEOUS MANIFESTATIONS OF CORYNEBACTERIUM GROUP JK SEPSIS

Authors
JERDAN, MS; SHAPIRO, RS; SMITH, NB; VIRSHUP, DM; HOOD, AF
MLA Citation
JERDAN, MS, SHAPIRO, RS, SMITH, NB, VIRSHUP, DM, and HOOD, AF. "CUTANEOUS MANIFESTATIONS OF CORYNEBACTERIUM GROUP JK SEPSIS." JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY 16.2 (February 1987): 444-447.
Source
wos-lite
Published In
Journal of The American Academy of Dermatology
Volume
16
Issue
2
Publish Date
1987
Start Page
444
End Page
447
DOI
10.1016/S0190-9622(87)70058-9

Cutaneous manifestations of Corynebacterium group JK sepsis

A 14-year-old boy developed group JK corynebacteria sepsis and a generalized erythematous macular and papular skin eruption following chemotherapy for relapse of acute lymphocytic leukemia. Lesional skin biopsy demonstrated effacement of eccrine glands by numerous pleomorphic gram-positive bacilli, morphologically consistent with Corynebacterium and confirmed by culture. This is the first known report documenting the generalized skin manifestations and histopathologic features associated with Corynebacterium sepsis.

Authors
Jerdan, MS; Shapiro, RS; Smith, NB; Virshup, DM; Hood, AF
MLA Citation
Jerdan, MS, Shapiro, RS, Smith, NB, Virshup, DM, and Hood, AF. "Cutaneous manifestations of Corynebacterium group JK sepsis." Journal of the American Academy of Dermatology 16.2 II SUPPL. (1987): 444-447.
Source
scival
Published In
Journal of The American Academy of Dermatology
Volume
16
Issue
2 II SUPPL.
Publish Date
1987
Start Page
444
End Page
447
DOI
10.1016/S0190-9622(87)70058-9

CLATHRIN-COATED VESICLE ASSEMBLY POLYPEPTIDES - PHYSICAL-PROPERTIES AND IDENTIFICATION OF A NEW SUBUNIT

Authors
VIRSHUP, DM; BENNETT, V
MLA Citation
VIRSHUP, DM, and BENNETT, V. "CLATHRIN-COATED VESICLE ASSEMBLY POLYPEPTIDES - PHYSICAL-PROPERTIES AND IDENTIFICATION OF A NEW SUBUNIT." JOURNAL OF CELL BIOLOGY 103.5 (November 1986): A53-A53.
Source
wos-lite
Published In
The Journal of Cell Biology
Volume
103
Issue
5
Publish Date
1986
Start Page
A53
End Page
A53

Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes.

Two new vascular smooth muscle relaxants, bepridil and cetiedil, were found to possess specific CaM-inhibitory properties which resembled those of trifluoperazine. Trifluoperazine, bepridil, and cetiedil inhibited Ca2+-dependent 125I-CaM binding to erythrocyte membranes and CaM activation of membrane Ca2+-ATPase with IC50 values of approximately 12, approximately 17, and approximately 40 microM, respectively. This does not appear to be the result of a nonspecific hydrophobic interaction since inhibition was not observed with micromolar concentrations of many other hydrophobic agents. The predominant inhibition of binding and Ca2+-ATPase activation was competitive with respect to CaM. Bepridil and cetiedil bind directly to CaM since these drugs displaced [3H]trifluoperazine from sites on CaM. Inhibition of Ca2+-ATPase and binding by the drugs was not due to interference with the catalytic activity of this enzyme since: (a) neither inhibition of CaM-independent basal Ca2+-ATPase activity nor inhibition of proteolytically-activated Ca2+-ATPase activities were produced by these agents, and (b) no drug-induced inhibition of CaM binding was detected when membranes were preincubated with these agents but washed prior to addition of 125I-CaM. Thus, bepridil and cetiedil competitively inhibit Ca2+-dependent interactions of CaM with erythrocyte membranes, most likely by a direct interaction between these drugs and CaM. The principal clinical actions of these drugs may be explained by their interactions with CaM or CaM-related proteins leading to reduced activation of Ca2+-regulated enzymes in certain other tissues, such as myosin light chain kinase in vascular smooth muscle.

Authors
Agre, P; Virshup, D; Bennett, V
MLA Citation
Agre, P, Virshup, D, and Bennett, V. "Bepridil and cetiedil. Vasodilators which inhibit Ca2+-dependent calmodulin interactions with erythrocyte membranes." J Clin Invest 74.3 (September 1984): 812-820.
PMID
6088585
Source
pubmed
Published In
Journal of Clinical Investigation
Volume
74
Issue
3
Publish Date
1984
Start Page
812
End Page
820
DOI
10.1172/JCI111497

Unique sensitivity of Hb Zürich to oxidative injury by phenazopyridine: reversal of the effects by elevating carboxyhemoglobin levels in vivo and in vitro.

Phenazopyridine (PAP) causes a hemolytic anemia in normal individuals who receive an overdose or in patients with decreased renal function given therapeutic doses. There are no reports of PAP-induced hemolysis in individuals with unstable hemoglobins. Therapeutic doses of PAP administered to a subject with Hb Zürich (His E7 (63) beta leads to Arg) caused a severe hemolytic anemia with many large Heinz bodies appearing in the red cells. Incubation of whole blood from three asymptomatic Hb Zürich subjects with PAP at a molar ratio of PAP/Hb of 1.3/1 produced a moderate to marked increase in methemoglobin (MHb) and Heinz body formation accompanied by a slight to moderate decrease in levels of reduced glutathione. The rates of MHb formation were proportional to the concentration of PAP. In two of the subjects the rates of formation of Heinz bodies and MHb were substantially higher than in the third subject. Red cells from five normal adults were not affected. In Hb Zürich red cells the affinity of the abnormal beta chain for carbon monoxide is much greater than that of the normal alpha and beta chains. The two subjects with higher rates of MHb and Heinz body formation were nonsmokers with in vivo carboxyhemoglobin (HbCO) levels of 4-6%. The third subject with low rates of MHb and Heinz body formation was a smoker with in vivo HbCO levels of 15-18%. Increasing levels of HbCO from 8.2% to 14.3% by the in vitro addition of CO caused a marked reduction in the rate of Heinz body formation and a moderate decrease in MHb formation. Rates of MHb formation similar to those observed in normal red cells occurred at HbCO percentages of 89.2 and 99.2. Red cells containing Hb Zürich are extremely sensitive to oxidative injury by PAP, either in vivo or in vitro. The degree of oxidative injury diminishes as the level of HbCO increases, a phenomenon that is enhanced by preferential binding of CO to the abnormal beta subunit of Hb Zürich.

Authors
Virshup, DM; Zinkham, WH; Sirota, RL; Caughey, WS
MLA Citation
Virshup, DM, Zinkham, WH, Sirota, RL, and Caughey, WS. "Unique sensitivity of Hb Zürich to oxidative injury by phenazopyridine: reversal of the effects by elevating carboxyhemoglobin levels in vivo and in vitro." Am J Hematol 14.4 (June 1983): 315-324.
PMID
6859031
Source
pubmed
Published In
American Journal of Hematology
Volume
14
Issue
4
Publish Date
1983
Start Page
315
End Page
324

Unique sensitivity of HB Zurich to oxidative injury by phenazopyridine: Reversal of the effect by elevating carboxyhemoglobin levels in vivo and in vitro

Authors
Virshup, DM; Wm, HZ; Sirota, RL; Caughey, WS
MLA Citation
Virshup, DM, Wm, HZ, Sirota, RL, and Caughey, WS. "Unique sensitivity of HB Zurich to oxidative injury by phenazopyridine: Reversal of the effect by elevating carboxyhemoglobin levels in vivo and in vitro." Pediatric Research 16.4 II (1982): No.-830.
Source
scival
Published In
Pediatric Research
Volume
16
Issue
4 II
Publish Date
1982
Start Page
No.
End Page
830
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