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Zhang, Weiguo

Overview:


Activation via the T-cell antigen receptor (TCR) triggers a cascade of intracellular biochemical events eventually leading to T-cell proliferation and effector functions. One of the earliest events is the activation of the Src family tyrosine kinases Fyn and Lck. The activated Src family kinases phosphorylate the CD3 subunits and TCRζ chains. ZAP-70 tyrosine kinase is recruited to the antigen receptors via the binding to CD3 and TCRζ. ZAP-70 is then tyrosine phosphorylated by these Src family kinases and thus activated. These activated tyrosine kinases further phosphorylate a number of intracellular proteins, such as PLC-γ1, Vav, Cbl, SLP-76, and LAT, and activate downstream signaling pathways including the Ras-MAPK pathway and Ca2+flux. Activation of these two pathways is required for AP-1 and NFAT-mediated transcription, IL-2 production, and T-cell proliferation.

Our primary interest of the laboratory is to understand the role of membrane-associated adaptor proteins in lymphocyte activation, development, and immune response. One of these proteins is LAT (Linker for Activation of T-cells). LAT is tyrosine phosphorylated upon T-cell activation and associates with several signaling molecules including Grb2, Gads, and PLC-γ1. LAT-deficient T-cells are defective in the Ras-MAPK activation and Ca2+ flux after the TCR engagement. LAT knockout mice have an early block in thymocyte development. Interestingly, mice with a mutation in LAT develop a severe autoimmune disease. We are investigating how LAT interacts with other signaling proteins and how LAT regulates T cell activation and immune responses.

In addition to LAT, we are working on two LAT-like molecules, LAB and LAX. We have generated mice deficient in these proteins and are analyzing the phenotypes of these mice to determine the role of these proteins in lymphocyte signaling and immune responses.

We are also interested in FcεRI-mediated signaling. We are working on the role of LAT, LAB, and RasGRP1 in FcεRI-mediated signaling, mast cell function, and allergic responses.

Our long-term goal is to understand the details of immunoreceptor-mediated signaling pathways. Understanding these signaling pathways may identify therapeutic targets that could facilitate the development of drugs that suppress, modify, or augment immune responses in autoimmunity, transplantation, allergy, and cancer.

Positions:

Associate Professor of Immunology

Immunology
School of Medicine

Director of Undergraduate Studies

Immunology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1994

Ph.D. — Yeshiva University New York

Postdoctoral Fellow, Microbiology And Immunology

Yeshiva University New York

Postdoctoral Fellow, Cell Biology And Metabolism Branch

National Institutes of Health

Grants:

Basic Immunology Training Program

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 2002
End Date
June 30, 2019

Training Program in Inflammatory and Immunological Diseases

Administered By
Medicine, Rheumatology and Immunology
AwardedBy
National Institutes of Health
Role
Preceptor
Start Date
September 30, 1980
End Date
August 31, 2017

Phospholipase D proteins in immunoreceptor-mediated signaling

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
February 16, 2011
End Date
January 31, 2017

Analysis of adaptor protein (LAT) in TCR signaling

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
January 01, 2001
End Date
August 31, 2014

Regulation of thymocyte maturation and mature T lymphocyte homeostasis by c-FLIP

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
March 01, 2008
End Date
May 31, 2013

Adaptor molecules in lymphocyte signaling

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
June 15, 2003
End Date
August 31, 2012

A novel pathway regulating cytokine production and asthma development

Administered By
Immunology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
May 15, 2009
End Date
April 30, 2011

Development And Persistence Of Immunity In SCID Chimeras

Administered By
Pediatrics, Allergy and Immunology
AwardedBy
National Institutes of Health
Role
Consultant
Start Date
December 01, 1998
End Date
April 30, 2010
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Publications:

Tonic LAT-HDAC7 Signals Sustain Nur77 and Irf4 Expression to Tune Naive CD4 T Cells.

CD4+ T cells differentiate into T helper cell subsets in feedforward manners with synergistic signals from the T cell receptor (TCR), cytokines, and lineage-specific transcription factors. Naive CD4+ T cells avoid spontaneous engagement of feedforward mechanisms but retain a prepared state. T cells lacking the adaptor molecule LAT demonstrate impaired TCR-induced signals yet cause a spontaneous lymphoproliferative T helper 2 (TH2) cell syndrome in mice. Thus, LAT constitutes an unexplained maintenance cue. Here, we demonstrate that tonic signals through LAT constitutively export the repressor HDAC7 from the nucleus of CD4+ T cells. Without such tonic signals, HDAC7 target genes Nur77 and Irf4 are repressed. We reveal that Nur77 suppresses CD4+ T cell proliferation and uncover a suppressive role for Irf4 in TH2 polarization; halving Irf4 gene-dosage leads to increases in GATA3+ and IL-4+ cells. Our studies reveal that naive CD4+ T cells are dynamically tuned by tonic LAT-HDAC7 signals.

Authors
Myers, DR; Lau, T; Markegard, E; Lim, HW; Kasler, H; Zhu, M; Barczak, A; Huizar, JP; Zikherman, J; Erle, DJ; Zhang, W; Verdin, E; Roose, JP
MLA Citation
Myers, DR, Lau, T, Markegard, E, Lim, HW, Kasler, H, Zhu, M, Barczak, A, Huizar, JP, Zikherman, J, Erle, DJ, Zhang, W, Verdin, E, and Roose, JP. "Tonic LAT-HDAC7 Signals Sustain Nur77 and Irf4 Expression to Tune Naive CD4 T Cells." Cell reports 19.8 (May 2017): 1558-1571.
PMID
28538176
Source
epmc
Published In
Cell Reports
Volume
19
Issue
8
Publish Date
2017
Start Page
1558
End Page
1571
DOI
10.1016/j.celrep.2017.04.076

Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function.

Phospholipase D (PLD) proteins are enzymes that catalyze the hydrolysis of phosphatidylcholine to generate an important signaling lipid, phosphatidic acid. Phosphatidic acid is a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Previous studies using inhibitors and overexpression of PLD proteins indicate that PLD1 and PLD2 play positive roles in FcεRI-mediated signaling and mast cell function. We used mice deficient in PLD1, PLD2, or both to study the function of these enzymes in mast cells. In contrast to published studies, we found that PLD1 deficiency impaired FcεRI-mediated mast cell degranulation; however, PLD2 deficiency enhanced it. Biochemical analysis showed that PLD deficiency affected activation of the PI3K pathway and RhoA. Furthermore, our data indicated that, although PLD1 deficiency impaired F-actin disassembly, PLD2 deficiency enhanced microtubule formation. Together, our results suggested that PLD1 and PLD2, two proteins that catalyze the same enzymatic reaction, regulate different steps in mast cell degranulation.

Authors
Zhu, M; Zou, J; Li, T; O'Brien, SA; Zhang, Y; Ogden, S; Zhang, W
MLA Citation
Zhu, M, Zou, J, Li, T, O'Brien, SA, Zhang, Y, Ogden, S, and Zhang, W. "Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function." Journal of immunology (Baltimore, Md. : 1950) 195.9 (November 2015): 4492-4502.
PMID
26392467
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
195
Issue
9
Publish Date
2015
Start Page
4492
End Page
4502
DOI
10.4049/jimmunol.1500665

Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells.

Dendritic epidermal T cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout postnatal life of the mouse. DETCs have restricted antigenic specificity as a result of their exclusive usage of a canonical TCR. Although the importance of the TCR in DETC development has been well established, the exact role of TCR signaling in DETC homeostasis and function remains incompletely defined. In this study, we investigated TCR signaling in fully matured DETCs by lineage-restricted deletion of the Lat gene, an essential signaling molecule downstream of the TCR. We found that Lat deletion impaired TCR-dependent cytokine gene activation and the ability of DETCs to undergo proliferative expansion. However, linker for activation of T cells-deficient DETCs were able to maintain long-term population homeostasis, although with a reduced proliferation rate. Mice with Lat deletion in DETCs exhibited delayed wound healing accompanied by impaired clonal expansion within the wound area. Our study revealed differential requirements for TCR signaling in homeostatic maintenance of DETCs and in their effector function during wound healing.

Authors
Zhang, B; Wu, J; Jiao, Y; Bock, C; Dai, M; Chen, B; Chao, N; Zhang, W; Zhuang, Y
MLA Citation
Zhang, B, Wu, J, Jiao, Y, Bock, C, Dai, M, Chen, B, Chao, N, Zhang, W, and Zhuang, Y. "Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells." Journal of immunology (Baltimore, Md. : 1950) 195.9 (November 2015): 4282-4291.
PMID
26408667
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
195
Issue
9
Publish Date
2015
Start Page
4282
End Page
4291
DOI
10.4049/jimmunol.1501220

The Importance of IL-6 in the Development of LAT-Mediated Autoimmunity.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is highly tyrosine phosphorylated upon engagement of the TCR. Phosphorylated LAT binds Grb2, Gads, and phospholipase C (PLC)γ1 to mediate T cell activation, proliferation, and cytokine production. T cells from mice harboring a mutation at the PLCγ1 binding site of LAT (Y136F) have impaired calcium flux and Erk activation. Interestingly, these T cells are highly activated, resulting in the development of a lymphoproliferative syndrome in these mice. CD4(+) T cells in LATY136F mice are Th2 skewed, producing large amounts of IL-4. In this study, we showed that the LATY136F T cells could also overproduce IL-6 due to activated NF-κB, AKT, and p38 pathways. By crossing LATY136F mice with IL-6-deficient mice, we demonstrated that IL-6 is required for uncontrolled T cell expansion during the early stage of disease development. Reduced CD4(+) T cell expansion was not due to a further block in thymocyte development or an increase in the number of regulatory T cells, but was caused by reduction in cell survival. In aged IL-6(-/-) LATY136F mice, CD4(+) T cells began to hyperproliferate and induced splenomegaly; however, isotype switching and autoantibody production were diminished. Our data indicated that the LAT-PLCγ1 interaction is important for controlling IL-6 production by T cells and demonstrated a critical role of IL-6 in the development of this lymphoproliferative syndrome.

Authors
O'Brien, SA; Zhu, M; Zhang, W
MLA Citation
O'Brien, SA, Zhu, M, and Zhang, W. "The Importance of IL-6 in the Development of LAT-Mediated Autoimmunity." Journal of immunology (Baltimore, Md. : 1950) 195.2 (July 2015): 695-705.
PMID
26034173
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
195
Issue
2
Publish Date
2015
Start Page
695
End Page
705
DOI
10.4049/jimmunol.1403187

LAT-mediated TCR signaling is required for optimal function and clonal expansion but not long-term survival and homeostatic turnover of DETCs

Authors
Zhang, B; Bock, C; Wu, J; Jiao, Y; Chen, B; Chao, N; Zhang, W; Zhuang, Y
MLA Citation
Zhang, B, Bock, C, Wu, J, Jiao, Y, Chen, B, Chao, N, Zhang, W, and Zhuang, Y. "LAT-mediated TCR signaling is required for optimal function and clonal expansion but not long-term survival and homeostatic turnover of DETCs." May 1, 2015.
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
194
Publish Date
2015

Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is recently identified as a cytosolic DNA sensor and generates a non-canonical cGAMP that contains G(2',5')pA and A(3',5')pG phosphodiester linkages. cGAMP activates STING which triggers innate immune responses in mammals. However, the evolutionary functions and origins of cGAS and STING remain largely elusive. Here, we carried out comprehensive evolutionary analyses of the cGAS-STING pathway. Phylogenetic analysis of cGAS and STING families showed that their origins could be traced back to a choanoflagellate Monosiga brevicollis. Modern cGAS and STING may have acquired structural features, including zinc-ribbon domain and critical amino acid residues for DNA binding in cGAS as well as carboxy terminal tail domain for transducing signals in STING, only recently in vertebrates. In invertebrates, cGAS homologs may not act as DNA sensors. Both proteins cooperate extensively, have similar evolutionary characteristics, and thus may have co-evolved during metazoan evolution. cGAS homologs and a prokaryotic dinucleotide cyclase for canonical cGAMP share conserved secondary structures and catalytic residues. Therefore, non-mammalian cGAS may function as a nucleotidyltransferase and could produce cGAMP and other cyclic dinucleotides. Taken together, assembling signaling components of the cGAS-STING pathway onto the eukaryotic evolutionary map illuminates the functions and origins of this innate immune pathway.

Authors
Wu, X; Wu, F-H; Wang, X; Wang, L; Siedow, JN; Zhang, W; Pei, Z-M
MLA Citation
Wu, X, Wu, F-H, Wang, X, Wang, L, Siedow, JN, Zhang, W, and Pei, Z-M. "Molecular evolutionary and structural analysis of the cytosolic DNA sensor cGAS and STING." Nucleic acids research 42.13 (July 2014): 8243-8257.
PMID
24981511
Source
epmc
Published In
Nucleic Acids Research
Volume
42
Issue
13
Publish Date
2014
Start Page
8243
End Page
8257
DOI
10.1093/nar/gku569

Linker for activation of T cells (LAT) in the regulation of T cell activation and autoimmunity

Authors
Zhang, W; Sullivan, S; Zhu, M; Lewis, C
MLA Citation
Zhang, W, Sullivan, S, Zhu, M, and Lewis, C. "Linker for activation of T cells (LAT) in the regulation of T cell activation and autoimmunity." May 1, 2014.
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
192
Publish Date
2014

The role of LAT-PLCγ1 interaction in γδ T cell development and homeostasis.

LAT is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals to modulate T cell development, activation, and proliferation. Upon T cell activation, LAT is phosphorylated and associates with Grb2, Gads, and PLCγ1 through its four distal tyrosine residues. Mutation of one of these tyrosines, Y136, abolishes LAT binding to PLCγ1. This results in impaired TCR-mediated calcium mobilization and Erk activation. CD4 αβ T cells in LATY136F knock-in mice undergo uncontrolled expansion, resulting in a severe autoimmune syndrome. In this study, we investigated the importance of the LAT-PLCγ1 interaction in γδ T cells by crossing LATY136F mice with TCRβ(-/-) mice. Our data showed that the LATY136F mutation had no major effect on homeostasis of epithelial γδ T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4(+) γδ T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL-4, resulting in an autoimmune syndrome similar to that caused by αβ T cells in LATY136F mice. Development of these hyperproliferative γδ T cells was not dependent on MHC class II expression or CD4, and their proliferation could be suppressed, in part, by regulatory T cells. Our data indicated that a unique subset of CD4 γδ T cells can hyperproliferate in LATY136F mice and suggested that LAT-PLCγ1 signaling may function differently in various subsets of γδ T cells.

Authors
Sullivan, SA; Zhu, M; Bao, S; Lewis, CA; Ou-Yang, C-W; Zhang, W
MLA Citation
Sullivan, SA, Zhu, M, Bao, S, Lewis, CA, Ou-Yang, C-W, and Zhang, W. "The role of LAT-PLCγ1 interaction in γδ T cell development and homeostasis." Journal of immunology (Baltimore, Md. : 1950) 192.6 (March 2014): 2865-2874.
PMID
24523509
Source
epmc
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
192
Issue
6
Publish Date
2014
Start Page
2865
End Page
2874
DOI
10.4049/jimmunol.1302493

Molecular editing of cellular responses by the high-affinity receptor for IgE.

Cellular responses elicited by cell surface receptors differ according to stimulus strength. We investigated how the high-affinity receptor for immunoglobulin E (IgE) modulates the response of mast cells to a high- or low-affinity stimulus. Both high- and low-affinity stimuli elicited similar receptor phosphorylation; however, differences were observed in receptor cluster size, mobility, distribution, and the cells' effector responses. Low-affinity stimulation increased receptor association with the Src family kinase Fgr and shifted signals from the adapter LAT1 to the related adapter LAT2. LAT1-dependent calcium signals required for mast cell degranulation were dampened, but the role of LAT2 in chemokine production was enhanced, altering immune cell recruitment at the site of inflammation. These findings uncover how receptor discrimination of stimulus strength can be interpreted as distinct in vivo outcomes.

Authors
Suzuki, R; Leach, S; Liu, W; Ralston, E; Scheffel, J; Zhang, W; Lowell, CA; Rivera, J
MLA Citation
Suzuki, R, Leach, S, Liu, W, Ralston, E, Scheffel, J, Zhang, W, Lowell, CA, and Rivera, J. "Molecular editing of cellular responses by the high-affinity receptor for IgE." Science (New York, N.Y.) 343.6174 (February 6, 2014): 1021-1025.
PMID
24505132
Source
epmc
Published In
Science
Volume
343
Issue
6174
Publish Date
2014
Start Page
1021
End Page
1025
DOI
10.1126/science.1246976

Molecular editing of cellular responses by the high-affinity receptor for IgE

Authors
Suzuki, R; Leach, S; Liu, W; Ralston, E; Scheffel, J; Zhang, W; Lowell, CA; Rivera, J
MLA Citation
Suzuki, R, Leach, S, Liu, W, Ralston, E, Scheffel, J, Zhang, W, Lowell, CA, and Rivera, J. "Molecular editing of cellular responses by the high-affinity receptor for IgE." Science 343.6174 (2014): 1021-1025.
Source
scopus
Published In
Science
Volume
343
Issue
6174
Publish Date
2014
Start Page
1021
End Page
1025

Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function.

Signaling pathways leading to natural killer (NK)-cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family-independent SLP-76-dependent signaling pathway was identified. The LAT family-independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family-dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76-dependent events, including phosphorylation of AKT and extracellular signal-related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function.

Authors
May, RM; Okumura, M; Hsu, C-J; Bassiri, H; Yang, E; Rak, G; Mace, EM; Philip, NH; Zhang, W; Baumgart, T; Orange, JS; Nichols, KE; Kambayashi, T
MLA Citation
May, RM, Okumura, M, Hsu, C-J, Bassiri, H, Yang, E, Rak, G, Mace, EM, Philip, NH, Zhang, W, Baumgart, T, Orange, JS, Nichols, KE, and Kambayashi, T. "Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function." Blood 121.16 (April 2013): 3135-3146.
PMID
23407547
Source
epmc
Published In
Blood
Volume
121
Issue
16
Publish Date
2013
Start Page
3135
End Page
3146
DOI
10.1182/blood-2012-12-474361

The requirement of linker for activation of T cells in the primary and memory responses of CD8 T cells.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that links TCR engagement to downstream signaling events. Although it is clear that LAT is essential in thymocyte development and initiation of T cell activation, its function during T cell expansion, contraction, and memory formation remains unknown. To study the role of TCR-mediated signaling in CD8 T cells during the course of pathogen infection, we used an inducible mouse model to delete LAT in Ag-specific CD8 T cells at different stages of Listeria infection and analyzed the effect of deletion on T cell responses. Our data showed that LAT is important for maintaining CD8 T cell expansion during the priming phase; however, it is not required for CD8 T cell contraction and memory maintenance. Moreover, LAT deficiency accelerates memory differentiation during the effector-to-memory transition, leading to a higher frequency of KLRG1(low)IL-7R(high)CD62L(high) memory T cells. Nonetheless, these LAT-deficient memory T cells were unable to proliferate or produce cytokines upon secondary infection. Our data demonstrated that, although TCR-mediated signaling is dispensable for contraction and memory maintenance, it regulates CD8 T cell memory differentiation and is essential for the memory response against pathogens.

Authors
Ou-Yang, C-W; Zhu, M; Sullivan, SA; Fuller, DM; Zhang, W
MLA Citation
Ou-Yang, C-W, Zhu, M, Sullivan, SA, Fuller, DM, and Zhang, W. "The requirement of linker for activation of T cells in the primary and memory responses of CD8 T cells." J Immunol 190.6 (March 15, 2013): 2938-2947.
PMID
23401587
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
190
Issue
6
Publish Date
2013
Start Page
2938
End Page
2947
DOI
10.4049/jimmunol.1203163

LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear β-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and β-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

Authors
Orr, SJ; Burg, AR; Chan, T; Quigley, L; Jones, GW; Ford, JW; Hodge, D; Razzook, C; Sarhan, J; Jones, YL; Whittaker, GC; Boelte, KC; Lyakh, L; Cardone, M; O'Connor, GM; Tan, C; Li, H; Anderson, SK; Jones, SA; Zhang, W; Taylor, PR; Trinchieri, G; McVicar, DW
MLA Citation
Orr, SJ, Burg, AR, Chan, T, Quigley, L, Jones, GW, Ford, JW, Hodge, D, Razzook, C, Sarhan, J, Jones, YL, Whittaker, GC, Boelte, KC, Lyakh, L, Cardone, M, O'Connor, GM, Tan, C, Li, H, Anderson, SK, Jones, SA, Zhang, W, Taylor, PR, Trinchieri, G, and McVicar, DW. "LAB/NTAL Facilitates Fungal/PAMP-induced IL-12 and IFN-γ Production by Repressing β-Catenin Activation in Dendritic Cells." PLoS Pathogens 9.5 (2013).
PMID
23675302
Source
scival
Published In
PLoS pathogens
Volume
9
Issue
5
Publish Date
2013
DOI
10.1371/journal.ppat.1003357

The importance of the Erk pathway in the development of linker for activation of T cells-mediated autoimmunity.

The ability of the transmembrane adaptor protein linker for activation of T cells (LAT) to regulate T cell development, activation, survival, and homeostasis depends upon phosphorylation of its multiple tyrosine residues. The mutation of tyrosine 136 on LAT abrogates its interaction with phospholipase C-γ1, causing severe ramifications on TCR-mediated signaling. Mice harboring this mutation, LATY136F mice, have significantly impaired thymocyte development; however, they rapidly develop a fatal lymphoproliferative disease marked by the uncontrolled expansion of Th2-skewed CD4(+) T cells, high levels of IgE and IgG1, and autoantibody production. In this study, we assessed the contribution of multiple signaling pathways in LATY136F disease development. The deletion of the critical signaling proteins Gads and RasGRP1 caused a further block in thymocyte development, but, over time, could not prevent CD4(+) T cell hyperproliferation. Also, restoring signaling through the NF-κB and NFAT pathways was unable to halt the development of disease. However, expression of a constitutively active Raf transgene enhanced lymphoproliferation, indicating a role for the Ras-MAPK pathway in LAT-mediated disease.

Authors
Fuller, DM; Zhu, M; Koonpaew, S; Nelson, MI; Zhang, W
MLA Citation
Fuller, DM, Zhu, M, Koonpaew, S, Nelson, MI, and Zhang, W. "The importance of the Erk pathway in the development of linker for activation of T cells-mediated autoimmunity." J Immunol 189.8 (October 15, 2012): 4005-4013.
PMID
22984075
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
189
Issue
8
Publish Date
2012
Start Page
4005
End Page
4013
DOI
10.4049/jimmunol.1201380

Differential requirement of RasGRP1 for γδ T cell development and activation.

γδ T (γδT) cells belong to a distinct T cell lineage that performs immune functions different from αβ T (αβT) cells. Previous studies established that Erk1/2 MAPKs are critical for positive selection of αβT cells. Additional evidence suggests that increased Erk1/2 activity promotes γδT cell generation. RasGRP1, a guanine nucleotide-releasing factor for Ras, plays an important role in positive selection of αβT cells by activating the Ras-Erk1/2 pathway. In this article, we demonstrate that RasGRP1 is critical for TCR-induced Erk1/2 activation in γδT cells, but it exerts different roles for γδT cell generation and activation. Deficiency of RasGRP1 does not obviously affect γδT cell numbers in the thymus, but it leads to increased γδT cells, particularly CD4(-)CD8(+) γδT cells, in the peripheral lymphoid organs. The virtually unhindered γδT cell development in the RasGRP1(-/-) thymus proved to be cell intrinsic, whereas the increase in CD8(+) γδT cells is caused by non-cell-intrinsic mechanisms. Our data provide genetic evidence that decreased Erk1/2 activation in the absence of RasGRP1 is compatible with γδT cell generation. Although RasGRP1 is dispensable for γδT cell generation, RasGRP1-deficient γδT cells are defective in proliferation following TCR stimulation. Additionally, RasGRP1-deficient γδT cells are impaired to produce IL-17 but not IFNγ. Together, these observations revealed that RasGRP1 plays differential roles for γδ and αβ T cell development but is critical for γδT cell proliferation and production of IL-17.

Authors
Chen, Y; Ci, X; Gorentla, B; Sullivan, SA; Stone, JC; Zhang, W; Pereira, P; Lu, J; Zhong, X-P
MLA Citation
Chen, Y, Ci, X, Gorentla, B, Sullivan, SA, Stone, JC, Zhang, W, Pereira, P, Lu, J, and Zhong, X-P. "Differential requirement of RasGRP1 for γδ T cell development and activation." J Immunol 189.1 (July 1, 2012): 61-71.
PMID
22623331
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
189
Issue
1
Publish Date
2012
Start Page
61
End Page
71
DOI
10.4049/jimmunol.1103272

Role of LAT in the granule-mediated cytotoxicity of CD8 T cells.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is essential to bridge T cell receptor (TCR) engagement to downstream signaling events. The indispensable role of LAT in thymocyte development and T cell activation has been well characterized; however, the function of LAT in cytotoxic-T-lymphocyte (CTL) cytotoxicity remains unknown. We show here that LAT-deficient CTLs failed to upregulate FasL and produce gamma interferon after engagement with target cells and had impaired granule-mediated killing. We further dissected the effect of the LAT deletion on each step of granule exocytosis. LAT deficiency led to altered synapse formation, subsequently causing unstable T cell-antigen-presenting cell (APC) conjugates. Microtubule organizing center polarization and granule reorientation were also impaired by LAT deficiency, leading to reduced granule delivery. Despite these defects, granule release was still observed in LAT-deficient CTLs due to residual calcium flux and phospholipase C (PLC) activity. Our data demonstrated that LAT-mediated signaling intricately regulates CTL cytotoxicity at multiple steps.

Authors
Ou-Yang, C-W; Zhu, M; Fuller, DM; Sullivan, SA; Chuck, MI; Ogden, S; Li, Q-J; Zhang, W
MLA Citation
Ou-Yang, C-W, Zhu, M, Fuller, DM, Sullivan, SA, Chuck, MI, Ogden, S, Li, Q-J, and Zhang, W. "Role of LAT in the granule-mediated cytotoxicity of CD8 T cells." Mol Cell Biol 32.14 (July 2012): 2674-2684.
PMID
22566687
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
32
Issue
14
Publish Date
2012
Start Page
2674
End Page
2684
DOI
10.1128/MCB.00356-12

Tyrosine phosphorylation-independent regulation of lipopolysaccharide-mediated response by the transmembrane adaptor protein LAB.

Linker for activation of B cells (LAB)/non-T cell activation linker is a transmembrane adaptor protein that functions in immunoreceptor-mediated signaling. Published studies have shown that LAB has both positive and negative roles in regulating TCR and high-affinity Fc receptor-mediated signaling and cellular function. In this study, we showed that LAB was also expressed in dendritic cells and that LAB deficiency affected LPS-mediated signaling and cytokine production. LPS-mediated MAPK activation was enhanced in LAB(-/-) bone marrow-derived dendritic cells. These bone marrow-derived dendritic cells also produced more TNF-α, IL-6, and IL-10 than wild-type cells. Moreover, LAB(-/-) mice were hyperresponsive to LPS-induced septic shock. These data indicated that LAB has a negative role in LPS-mediated responses. By using LAB knockin mice, which harbor mutations at five membrane-distal tyrosines, we further showed that, in contrast to its role in immunoreceptor-mediated signaling, LAB function in LPS-mediated signaling pathway did not depend on its tyrosine phosphorylation. Our study suggested a novel mechanism by which LAB functions in the regulation of innate immunity.

Authors
Zhu, M; Fuller, DM; Ou-Yang, C-W; Sullivan, SA; Zhang, W
MLA Citation
Zhu, M, Fuller, DM, Ou-Yang, C-W, Sullivan, SA, and Zhang, W. "Tyrosine phosphorylation-independent regulation of lipopolysaccharide-mediated response by the transmembrane adaptor protein LAB." J Immunol 188.6 (March 15, 2012): 2733-2741.
PMID
22308309
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
188
Issue
6
Publish Date
2012
Start Page
2733
End Page
2741
DOI
10.4049/jimmunol.1101581

The role of Ras guanine nucleotide releasing protein 4 in Fc epsilonRI-mediated signaling, mast cell function, and T cell development.

The RasGRP (Ras guanine nucleotide-releasing protein) family proteins are guanine nucleotide exchange factors that activate Ras GTPases, ultimately leading to MAPK activation and many cellular processes. The RasGRP family has four members. Published studies demonstrate that RasGRP1, RasGRP2, and RasGRP3 play critical roles in T cells, platelets, and B cells, respectively. RasGRP4 is highly expressed in mast cells. Although previous data suggest that it is important in mast cell development and function, the role of RasGRP4 in mast cells and allergic responses has not been clearly demonstrated. In this study, we generated RasGRP4(-/-) mice to examine the function of RasGRP4. Analyses of these mice showed that mast cells were able to develop normally in vivo and in vitro. Despite high levels of RasGRP4 expression in mast cells, RasGRP4 deficiency led to only a modest reduction in FcεRI-mediated degranulation and cytokine production. Interestingly, mast cells deficient in both RasGRP1 and RasGRP4 had a much more severe block in FcεRI-mediated signaling and mast cell function. We also made the unexpected finding that RasGRP4 functions during thymocyte development. Our data suggest that after the engagement of immunoreceptors, immune cells likely employ multiple members of the RasGRP family to transduce critical signals.

Authors
Zhu, M; Fuller, DM; Zhang, W
MLA Citation
Zhu, M, Fuller, DM, and Zhang, W. "The role of Ras guanine nucleotide releasing protein 4 in Fc epsilonRI-mediated signaling, mast cell function, and T cell development." J Biol Chem 287.11 (March 9, 2012): 8135-8143.
PMID
22262848
Source
pubmed
Published In
The Journal of biological chemistry
Volume
287
Issue
11
Publish Date
2012
Start Page
8135
End Page
8143
DOI
10.1074/jbc.M111.320580

Regulation of RasGRP1 function in T cell development and activation by its unique tail domain.

The Ras-guanyl nucleotide exchange factor RasGRP1 plays a critical role in T cell receptor-mediated Erk activation. Previous studies have emphasized the importance of RasGRP1 in the positive selection of thymocytes, activation of T cells, and control of autoimmunity. RasGRP1 consists of a number of well-characterized domains, which it shares with its other family members; however, RasGRP1 also contains an ~200 residue-long tail domain, the function of which is unknown. To elucidate the physiological role of this domain, we generated knock-in mice expressing RasGRP1 without the tail domain. Further analysis of these knock-in mice showed that thymocytes lacking the tail domain of RasGRP1 underwent aberrant thymic selection and, following TCR stimulation, were unable to activate Erk. Furthermore, the deletion of the tail domain led to enhanced CD4(+) T cell expansion in aged mice, as well as the production of autoantibodies. Mechanistically, the tail-deleted form of RasGRP1 was not able to traffic to the cell membrane following stimulation, indicating a potential reason for its inability to activate Erk. While the DAG-binding C1 domain of RasGRP1 has long been recognized as an important factor mediating Erk activation, we have revealed the physiological relevance of the tail domain in RasGRP1 function and control of Erk signaling.

Authors
Fuller, DM; Zhu, M; Song, X; Ou-Yang, C-W; Sullivan, SA; Stone, JC; Zhang, W
MLA Citation
Fuller, DM, Zhu, M, Song, X, Ou-Yang, C-W, Sullivan, SA, Stone, JC, and Zhang, W. "Regulation of RasGRP1 function in T cell development and activation by its unique tail domain." PLoS One 7.6 (2012): e38796-.
PMID
22719950
Source
pubmed
Published In
PloS one
Volume
7
Issue
6
Publish Date
2012
Start Page
e38796
DOI
10.1371/journal.pone.0038796

Regulation of Erk activation in T cells through the tail domain of RasGRP1

Authors
Fuller, D; Zhang, W
MLA Citation
Fuller, D, and Zhang, W. "Regulation of Erk activation in T cells through the tail domain of RasGRP1." April 2011.
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
186
Publish Date
2011

LAT-independent triggering of granule-mediated cytotoxicity of CD8 T cells

Authors
Ouyang, CC; Zhu, M; Li, Q-J; Zhang, W
MLA Citation
Ouyang, CC, Zhu, M, Li, Q-J, and Zhang, W. "LAT-independent triggering of granule-mediated cytotoxicity of CD8 T cells." April 2011.
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
186
Publish Date
2011

A tale of two TRAPs: LAT and LAB in the regulation of lymphocyte development, activation, and autoimmunity.

Transmembrane adaptor proteins (TRAPs) link antigen receptor engagement to downstream cellular processes. Although these proteins typically lack intrinsic enzymatic activity, they are phosphorylated on multiple tyrosine residues following lymphocyte activation, allowing them to function as scaffolds for the assembly of multi-molecular signaling complexes. Among the many TRAPs that have been discovered in recent years, the LAT (linker for activation of T cells) family of adaptor proteins plays an important role in the positive and negative regulation of lymphocyte maturation, activation, and differentiation. Of the two members in this family, LAT is an indispensable component controlling T cell and mast cell activation and function; LAB (linker for activation of B cells), also called NTAL, is necessary to fine-tune lymphocyte activation and may be a key regulator of innate immune responses. Here, we review recent advances on the function of LAT and LAB in the regulation of development and activation of immune cells.

Authors
Fuller, DM; Zhu, M; Ou-Yang, C-W; Sullivan, SA; Zhang, W
MLA Citation
Fuller, DM, Zhu, M, Ou-Yang, C-W, Sullivan, SA, and Zhang, W. "A tale of two TRAPs: LAT and LAB in the regulation of lymphocyte development, activation, and autoimmunity." Immunol Res 49.1-3 (April 2011): 97-108. (Review)
PMID
21136199
Source
pubmed
Published In
Immunologic Research
Volume
49
Issue
1-3
Publish Date
2011
Start Page
97
End Page
108
DOI
10.1007/s12026-010-8197-3

Basal LAT-diacylglycerol-rasGRP1 signals in t cells maintain TCRα gene expression

In contrast to the well-characterized T cell receptor (TCR) signaling pathways that induce genes that drive T cell development or polarization of naïve CD4 T cells into the diverse T H1, T H2, T H17 and T reg lineages, it is unclear what signals maintain specific gene expression in mature resting T cells. Resting T cells residing in peripheral lymphoid organs exhibit low-level constitutive signaling. Whereas tonic signals in B cells are known to be critical for survival, the roles of tonic signals in peripheral T cells are unknown. Here we demonstrate that constitutive signals in Jurkat T cell lines are transduced via the adapter molecule LAT and the Ras exchange factor RasGRP1 to maintain expression of TCRα mRNA and surface expression of the TCR/CD3 complex. Independent approaches of reducing basal activity through the LAT-diacylglycerol-RasGRP pathway led to reduced constitutive Ras-MEK-ERK signals and decreased TCRα mRNA and surface TCR expression in Jurkat cells. However, loss of TCR expression takes several days in these cell line experiments. In agreement with these in vitro approaches, inducible deletion of Lat in vivo results in reduced TCRα mRNA- and surface TCR- expression in a delayed temporal manner as well. Lastly, we demonstrate that loss of basal LAT-RasGRP signals appears to lead to silencing or repression of TCRα transcription. We postulate that basal LAT-diacylglycerol-RasGRP signals fulfill a regulatory function in peripheral T lymphocytes by maintaining proper gene expression programs. © 2011 Markegard et al.

Authors
Markegard, E; Trager, E; Yang, C-WO; Zhang, W; Weiss, A; Roose, JP
MLA Citation
Markegard, E, Trager, E, Yang, C-WO, Zhang, W, Weiss, A, and Roose, JP. "Basal LAT-diacylglycerol-rasGRP1 signals in t cells maintain TCRα gene expression." PLoS ONE 6.9 (2011).
PMID
21966541
Source
scival
Published In
PloS one
Volume
6
Issue
9
Publish Date
2011
DOI
10.1371/journal.pone.0025540

The importance of LAT in the activation, homeostasis, and regulatory function of T cells.

LAT (linker for activation of T cells) is a transmembrane adaptor protein that plays an essential role in TCR-mediated signaling and thymocyte development. Because LAT-deficient mice have an early block in thymocyte development, we utilized an inducible system to delete LAT in primary T cells to study LAT function in T cell activation, homeostasis, and survival. Deletion of LAT caused primary T cells to become unresponsive to stimulation from the TCR and impaired T cell homeostatic proliferation and long term survival. Furthermore, deletion of LAT led to reduced expression of Foxp3, CTLA-4, and CD25 in T(reg) cells and impaired their function. Consequently, mice with LAT deleted developed a lymphoproliferative syndrome similar to that in LATY136F mice, although less severe. Our data implicate that LAT has positive and negative roles in the regulation of mature T cells.

Authors
Shen, S; Chuck, MI; Zhu, M; Fuller, DM; Yang, C-WO; Zhang, W
MLA Citation
Shen, S, Chuck, MI, Zhu, M, Fuller, DM, Yang, C-WO, and Zhang, W. "The importance of LAT in the activation, homeostasis, and regulatory function of T cells." J Biol Chem 285.46 (November 12, 2010): 35393-35405.
PMID
20837489
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
46
Publish Date
2010
Start Page
35393
End Page
35405
DOI
10.1074/jbc.M110.145052

Independent and cooperative roles of adaptor molecules in proximal signaling during FcepsilonRI-mediated mast cell activation.

Activation through FcepsilonRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1(-/-) mast cells and that SLP-76(-/-) and LAT1(-/-) mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1(-/-) mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cgamma (PLCgamma). Comparison of SLP-76(-/-) LAT1(-/-) and SLP-76(-/-) mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcepsilonRI in mast cells.

Authors
Kambayashi, T; Okumura, M; Baker, RG; Hsu, C-J; Baumgart, T; Zhang, W; Koretzky, GA
MLA Citation
Kambayashi, T, Okumura, M, Baker, RG, Hsu, C-J, Baumgart, T, Zhang, W, and Koretzky, GA. "Independent and cooperative roles of adaptor molecules in proximal signaling during FcepsilonRI-mediated mast cell activation." Mol Cell Biol 30.17 (September 2010): 4188-4196.
PMID
20606011
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
30
Issue
17
Publish Date
2010
Start Page
4188
End Page
4196
DOI
10.1128/MCB.00305-10

LAB/NTAL regulates zymosan-mediated inflammatory responses in Bone Marrow Derived Dendritic Cells (BMDC)

Authors
Orr, S; Quigley, L; Roessler, S; Hodge, D; Razzook, C; Cardone, M; Zhang, W; Trinchieri, G; Lyakh, L; McVicar, D
MLA Citation
Orr, S, Quigley, L, Roessler, S, Hodge, D, Razzook, C, Cardone, M, Zhang, W, Trinchieri, G, Lyakh, L, and McVicar, D. "LAB/NTAL regulates zymosan-mediated inflammatory responses in Bone Marrow Derived Dendritic Cells (BMDC)." April 1, 2010.
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
184
Publish Date
2010

The role of the LAT-PLC-gamma1 interaction in T regulatory cell function.

The interaction between the linker for activation of T cells (LAT) with PLC-gamma1 is important for TCR-mediated Ca(2+) signaling and MAPK activation. Knock-in mice harboring a mutation at the PLC-gamma1 binding site (Y136) of LAT develop a severe lymphoproliferative syndrome. These mice have defective thymic development and selection and lack natural regulatory T cells, implicating a breakdown of both central and peripheral tolerance. To bypass this developmental defect, we developed a conditional knock-in line in which only LATY136F is expressed in mature T cells after deletion of the wild type LAT allele. Analysis of LATY136F T cells indicated that the interaction between LAT and PLC-gamma1 plays an important role in TCR-mediated signaling, proliferation, and IL-2 production. Furthermore, the deletion of LAT induced development of the lymphoproliferative syndrome in these mice. Although Foxp3(+) natural Treg cells were present in these mice after deletion, they were unable to suppress the proliferation of conventional T cells. Our data indicate that the binding of LAT to PLC-gamma1 is essential for the suppressive function of CD4(+)CD25(+) regulatory T cells.

Authors
Chuck, MI; Zhu, M; Shen, S; Zhang, W
MLA Citation
Chuck, MI, Zhu, M, Shen, S, and Zhang, W. "The role of the LAT-PLC-gamma1 interaction in T regulatory cell function." J Immunol 184.5 (March 1, 2010): 2476-2486.
PMID
20130215
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
184
Issue
5
Publish Date
2010
Start Page
2476
End Page
2486
DOI
10.4049/jimmunol.0902876

The linker for activation of B cells (LAB)/non-T cell activation linker (NTAL) regulates triggering receptor expressed on myeloid cells (TREM)-2 signaling and macrophage inflammatory responses independently of the linker for activation of T cells.

Triggering receptor expressed on myeloid cells-2 (TREM-2) is rapidly emerging as a key regulator of the innate immune response via its regulation of macrophage inflammatory responses. Here we demonstrate that proximal TREM-2 signaling parallels other DAP12-based receptor systems in its use of Syk and Src-family kinases. However, we find that the linker for activation of T cells (LAT) is severely reduced as monocytes differentiate into macrophages and that TREM-2 exclusively uses the linker for activation of B cells (LAB encoded by the gene Lat2(-/-)) to mediate downstream signaling. LAB is required for TREM-2-mediated activation of Erk1/2 and dampens proximal TREM-2 signals through a novel LAT-independent mechanism resulting in macrophages with proinflammatory properties. Thus, Lat2(-/-) macrophages have increased TREM-2-induced proximal phosphorylation, and lipopolysaccharide stimulation of these cells leads to increased interleukin-10 (IL-10) and decreased IL-12p40 production relative to wild type cells. Together these data identify LAB as a critical, LAT-independent regulator of TREM-2 signaling and macrophage development capable of controlling subsequent inflammatory responses.

Authors
Whittaker, GC; Orr, SJ; Quigley, L; Hughes, L; Francischetti, IMB; Zhang, W; McVicar, DW
MLA Citation
Whittaker, GC, Orr, SJ, Quigley, L, Hughes, L, Francischetti, IMB, Zhang, W, and McVicar, DW. "The linker for activation of B cells (LAB)/non-T cell activation linker (NTAL) regulates triggering receptor expressed on myeloid cells (TREM)-2 signaling and macrophage inflammatory responses independently of the linker for activation of T cells." J Biol Chem 285.5 (January 29, 2010): 2976-2985.
PMID
19948717
Source
pubmed
Published In
The Journal of biological chemistry
Volume
285
Issue
5
Publish Date
2010
Start Page
2976
End Page
2985
DOI
10.1074/jbc.M109.038398

Vav and Rac activation in B cell antigen receptor endocytosis involves Vav recruitment to the adapter protein LAB.

The signal transduction events supporting B cell antigen receptor (BCR) endocytosis are not well understood. We have identified a pathway supporting BCR internalization that begins with tyrosine phosphorylation of the adapter protein LAB. Phosphorylated LAB recruits a complex of Grb2-dynamin and the guanine nucleotide exchange factor Vav. Vav is required for activation of the small GTPases Rac1 and Rac2. All these proteins contribute to (and dynamin, Vav, and Rac1/2 are required for) BCR endocytosis and presentation of antigen to T cells. This is the first description of a sequential signal transduction pathway from BCR to internalization and antigen presentation.

Authors
Malhotra, S; Kovats, S; Zhang, W; Coggeshall, KM
MLA Citation
Malhotra, S, Kovats, S, Zhang, W, and Coggeshall, KM. "Vav and Rac activation in B cell antigen receptor endocytosis involves Vav recruitment to the adapter protein LAB." J Biol Chem 284.52 (December 25, 2009): 36202-36212.
PMID
19858206
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
52
Publish Date
2009
Start Page
36202
End Page
36212
DOI
10.1074/jbc.M109.040089

Regulation of lymphocyte development and activation by the LAT family of adapter proteins.

Transmembrane adapter proteins (TRAPs) are critical components of signaling pathways in lymphocytes, linking antigen receptor engagement to downstream cellular processes. While these proteins lack intrinsic enzymatic activity, their phosphorylation following receptor ligation allows them to function as scaffolds for the assembly of multi-molecular signaling complexes. Many TRAPs have recently been discovered, and numerous studies demonstrate their roles in the positive and negative regulation of lymphocyte maturation, activation, and differentiation. One such example is the linker for activation of T cells (LAT) family of adapter proteins. While LAT has been shown to play an indispensable role in T-cell and mast cell function, the other family members, linker for activation of B cells (LAB) and linker for activation of X cells (LAX), are necessary to fine-tune immune responses. In addition to its well-established role in the positive regulation of lymphocyte activation, LAT exerts an inhibitory effect on T-cell receptor-mediated signaling. Furthermore, LAT, along with LAB and LAX, plays a crucial role in establishing and maintaining tolerance. Here, we review recent data concerning the regulation of lymphocyte development and activation by the LAT family of proteins.

Authors
Fuller, DM; Zhang, W
MLA Citation
Fuller, DM, and Zhang, W. "Regulation of lymphocyte development and activation by the LAT family of adapter proteins." Immunol Rev 232.1 (November 2009): 72-83. (Review)
PMID
19909357
Source
pubmed
Published In
Immunological Reviews
Volume
232
Issue
1
Publish Date
2009
Start Page
72
End Page
83
DOI
10.1111/j.1600-065X.2009.00828.x

B cell antigen receptor endocytosis and antigen presentation to T cells require Vav and dynamin.

Antigen binding to the B cell antigen receptor (BCR) initiates an array of signaling events. These include endocytosis of ligand-receptor complexes via clathrin-coated pits, trafficking of the internalized ligand to lysosomes, degradation of the associated proteins to peptides, and peptide presentation on nascent major histocompatibility complex class II to T cells. The signal transduction events supporting BCR internalization are not well understood. We have identified a pathway supporting BCR internalization that includes the Vav1 and/or Vav3 isoforms and the GTPase dynamin. Vav1 and -3 are not required for B cell development and maturation, nor for a variety of BCR-induced signaling events nor for BCR signaling leading to major histocompatibility complex class II and CD80 expression, but Vav1 and/or -3 are absolutely required for BCR endocytosis and BCR-induced Rac-GTP loading. This is the first demonstration of a link between Vav and Rac in BCR internalization leading to antigen presentation to T cells.

Authors
Malhotra, S; Kovats, S; Zhang, W; Coggeshall, KM
MLA Citation
Malhotra, S, Kovats, S, Zhang, W, and Coggeshall, KM. "B cell antigen receptor endocytosis and antigen presentation to T cells require Vav and dynamin." J Biol Chem 284.36 (September 4, 2009): 24088-24097.
PMID
19586920
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
36
Publish Date
2009
Start Page
24088
End Page
24097
DOI
10.1074/jbc.M109.014209

Palmitoylation-dependent plasma membrane transport but lipid raft-independent signaling by linker for activation of T cells.

Linker for activation of T cells (LAT) is a dually palmitoylated transmembrane adaptor protein essential for T cell development and activation. However, whether LAT palmitoylation and/or lipid raft localization are required for its function is controversial. To address this question, we used a combination of biochemical, imaging, and genetic approaches, including LAT retrovirus-transduced mouse T cells and bone marrow chimeric mice. A nonpalmitoylated, non-lipid raft-residing mutant of transmembrane LAT could not reconstitute T cell development in bone marrow chimeric mice. This mutant was absent from the plasma membrane (PM) and was restricted mainly to the Golgi apparatus. A chimeric, nonpalmitoylated LAT protein consisting of the PM-targeting N-terminal sequence of Src kinase and the LAT cytoplasmic domain (Src-LAT) localized as a peripheral membrane protein in the PM, but outside lipid rafts. Nevertheless, Src-LAT restored T cell development and activation. Lastly, monopalmitoylation of LAT on Cys(26) (but not Cys(29)) was required and sufficient for its PM transport and function. Thus, the function of LAT in T cells requires its PM, but not raft, localization, even when expressed as a peripheral membrane protein. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its PM transport.

Authors
Hundt, M; Harada, Y; De Giorgio, L; Tanimura, N; Zhang, W; Altman, A
MLA Citation
Hundt, M, Harada, Y, De Giorgio, L, Tanimura, N, Zhang, W, and Altman, A. "Palmitoylation-dependent plasma membrane transport but lipid raft-independent signaling by linker for activation of T cells." J Immunol 183.3 (August 1, 2009): 1685-1694.
PMID
19592663
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
183
Issue
3
Publish Date
2009
Start Page
1685
End Page
1694
DOI
10.4049/jimmunol.0803921

The importance of Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons sterile-alpha motif domain in thymic selection and T-cell activation.

The Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76) is a cytosolic adaptor protein essential for thymocyte development and T-cell activation. It contains a sterile-alpha motif (SAM) domain, 3 phosphotyrosine motifs, a proline-rich region, and a Src homology 2 domain. Whereas the other domains have been extensively studied, the role of the SAM domain in SLP-76 function is not known. To understand the function of this domain, we generated SLP-76 knockin mice with the SAM domain deleted. Analysis of these mice showed that thymocyte development was partially blocked at the double-positive to single-positive transition. Positive and negative thymic selection was also impaired. In addition, we analyzed T-cell receptor (TCR)-mediated signaling in T cells from these mutant mice. TCR-mediated inositol 1,4,5-triphosphate production, calcium flux, and extracellular signal-regulated kinase activation were decreased, leading to defective interleukin-2 production and proliferation. Moreover, despite normal association between Gads and SLP-76, TCR-mediated formation of SLP-76 microclusters was impaired by the deletion of the SAM domain. Altogether, our data demonstrated that the SAM domain is indispensable for optimal SLP-76 signaling.

Authors
Shen, S; Lau, J; Zhu, M; Zou, J; Fuller, D; Li, Q-J; Zhang, W
MLA Citation
Shen, S, Lau, J, Zhu, M, Zou, J, Fuller, D, Li, Q-J, and Zhang, W. "The importance of Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons sterile-alpha motif domain in thymic selection and T-cell activation." Blood 114.1 (July 2, 2009): 74-84.
PMID
19401562
Source
pubmed
Published In
Blood
Volume
114
Issue
1
Publish Date
2009
Start Page
74
End Page
84
DOI
10.1182/blood-2008-09-177832

The essential role of LAT in thymocyte development during transition from the double-positive to single-positive stage.

The linker for activation of T cells (LAT) is an adaptor protein that couples TCR engagement to downstream signaling cascades. LAT is important in early thymocyte development as LAT-deficient mice have a complete block at the double-negative (DN) 3 stage. To study the role of LAT beyond the DN3 stage, we generated mice in which the lat gene could be deleted by the Cre recombinase. Analysis of these mice showed that deletion of LAT after the DN3 stage allowed thymocytes to develop past the DN3 to DN4 checkpoint and to generate double-positive thymocytes. However, LAT-deficient DP thymocytes were severely defective in responding to stimulation via the TCR and failed to differentiate into single-positive thymocytes efficiently. Consequently, few LAT-deficient mature T cells could be found in the periphery. These T cells had undergone extensive homeostatic proliferation and expressed low levels of the TCR on their surface. Collectively, our data indicate that in addition to its role in pre-TCR signaling, LAT also plays an essential role in thymocyte development during transition from the double-positive to single-positive stage.

Authors
Shen, S; Zhu, M; Lau, J; Chuck, M; Zhang, W
MLA Citation
Shen, S, Zhu, M, Lau, J, Chuck, M, and Zhang, W. "The essential role of LAT in thymocyte development during transition from the double-positive to single-positive stage." J Immunol 182.9 (May 1, 2009): 5596-5604.
PMID
19380807
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
182
Issue
9
Publish Date
2009
Start Page
5596
End Page
5604
DOI
10.4049/jimmunol.0803170

Negative regulation of TCR signaling by linker for activation of X cells via phosphotyrosine-dependent and -independent mechanisms.

The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.

Authors
Shapiro, MJ; Nguyen, CT; Aghajanian, H; Zhang, W; Shapiro, VS
MLA Citation
Shapiro, MJ, Nguyen, CT, Aghajanian, H, Zhang, W, and Shapiro, VS. "Negative regulation of TCR signaling by linker for activation of X cells via phosphotyrosine-dependent and -independent mechanisms." J Immunol 181.10 (November 15, 2008): 7055-7061.
PMID
18981125
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
181
Issue
10
Publish Date
2008
Start Page
7055
End Page
7061

Analysis of the linker for activation of T cells and the linker for activation of B cells in natural killer cells reveals a novel signaling cassette, dual usage in ITAM signaling, and influence on development of the Ly49 repertoire.

The linker for activation of T cells (LAT) and the linker for activation of B cells (LAB/NTAL/LAT2) are integral proteins in receptor coupling to downstream events. Both proteins are expressed in natural killer (NK) cells and LAT is phosphorylated during target cell interactions or ligation of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled CD16. Regardless, Lat(-/-) mice exhibit normal natural and antibody-mediated killing. Here we place both LAT and LAB in the DAP12 pathway of NK cells. Moreover, we unveil a LAT-independent pathway that requires expression of Syk. Mice lacking either LAT or LAB have a skewed Ly49 repertoire, and activated NK cells from Lat(-/-) mice have reduced responses to the ITAM-coupled receptor NK1.1. In contrast, resting Lat(-/-) NK cells show intact NK1.1 responses, whereas NK cells without LAB are hyperactive. Elimination of both adaptors severely reduces NK1.1 signaling under both conditions. Together these data show that NK ITAMs preferentially use a signaling cassette regulated by interplay between LAT and LAB. Activation by interleukin-2 causes a shift to greater dependency on LAT due to suppression of Syk signaling. The overlapping use of multiple adaptors permits fine-tuning of NK-cell ITAM responses over the course of an immune response.

Authors
Whittaker, GC; Burshtyn, DN; Orr, SJ; Quigley, L; Hodge, DL; Pascal, V; Zhang, W; McVicar, DW
MLA Citation
Whittaker, GC, Burshtyn, DN, Orr, SJ, Quigley, L, Hodge, DL, Pascal, V, Zhang, W, and McVicar, DW. "Analysis of the linker for activation of T cells and the linker for activation of B cells in natural killer cells reveals a novel signaling cassette, dual usage in ITAM signaling, and influence on development of the Ly49 repertoire." Blood 112.7 (October 1, 2008): 2869-2877.
PMID
18645037
Source
pubmed
Published In
Blood
Volume
112
Issue
7
Publish Date
2008
Start Page
2869
End Page
2877
DOI
10.1182/blood-2007-11-121590

Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells.

Transmembrane adaptor proteins couple antigen receptor engagement to downstream signaling cascades in lymphocytes. One example of these proteins is the linker for activation of T cells (LAT), which plays an indispensable role in T cell activation and development. Here, we report identification of a new transmembrane adaptor molecule, namely growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT), which is expressed in B cells and myeloid cells. Similar to LAT, GAPT has an extracellular domain, a transmembrane domain, and a cytoplasmic tail with multiple Grb2-binding motifs. In contrast to other transmembrane adaptor proteins, GAPT is not phosphorylated upon BCR ligation but associates with Grb2 constitutively through its proline-rich region. Targeted disruption of the gapt gene in mice affects neither B cell development nor a nitrophenylacetyl-specific antibody response. However, in the absence of GAPT, B cell proliferation after BCR cross-linking is enhanced. In aged GAPT(-/-) mice, the number of marginal zone (MZ) B cells is increased, and other B cell subsets are normal. The serum concentrations of IgM, IgG2b, and IgG3 are also elevated in these mice. These data indicate that GAPT might play an important role in control of B cell activation and proper maintenance of MZ B cells.

Authors
Liu, Y; Zhang, W
MLA Citation
Liu, Y, and Zhang, W. "Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells." J Leukoc Biol 84.3 (September 2008): 842-851.
PMID
18559951
Source
pubmed
Published In
Journal of leukocyte biology
Volume
84
Issue
3
Publish Date
2008
Start Page
842
End Page
851
DOI
10.1189/jlb.0208087

Palmitoylation-dependent plasma membrane transport, but lipid raft-independent signaling function, of linker for activation of T cells (LAT)

Authors
Hundt, M; Harada, Y; Hayashi, K; De Giorgio, L; Tanimura, N; Zhang, W; Altman, A
MLA Citation
Hundt, M, Harada, Y, Hayashi, K, De Giorgio, L, Tanimura, N, Zhang, W, and Altman, A. "Palmitoylation-dependent plasma membrane transport, but lipid raft-independent signaling function, of linker for activation of T cells (LAT)." FASEB JOURNAL 22 (April 2008).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
22
Publish Date
2008

LAT Plays Important Role in Thymocyte Survival and Mature T Cell Activation

Authors
Shen, S; Zhu, M; Zhang, W
MLA Citation
Shen, S, Zhu, M, and Zhang, W. "LAT Plays Important Role in Thymocyte Survival and Mature T Cell Activation." April 1, 2007.
Source
wos-lite
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
178
Publish Date
2007

Mouse TCRalphabeta+CD8alphaalpha intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses.

Mouse small intestine intraepithelial lymphocytes (IEL) that express alphabetaTCR and CD8alphaalpha homodimers are an enigmatic T cell subset, as their specificity and in vivo function remain to be defined. To gain insight into the nature of these cells, we performed global gene expression profiling using microarray analysis combined with real-time quantitative PCR and flow cytometry. Using these methods, TCRalphabeta(+)CD8alphaalpha IEL were compared with their TCRalphabeta(+)CD8beta(+) and TCRgammadelta(+) counterparts. Interestingly, TCRalphabeta(+)CD8alphaalpha IEL were found to preferentially express genes that would be expected to down-modulate their reactivity. They have a unique expression pattern of members of the Ly49 family of NK receptors and tend to express inhibitory receptors, along with some activating receptors. The signaling machinery of both TCRalphabeta(+)CD8alphaalpha and TCRgammadelta(+) IEL is constructed differently than other IEL and peripheral T cells, as evidenced by their low-level expression of the linker for activation of T cells and high expression of the non-T cell activation linker, which suppresses T cell activation. The TCRalphabeta(+)CD8alphaalpha IEL subset also has increased expression of genes that could be involved in immune regulation, including TGF-beta(3) and lymphocyte activation gene-3. Collectively, these data underscore the fact that, while TCRalphabeta(+)CD8alphaalpha IEL resemble TCRgammadelta(+) IEL, they are a unique population of cells with regulated Ag reactivity that could have regulatory function.

Authors
Denning, TL; Granger, SW; Mucida, D; Graddy, R; Leclercq, G; Zhang, W; Honey, K; Rasmussen, JP; Cheroutre, H; Rudensky, AY; Kronenberg, M
MLA Citation
Denning, TL, Granger, SW, Mucida, D, Graddy, R, Leclercq, G, Zhang, W, Honey, K, Rasmussen, JP, Cheroutre, H, Rudensky, AY, and Kronenberg, M. "Mouse TCRalphabeta+CD8alphaalpha intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses." J Immunol 178.7 (April 1, 2007): 4230-4239.
PMID
17371979
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
178
Issue
7
Publish Date
2007
Start Page
4230
End Page
4239

An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response.

Cross-linking of the FcepsilonRI activates the phosphatidyl inositol 3 kinase (PI3K) and mitogen-activated protein kinase pathways. Previous studies demonstrate that Ras guanyl nucleotide-releasing protein (RasGRP)1 is essential in T cell receptor-mediated Ras-Erk activation. Here, we report that RasGRP1 plays an important role in FcepsilonRI-mediated PI3K activation and mast cell function. RasGRP1-deficient mice failed to mount anaphylactic allergic reactions. RasGRP1-/- mast cells had markedly reduced degranulation and cytokine production. Although FcepsilonRI-mediated Erk activation was normal, PI3K activation was diminished. Consequently, activation of Akt, PIP3-dependent kinase, and protein kinase C delta was defective. Expression of a constitutively active form of N-Ras could rescue the degranulation defect and Akt activation. We further demonstrated that RasGRP1-/- mast cells were defective in granule translocation, microtubule formation, and RhoA activation. Our results identified RasGRP1 as an essential regulator of mast cell function.

Authors
Liu, Y; Zhu, M; Nishida, K; Hirano, T; Zhang, W
MLA Citation
Liu, Y, Zhu, M, Nishida, K, Hirano, T, and Zhang, W. "An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response." J Exp Med 204.1 (January 22, 2007): 93-103.
PMID
17190838
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
204
Issue
1
Publish Date
2007
Start Page
93
End Page
103
DOI
10.1084/jem.20061598

Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor.

Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation. Both adaptors were distributed predominantly at the plasma membrane in resting cells and co-clustered with other raft-associated proteins; however, they distributed differently in buoyant membranes isolated by either detergent resistance or non-detergent methods, indicating that they might localize to distinct rafts. After activation, LAB/NTAL was internalized and co-localized with the BCR while PAG/Cbp remained on the cell surface. BCR internalization was reduced in LAB/NTAL-deficient murine B cells, suggesting a regulatory role for LAB/NTAL in activation-induced internalization of the BCR. The cytoplasmic domain of LAB/NTAL, and not the transmembrane/juxtamembrane region, was found to be essential for its internalization.

Authors
Mutch, CM; Sanyal, R; Unruh, TL; Grigoriou, L; Zhu, M; Zhang, W; Deans, JP
MLA Citation
Mutch, CM, Sanyal, R, Unruh, TL, Grigoriou, L, Zhu, M, Zhang, W, and Deans, JP. "Activation-induced endocytosis of the raft-associated transmembrane adaptor protein LAB/NTAL in B lymphocytes: evidence for a role in internalization of the B cell receptor." Int Immunol 19.1 (January 2007): 19-30.
PMID
17090619
Source
pubmed
Published In
International Immunology
Volume
19
Issue
1
Publish Date
2007
Start Page
19
End Page
30
DOI
10.1093/intimm/dxl118

Erratum: Mouse TCRαβ+CD8αα intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses (Journal of Immunology (2007) 178, (4230-4239))

Authors
Denning, TL; Granger, S; Mucida, D; Graddy, R; Leclercq, G; Zhang, W; Honey, K; Rasmussen, JP; Cheroutre, H; Rudensky, AY; Kronenberg, M
MLA Citation
Denning, TL, Granger, S, Mucida, D, Graddy, R, Leclercq, G, Zhang, W, Honey, K, Rasmussen, JP, Cheroutre, H, Rudensky, AY, and Kronenberg, M. "Erratum: Mouse TCRαβ+CD8αα intraepithelial lymphocytes express genes that down-regulate their antigen reactivity and suppress immune responses (Journal of Immunology (2007) 178, (4230-4239))." Journal of Immunology 178.10 (2007): 6654--.
Source
scival
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
178
Issue
10
Publish Date
2007
Start Page
6654-

Negative regulation of T cell activation and autoimmunity by the transmembrane adaptor protein LAB.

LAB (linker for activation of B cells), also known as NTAL (non-T cell activation linker), is a LAT (linker for activation of T cells)-like adaptor protein that is expressed in B, NK, and mast cells. Its role in lymphocytes has not been clearly demonstrated. Here, we showed that aged LAB-deficient (Lat2(-/-)) mice developed an autoimmune syndrome. Lat2(-/-) T cells were hyperactivated and produced more cytokines than Lat2(+/+) T cells. Even though LAB was absent in naive T cells, LAB could be detected in activated Lat2(+/+) T cells. LAT-mediated signaling events were enhanced in Lat2(-/-) T cells; however, they were suppressed in T cells that overexpressed LAB. Mice with the Lat2 gene conditionally deleted from T cells also developed the autoimmune syndrome like Lat2(-/-) mice. Together, these data demonstrated an important role of LAB in limiting autoimmune response and exposed a mechanism regulating T cell activation.

Authors
Zhu, M; Koonpaew, S; Liu, Y; Shen, S; Denning, T; Dzhagalov, I; Rhee, I; Zhang, W
MLA Citation
Zhu, M, Koonpaew, S, Liu, Y, Shen, S, Denning, T, Dzhagalov, I, Rhee, I, and Zhang, W. "Negative regulation of T cell activation and autoimmunity by the transmembrane adaptor protein LAB." Immunity 25.5 (November 2006): 757-768.
PMID
17081783
Source
pubmed
Published In
Immunity
Volume
25
Issue
5
Publish Date
2006
Start Page
757
End Page
768
DOI
10.1016/j.immuni.2006.08.025

Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX.

LAX is a transmembrane adaptor protein that is expressed in both T and B cells. Upon stimulation via the antigen receptors, it is tyrosine-phosphorylated and binds Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. Disruption of the Lax gene causes hyperresponsiveness in T and B lymphocytes. Here, we showed that LAX was also expressed in mast cells. Upon engagement of the Fc epsilonRI, LAX was also phosphorylated and interacted with Grb2 and p85. LAX-deficient mast cells were hyperresponsive to stimulation via the Fc epsilonRI, as evidenced by enhanced degranulation, p38 MAPK, Akt, and phosphatidylinositol 3-kinase activation. This hyperresponsiveness was likely a consequence of reduced LAB expression after sensitization of mast cells with anti-dinitrophenyl IgE. In addition, Fc epsilonRI-mediated cytokine production and cell survival were also enhanced. These data suggested that LAX negatively regulates mast cell function.

Authors
Zhu, M; Rhee, I; Liu, Y; Zhang, W
MLA Citation
Zhu, M, Rhee, I, Liu, Y, and Zhang, W. "Negative regulation of Fc epsilonRI-mediated signaling and mast cell function by the adaptor protein LAX." J Biol Chem 281.27 (July 7, 2006): 18408-18413.
PMID
16672218
Source
pubmed
Published In
The Journal of biological chemistry
Volume
281
Issue
27
Publish Date
2006
Start Page
18408
End Page
18413
DOI
10.1074/jbc.M601535200

LAT-mediated signaling in CD4+CD25+ regulatory T cell development.

Engagement of the T cell receptor for antigen (TCR) induces formation of signaling complexes mediated through the transmembrane adaptor protein, the linker for activation of T cells (LAT). LAT plays an important role in T cell development, activation, and homeostasis. A knock-in mutation at Tyr136, which is the phospholipase C (PLC)-gamma1-binding site in LAT, leads to a severe autoimmune disease in mice. In this study, we show that CD4+CD25+ T reg cells that expressed Foxp3 transcription factor were nearly absent in both thymus and peripheral lymphoid organs of LAT(Y136F) mice. This defect was not a result of the autoimmune environment as LAT(Y136F) T reg cells also failed to develop in healthy LAT-/- mice that received mixed wild-type and LAT(Y136F) bone marrow cells. Moreover, adoptive transfer of normal CD4+CD25+ T reg cells protected neonatal LAT(Y136F) mice from developing this disease. These T reg cells effectively controlled expansion of CD4+ T cells in LAT(Y136F) mice likely via granzymes and/or TGF-beta-mediated suppression. Furthermore, ectopic expression of Foxp3 conferred a suppressive function in LAT(Y136F) T cells. Our data indicate that the LAT-PLC-gamma1 interaction plays a critical role in Foxp3 expression and the development of CD4+CD25+ T reg cells.

Authors
Koonpaew, S; Shen, S; Flowers, L; Zhang, W
MLA Citation
Koonpaew, S, Shen, S, Flowers, L, and Zhang, W. "LAT-mediated signaling in CD4+CD25+ regulatory T cell development." J Exp Med 203.1 (January 23, 2006): 119-129.
PMID
16380508
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
203
Issue
1
Publish Date
2006
Start Page
119
End Page
129
DOI
10.1084/jem.20050903

Negative regulation of lymphocyte activation by the adaptor protein LAX.

The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

Authors
Zhu, M; Granillo, O; Wen, R; Yang, K; Dai, X; Wang, D; Zhang, W
MLA Citation
Zhu, M, Granillo, O, Wen, R, Yang, K, Dai, X, Wang, D, and Zhang, W. "Negative regulation of lymphocyte activation by the adaptor protein LAX." J Immunol 174.9 (May 1, 2005): 5612-5619.
PMID
15843560
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
174
Issue
9
Publish Date
2005
Start Page
5612
End Page
5619

Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development.

It has been proposed that upon T cell activation, linker for activation of T cells (LAT), a transmembrane adaptor protein localized to lipid rafts, orchestrates formation of multiprotein complexes and activates signaling cascades in lipid rafts. However, whether lipid rafts really exist or function remains controversial. To address the importance of lipid rafts in LAT function, we generated a fusion protein to target LAT to nonraft fractions using the transmembrane domain from a nonraft protein, linker for activation of X cells (LAX). Surprisingly, this fusion protein functioned well in TCR signaling. It restored MAPK activation, calcium flux, and NFAT activation in LAT-deficient cells. To further study the function of this fusion protein in vivo, we generated transgenic mice that express this protein. Analysis of these mice indicated that it was fully capable of replacing LAT in thymocyte development and T cell function. Our results demonstrate that LAT localization to lipid rafts is not essential during normal T cell activation and development.

Authors
Zhu, M; Shen, S; Liu, Y; Granillo, O; Zhang, W
MLA Citation
Zhu, M, Shen, S, Liu, Y, Granillo, O, and Zhang, W. "Cutting Edge: Localization of linker for activation of T cells to lipid rafts is not essential in T cell activation and development." J Immunol 174.1 (January 1, 2005): 31-35.
PMID
15611224
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
174
Issue
1
Publish Date
2005
Start Page
31
End Page
35

Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.

Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.

Authors
Zhu, M; Liu, Y; Koonpaew, S; Granillo, O; Zhang, W
MLA Citation
Zhu, M, Liu, Y, Koonpaew, S, Granillo, O, and Zhang, W. "Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL." J Exp Med 200.8 (October 18, 2004): 991-1000.
PMID
15477350
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
200
Issue
8
Publish Date
2004
Start Page
991
End Page
1000
DOI
10.1084/jem.20041223

An important role of phospholipase Cgamma1 in pre-B-cell development and allelic exclusion.

Phospholipase Cgamma1 (PLCgamma1) has been reported to be expressed predominantly in T cells and to play an important role in T-cell receptor signaling. Here we show that PLCgamma1 is expressed throughout B-cell development, with high expression in B-cell progenitors, and is involved in pre-B-cell receptor (pre-BCR) signaling. Reduced expression of PLCgamma1, in the absence of PLCgamma2 (PLCgamma1+/-PLCgamma2-/-), impedes early B-cell development at the pro-B- to pre-B-cell transition and impairs immunoglobulin heavy chain allelic exclusion, hallmarks of defective pre-BCR signaling. In contrast, early B-cell development is largely normal, whereas late B-cell maturation is impaired in the absence of PLCgamma2 alone (PLCgamma2-/-) and overexpression of PLCgamma1 in PLCgamma2-/- mice fails to restore BCR-mediated B-cell proliferation and maturation. These studies reveal an essential role of PLCgamma1, distinct from that of PLCgamma2, in B-cell development.

Authors
Wen, R; Chen, Y; Schuman, J; Fu, G; Yang, S; Zhang, W; Newman, DK; Wang, D
MLA Citation
Wen, R, Chen, Y, Schuman, J, Fu, G, Yang, S, Zhang, W, Newman, DK, and Wang, D. "An important role of phospholipase Cgamma1 in pre-B-cell development and allelic exclusion." EMBO J 23.20 (October 13, 2004): 4007-4017.
PMID
15372077
Source
pubmed
Published In
EMBO Journal
Volume
23
Issue
20
Publish Date
2004
Start Page
4007
End Page
4017
DOI
10.1038/sj.emboj.7600405

Transmembrane adaptor proteins: organizers of immunoreceptor signalling.

Authors
Horejsí, V; Zhang, W; Schraven, B
MLA Citation
Horejsí, V, Zhang, W, and Schraven, B. "Transmembrane adaptor proteins: organizers of immunoreceptor signalling." Nat Rev Immunol 4.8 (August 2004): 603-616. (Review)
PMID
15286727
Source
pubmed
Published In
Nature Reviews Immunology
Volume
4
Issue
8
Publish Date
2004
Start Page
603
End Page
616
DOI
10.1038/nri1414

Requirement for Abl kinases in T cell receptor signaling.

BACKGROUND: The c-Abl and Arg proteins comprise a unique family of nonreceptor tyrosine kinases that have been implicated in the regulation of cell proliferation and survival, cytoskeletal reorganization, cell migration, and the response to oxidative stress and DNA damage. Targeted deletion or mutation of c-Abl in mice results in a variety of immune system phenotypes, including splenic and thymic atrophy, lymphopenia, and an increased susceptibility to infection. However, despite the generation of these mice over a decade ago, little is known regarding the mechanisms responsible for these phenotypes or the immune-related consequences of ablation of both the c-Abl and Arg kinases, which are coexpressed in lymphoid tissues. RESULTS: Here, we report that T cell receptor (TCR) stimulation results in activation of the endogenous Abl kinases. We demonstrate that Zap70 and the transmembrane adaptor linker for activation of T cells (LAT) are targets of the Abl kinases, and that loss of Abl kinase activity reduces TCR-induced Zap70 phosphorylation at tyrosine 319. This correlates with diminished LAT tyrosine phosphorylation, as well as reduced tyrosine phosphorylation and recruitment of phospholipase Cgamma1 to LAT. Significantly, we show that Abl kinase activity is required for maximal signaling leading to transcription of the IL-2 promoter, as well as TCR-induced IL-2 production and proliferation of primary T cells. CONCLUSIONS: We conclude that the Abl kinases have a role in the regulation of TCR-mediated signal transduction leading to IL-2 production and cell proliferation.

Authors
Zipfel, PA; Zhang, W; Quiroz, M; Pendergast, AM
MLA Citation
Zipfel, PA, Zhang, W, Quiroz, M, and Pendergast, AM. "Requirement for Abl kinases in T cell receptor signaling." Curr Biol 14.14 (July 27, 2004): 1222-1231.
PMID
15268851
Source
pubmed
Published In
Current Biology
Volume
14
Issue
14
Publish Date
2004
Start Page
1222
End Page
1231
DOI
10.1016/j.cub.2004.07.021

Linker for activation of B cells: a functional equivalent of a mutant linker for activation of T cells deficient in phospholipase C-gamma1 binding.

Adaptor proteins have important functions in coupling stimulation through immunoreceptors with downstream events. The adaptor linker for activation of B cells (LAB)/non-T cell activation linker (NTAL) is expressed in various immune cell types and has a similar domain structure as linker for activation of T cells (LAT). In this study we generated a LAB transgenic mouse to compare the functional differences between LAB and LAT. A LAB transgene expressed in LAT-deficient T cells was able to restore T cell development. However, these mice developed severe organomegaly with disorganized lymphoid tissues. Lymphocytes from these transgenic mice were hyperactivated, and T cells produced large amounts of type II cytokines. In addition, these activities appeared to be uncoupled from the TCR. An examination of the signaling capabilities of these T cells revealed that LAB resembled a LAT molecule unable to bind phospholipase C-gamma1.

Authors
Janssen, E; Zhu, M; Craven, B; Zhang, W
MLA Citation
Janssen, E, Zhu, M, Craven, B, and Zhang, W. "Linker for activation of B cells: a functional equivalent of a mutant linker for activation of T cells deficient in phospholipase C-gamma1 binding." J Immunol 172.11 (June 1, 2004): 6810-6819.
PMID
15153499
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
172
Issue
11
Publish Date
2004
Start Page
6810
End Page
6819

The importance of three membrane-distal tyrosines in the adaptor protein NTAL/LAB.

NTAL (non-T cell activation linker)/LAB (linker for activation of B cells) is a LAT (linker for activation of T cells)-like molecule that is expressed in B cells, mast cells, natural killer cells, and monocytes. Upon engagement of the B cell receptor or Fc receptors, it is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT(-/-) mice. In this study, we utilized various LAB Tyr to Phe mutants to map the phosphorylation and Grb2-binding sites of LAB. We also examined the function of these mutants by investigating their ability to rescue signaling defects in LAT-deficient Jurkat cells and thymocyte development in LAT(-/-) mice. Our results indicated that human LAB was primarily phosphorylated on three membrane-distal tyrosines, Tyr(136), Tyr(193), and Tyr(233). Mutation of these three tyrosines abolished Grb2 binding and LAB function. Our data suggested that these tyrosines are the most important tyrosines for LAB function.

Authors
Koonpaew, S; Janssen, E; Zhu, M; Zhang, W
MLA Citation
Koonpaew, S, Janssen, E, Zhu, M, and Zhang, W. "The importance of three membrane-distal tyrosines in the adaptor protein NTAL/LAB." J Biol Chem 279.12 (March 19, 2004): 11229-11235.
PMID
14722116
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
12
Publish Date
2004
Start Page
11229
End Page
11235
DOI
10.1074/jbc.M311394200

Erratum: Transmembrane adaptor proteins: Organizers of immunoreceptor signalling (Nature Reviews Immunology (2004) 4 (603-616))

Authors
Hořejší, V; Zhang, W; Schraven, B
MLA Citation
Hořejší, V, Zhang, W, and Schraven, B. "Erratum: Transmembrane adaptor proteins: Organizers of immunoreceptor signalling (Nature Reviews Immunology (2004) 4 (603-616))." Nature Reviews Immunology 4.9 (2004): 743--.
Source
scival
Published In
Nature Reviews Immunology
Volume
4
Issue
9
Publish Date
2004
Start Page
743-

Adaptor proteins in lymphocyte activation.

Adaptor proteins are unique, as they contain modular domains and lack intrinsic enzymatic activity. These proteins are scaffolds for the organization of macromolecular complexes and they recruit other proteins for correct localization during molecular signal transduction. Numerous recent advances have been made through the elucidation of new adaptor proteins and the recognition of novel functions for previously identified molecules. In addition, the roles of adaptors in both the positive and negative regulation of lymphocyte activation have been further clarified.

Authors
Janssen, E; Zhang, W
MLA Citation
Janssen, E, and Zhang, W. "Adaptor proteins in lymphocyte activation." Curr Opin Immunol 15.3 (June 2003): 269-276. (Review)
PMID
12787751
Source
pubmed
Published In
Current Opinion in Immunology
Volume
15
Issue
3
Publish Date
2003
Start Page
269
End Page
276

LAB: a new membrane-associated adaptor molecule in B cell activation.

The adaptor molecule, linker for activation of T cells (LAT), is essential in T cell activation and development; a similar molecule in B cells has not yet been identified. Here, we report the identification of a new adaptor protein, linker for activation of B cells (LAB). Like LAT, LAB was localized to lipid rafts. Upon activation via the B cell receptor (BCR), LAB was phosphorylated and interacted with the adaptor protein Grb2. Decreased LAB expression led to a reduction in BCR-mediated calcium flux and Erk activation. LAB rescued thymocyte development but not normal T cell activation in LAT(-/-) mice. Our data suggest that LAB links BCR engagement to downstream signaling pathways.

Authors
Janssen, E; Zhu, M; Zhang, W; Koonpaew, S; Zhang, W
MLA Citation
Janssen, E, Zhu, M, Zhang, W, Koonpaew, S, and Zhang, W. "LAB: a new membrane-associated adaptor molecule in B cell activation." Nat Immunol 4.2 (February 2003): 117-123.
PMID
12514734
Source
pubmed
Published In
Nature Immunology
Volume
4
Issue
2
Publish Date
2003
Start Page
117
End Page
123
DOI
10.1038/ni882

Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development.

Linker for activation of T cells (LAT) is a membrane-associated adaptor protein that is phosphorylated on multiple tyrosines upon TCR cross-linking. Previous studies show that LAT is essential for TCR-mediated signaling and thymocyte development. In this study, we expressed a series of LAT Tyr to Phe mutants in LAT-deficient J.CaM2.5 cells and examined their tyrosine phosphorylation; association with Grb2, Gads, and phospholipase C (PLC)-gamma1; and function in T cell activation. Our results showed that the five membrane-distal tyrosines were phosphorylated upon T cell activation. Grb2, Gads, and PLC-gamma1 associated with LAT preferentially via different sets of tyrosine residues; however, they failed to interact with LAT mutants containing only one tyrosine. We also determined the minimal requirement of LAT tyrosine residues in T cell activation and thymocyte development. Our results showed that a minimum of three tyrosines is required for LAT to function in T cell activation and thymocyte development. LAT mutants that were capable of binding Grb2 and PLC-gamma1 could reconstitute T cell activation in LAT-deficient cells and thymocyte development in LAT-deficient mice.

Authors
Zhu, M; Janssen, E; Zhang, W
MLA Citation
Zhu, M, Janssen, E, and Zhang, W. "Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development." J Immunol 170.1 (January 1, 2003): 325-333.
PMID
12496416
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
170
Issue
1
Publish Date
2003
Start Page
325
End Page
333

Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling.

Membrane-associated adaptors play an important role in coupling antigen receptor engagement to downstream signaling events, such as Ras-MAPK activation, Ca(2+) flux, and nuclear factor of activated T cells (NFAT) activation. Here we identified a novel membrane-associated adaptor protein, LAX. LAX is mainly expressed in B cells, T cells, and other lymphoid-specific cell types. It shares no overall sequence homology with LAT and is not localized to lipid rafts. However, like LAT, LAX has tyrosine motifs for binding Grb2, Gads, and the p85 subunit of phosphatidylinositol 3-kinase. Upon stimulation via the B or T cell receptors, LAX is rapidly phosphorylated by Src and Syk family tyrosine kinases and interacts with Grb2, Gads, and p85. Overexpression of LAX in Jurkat cells specifically inhibits T cell receptor-mediated p38 MAPK activation and NFAT/AP-1 transcriptional activation. Our data suggested that LAX functions to negatively regulate signaling in lymphocytes.

Authors
Zhu, M; Janssen, E; Leung, K; Zhang, W
MLA Citation
Zhu, M, Janssen, E, Leung, K, and Zhang, W. "Molecular cloning of a novel gene encoding a membrane-associated adaptor protein (LAX) in lymphocyte signaling." J Biol Chem 277.48 (November 29, 2002): 46151-46158.
PMID
12359715
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
48
Publish Date
2002
Start Page
46151
End Page
46158
DOI
10.1074/jbc.M208946200

Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development.

The integral membrane adapter protein linker for activation of T cells (LAT) performs a critical function in T cell antigen receptor (TCR) signal transduction by coupling the TCR to downstream signaling pathways. After TCR engagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites for multiple src homology 2-containing effector proteins. In the Jurkat T cell line, the distal four tyrosines of LAT bind PLCgamma-1, Grb2, and Gads. Mutation of these four tyrosine residues to phenylalanine (4YF) blocked TCR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-AT activation. In this study, we examined whether these four tyrosine residues were essential for T cell development by generating LAT "knock-in" mutant mice that express the 4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT(-/)- (null) mice; thymocyte development was arrested at the immature CD4(-)CD8(-) stage and no mature T cells were present. Knock-in mice expressing wild-type LAT protein, generated by a similar strategy, displayed a normal T cell developmental profile. These results demonstrate that the distal four tyrosine residues of LAT are essential for preTCR signaling and T cell development in vivo.

Authors
Sommers, CL; Menon, RK; Grinberg, A; Zhang, W; Samelson, LE; Love, PE
MLA Citation
Sommers, CL, Menon, RK, Grinberg, A, Zhang, W, Samelson, LE, and Love, PE. "Knock-in mutation of the distal four tyrosines of linker for activation of T cells blocks murine T cell development." J Exp Med 194.2 (July 16, 2001): 135-142.
PMID
11457888
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
194
Issue
2
Publish Date
2001
Start Page
135
End Page
142

Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT.

T cell activation induces functional changes in cell shape and cytoskeletal architecture. To facilitate the collection of dynamic, high-resolution images of activated T cells, we plated T cells on coverslips coated with antibodies to the T cell receptor (TCR). Using these images, we were able to quantitate the morphological responses of individual cells over time. Here, we show that TCR engagement triggers the formation and expansion of contacts bounded by continuously remodeled actin-rich rings. These processes are associated with the extension of lamellipodia and require actin polymerization, tyrosine kinase activation, cytoplasmic calcium increases, and LAT, an important hematopoietic adaptor. In addition, the maintenance of the resulting contact requires sustained calcium influxes, an intact microtubule cytoskeleton, and functional LAT.

Authors
Bunnell, SC; Kapoor, V; Trible, RP; Zhang, W; Samelson, LE
MLA Citation
Bunnell, SC, Kapoor, V, Trible, RP, Zhang, W, and Samelson, LE. "Dynamic actin polymerization drives T cell receptor-induced spreading: a role for the signal transduction adaptor LAT." Immunity 14.3 (March 2001): 315-329.
PMID
11290340
Source
pubmed
Published In
Immunity
Volume
14
Issue
3
Publish Date
2001
Start Page
315
End Page
329

Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8+ T cells

T cell responses against hapten-modified peptides play an important role in the pathogenesis of certain diseases, including contact dermatitis and allergy. However, the structural features of TCRs recognizing bulky, potentially mobile hapten groups remain poorly defined. To analyze the structural basis of TCR recognition of defined hapten-modified peptides, the immunodominant octapeptide derived from vesicular stomatitis virus nucleoprotein (VSV8) was modified with a trinitrophenyl (TNP) group at the primary TCR contact residues (position 4 or 6) and used for immunization of mice carrying either the TCR α- or β-chain of a VSV8 (unmodified)/H-2Kb-specific CTL clone as a transgene. Such mice allow independent analysis of one TCR chain by maintaining the other fixed. The TCR V gene usage of the responding T cell population was specifically altered depending upon the presence of the TNP group and its position on the peptide. The CDR3 sequences of the TNP-modified peptide-specific TCRs showed a preferential J region usage in both the CDR3α and β loops, indicating that the J regions of both CDR3s are critical for recognition of TNP-modified peptides. In contrast to our previous observations showing the prime importance of CDR3β residues encoded by D-segment or N-addition nucleotides for recognition of position 6 of unmodified VSV8, our studies of TNP-modified peptides demonstrate the importance of the Jβ region, while the Jα region was crucial for recognizing both TNP-modified and unmodified peptides. These data suggest that different structural strategies are utilized by the CDR3α and β loops to allow interaction with a haptenated peptide.

Authors
Honda, S; Zhang, W; Kalergis, AM; DiLorenzo, TP; Wang, F; Nathenson, SG
MLA Citation
Honda, S, Zhang, W, Kalergis, AM, DiLorenzo, TP, Wang, F, and Nathenson, SG. "Hapten addition to an MHC class I-binding peptide causes substantial adjustments of the TCR structure of the responding CD8+ T cells." Journal of Immunology 167.8 (2001): 4276-4285.
PMID
11591750
Source
scival
Published In
Journal of Immunology
Volume
167
Issue
8
Publish Date
2001
Start Page
4276
End Page
4285

Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation.

Activation of T cells can be initiated through cell surface molecules in addition to the T-cell receptor-CD3 (TCR-CD3) complex. In human T cells, ligation of the CD2 molecule by mitogenic pairs of anti-CD2 monoclonal antibodies activates T cells via biochemical signaling pathways similar but not identical to those elicited on TCR engagement. This study describes a key role for the p36/38 membrane adapter protein linker for T cell activation (LAT) in CD2-mediated T-cell activation. Following ligation of CD2 on the surface of the Jurkat T-cell line and human purified T cells, LAT was tyrosine phosphorylated and shown to associate in vivo with a number of other tyrosine phosphorylated proteins including PLCgamma-1, Grb-2, and SLP-76. Using Jurkat cell lines deficient in ZAP70/Syk (P116) or LAT (ANJ3) expression, CD2-dependent PLCgamma-1 and SLP-76 tyrosine phosphorylation required expression both of ZAP70 or Syk and of LAT. As predicted, the absence of either LAT or ZAP70/Syk kinases correlated with a defect in the induction of nuclear factor of activated T cells (NFAT) transcriptional activity, activation of the interleukin-2 promoter, and ERK phosphorylation following CD2 stimulation. These data suggest that LAT is an adapter protein important for the regulation of CD2-mediated T-cell activation.

Authors
Martelli, MP; Lin, H; Zhang, W; Samelson, LE; Bierer, BE
MLA Citation
Martelli, MP, Lin, H, Zhang, W, Samelson, LE, and Bierer, BE. "Signaling via LAT (linker for T-cell activation) and Syk/ZAP70 is required for ERK activation and NFAT transcriptional activation following CD2 stimulation." Blood 96.6 (September 15, 2000): 2181-2190.
PMID
10979964
Source
pubmed
Published In
Blood
Volume
96
Issue
6
Publish Date
2000
Start Page
2181
End Page
2190

Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling.

The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca(2+) flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are responsible for Grb2-binding; Tyr(171), and Tyr(191), but not Tyr(226), are necessary for Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma1 binding. Mutation of all three distal tyrosines also abolished PLC-gamma1 binding, suggesting there might be multiple binding sites for PLC-gamma1. Mutation of Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-gamma activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk and NF-AT activation.

Authors
Zhang, W; Trible, RP; Zhu, M; Liu, SK; McGlade, CJ; Samelson, LE
MLA Citation
Zhang, W, Trible, RP, Zhu, M, Liu, SK, McGlade, CJ, and Samelson, LE. "Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling." J Biol Chem 275.30 (July 28, 2000): 23355-23361.
PMID
10811803
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
30
Publish Date
2000
Start Page
23355
End Page
23361
DOI
10.1074/jbc.M000404200

LAT is essential for Fc(epsilon)RI-mediated mast cell activation.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow-derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of Fc(epsilon)RI, Syk, and Vav was intact in LAT-deficient BMMCs following Fc(epsilon)RI engagement, tyrosine phosphorylation of SLP-76, PLC-gamma1, and PLC-gamma2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after Fc(epsilon)RI cross-linking. These results show that LAT plays a critical role in Fc(epsilon)RI-mediated signaling in mast cells.

Authors
Saitoh, S; Arudchandran, R; Manetz, TS; Zhang, W; Sommers, CL; Love, PE; Rivera, J; Samelson, LE
MLA Citation
Saitoh, S, Arudchandran, R, Manetz, TS, Zhang, W, Sommers, CL, Love, PE, Rivera, J, and Samelson, LE. "LAT is essential for Fc(epsilon)RI-mediated mast cell activation." Immunity 12.5 (May 2000): 525-535.
PMID
10843385
Source
pubmed
Published In
Immunity
Volume
12
Issue
5
Publish Date
2000
Start Page
525
End Page
535

The role of membrane-associated adaptors in T cell receptor signalling.

Engagement of the T cell receptor leads to activation of several tyrosine kinases and phosphorylation of many intracellular proteins. This is followed by Ca2+ mobilization and activation of multiple biochemical pathways, including the Ras/MAPK cascade, and several downstream serine/threonine kinases. Membrane-associated adaptor proteins play an important role in T cell activation by coupling TCR ligation at the membrane to distal signalling cascades. Several new membrane associated adaptors have been identified in recent years. LAT (linker for activation of T cells) is an adaptor molecule, which following its phosphorylation associates with Grb2, Gads, PLC-gamma 1, and other signalling molecules. The functional importance of this molecule has been demonstrated by the study of LAT-deficient cell lines and LAT-deficient mice. Two other recently identified adaptor proteins, TRIM (T cell receptor interacting molecule) and SIT (SHP2-interacting transmembrane adaptor protein), which constitutively associate with several surface molecules, bind to PI3K and SHP2, respectively, after T cell activation and might also function in the TCR signalling pathway.

Authors
Zhang, W; Samelson, LE
MLA Citation
Zhang, W, and Samelson, LE. "The role of membrane-associated adaptors in T cell receptor signalling." Semin Immunol 12.1 (February 2000): 35-41. (Review)
PMID
10723796
Source
pubmed
Published In
Seminars in Immunology
Volume
12
Issue
1
Publish Date
2000
Start Page
35
End Page
41
DOI
10.1006/smim.2000.0205

LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI.

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.

Authors
Pasquet, JM; Gross, B; Quek, L; Asazuma, N; Zhang, W; Sommers, CL; Schweighoffer, E; Tybulewicz, V; Judd, B; Lee, JR; Koretzky, G; Love, PE; Samelson, LE; Watson, SP
MLA Citation
Pasquet, JM, Gross, B, Quek, L, Asazuma, N, Zhang, W, Sommers, CL, Schweighoffer, E, Tybulewicz, V, Judd, B, Lee, JR, Koretzky, G, Love, PE, Samelson, LE, and Watson, SP. "LAT is required for tyrosine phosphorylation of phospholipase cgamma2 and platelet activation by the collagen receptor GPVI." Mol Cell Biol 19.12 (December 1999): 8326-8334.
PMID
10567557
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
19
Issue
12
Publish Date
1999
Start Page
8326
End Page
8334

Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction.

Linker for activation of T cells (LAT) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the T cell receptor (TCR) signal. LAT phosphorylation is accomplished by the protein tyrosine kinase ZAP-70, but it is not at all clear how LAT (which is not associated with the TCR) encounters ZAP-70 (which is bound to the TCR). Here we show that LAT associates with surface CD4 and CD8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates Lck binding. In fact, LAT competes with Lck for binding to individual coreceptor molecules but differs from Lck in its preferential association with CD8 rather than CD4 in CD4(+)CD8(+) thymocytes. Importantly, as a consequence of LAT association with surface coreceptors, coengagement of the TCR with surface coreceptors induces LAT phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor-associated LAT molecules. These results point to a new function for CD4 and CD8 coreceptors in TCR signal transduction, namely to promote LAT phosphorylation by ZAP-70 by recruiting LAT to major histocompatibility complex-engaged TCR complexes.

Authors
Bosselut, R; Zhang, W; Ashe, JM; Kopacz, JL; Samelson, LE; Singer, A
MLA Citation
Bosselut, R, Zhang, W, Ashe, JM, Kopacz, JL, Samelson, LE, and Singer, A. "Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction." J Exp Med 190.10 (November 15, 1999): 1517-1526.
PMID
10562325
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
190
Issue
10
Publish Date
1999
Start Page
1517
End Page
1526

Requirement of the Src homology 2 domain protein Shb for T cell receptor-dependent activation of the interleukin-2 gene nuclear factor for activation of T cells element in Jurkat T cells.

Stimulation of the T cell antigen receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins. We have recently investigated the role of the adaptor protein Shb in the early events of T cell signaling and observed that Shb associates with Grb2, linker for activation of T cells (LAT) and the TCR zeta-chain in Jurkat cells. We now report that Shb also associates with phospholipase C-gamma1 (PLC-gamma1) in these cells. Overexpression of Src homology 2 domain defective Shb caused diminished phosphorylation of LAT and consequently the activation of mitogen-activated protein kinases was decreased upon TCR stimulation. In addition, the Shb mutant also blocked phosphorylation of PLC-gamma1 and the increase in cytoplasmic Ca(2+) following TCR stimulation. Nuclear factor for activation of T cells is a major target for Ras and calcium signaling pathways in T cells following TCR stimulation, and the overexpression of the mutant Shb prevented TCR-dependent activation of the nuclear factor for activation of T cells. Consequently, endogenous interleukin-2 production was decreased under these conditions. The results indicate a role for Shb as a link between the TCR and downstream signaling events involving LAT and PLC-gamma1 and resulting in the activation of transcription of the interleukin-2 gene.

Authors
Lindholm, CK; Gylfe, E; Zhang, W; Samelson, LE; Welsh, M
MLA Citation
Lindholm, CK, Gylfe, E, Zhang, W, Samelson, LE, and Welsh, M. "Requirement of the Src homology 2 domain protein Shb for T cell receptor-dependent activation of the interleukin-2 gene nuclear factor for activation of T cells element in Jurkat T cells." J Biol Chem 274.39 (September 24, 1999): 28050-28057.
PMID
10488157
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
39
Publish Date
1999
Start Page
28050
End Page
28057

Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line.

The adaptor molecule LAT (linker for activation of T cells) is a palmitoylated integral membrane protein that localizes to the glycolipid-enriched microdomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues and then binds several critical signaling molecules. Here, we describe the generation and characterization of a LAT-deficient cell line. Using this cell line, we demonstrate that LAT is required for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylation of phospholipase C-gamma1, Vav and SLP-76. LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcription. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys29, are required for LAT function and TCR signaling. These studies provide further evidence for the crucial role of the LAT molecule, and demonstrate the use of a LAT-deficient cell line for the analysis of LAT structure and function.

Authors
Zhang, W; Irvin, BJ; Trible, RP; Abraham, RT; Samelson, LE
MLA Citation
Zhang, W, Irvin, BJ, Trible, RP, Abraham, RT, and Samelson, LE. "Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line." Int Immunol 11.6 (June 1999): 943-950.
PMID
10360968
Source
pubmed
Published In
International Immunology
Volume
11
Issue
6
Publish Date
1999
Start Page
943
End Page
950

Linker for activation of T cells (LAT), a novel immunohistochemical marker for T cells, NK cells, mast cells, and megakaryocytes: evaluation in normal and pathological conditions.

LAT (linker for activation of T cells) is an integral membrane protein of 36-38 kd that plays an important role in T cell activation. Using a rabbit polyclonal antibody generated against the cytosolic portion of LAT, we investigated the immunohistochemical expression of LAT in normal and pathological hematolymphoid tissues. LAT reacts with human T cells in paraffin sections, including decalcified bone marrow trephines. LAT appears early in T cells at the thymocyte stage and before TdT expression in embryos, and is expressed in peripheral lymphoid tissues, without restriction to any T cell subpopulations. In addition to T cells, natural killer (NK) cells (evaluated with flow cytometry), megakaryocytes and mast cells are also LAT-positive, whereas B cells and other myeloid and monocytic derived cells are negative. Tested on a total of 264 paraffin-embedded tissue biopsies, LAT reacted with the great majority (96.8%) of T/NK-cell neoplasms, covering the full range of T cell maturation. Although antibodies to both LAT and CD3 had a similarly high sensitivity in the staining of T/NK-cell lymphomas, when used in conjunction, they successfully identified a higher number of cases (98.4%). Atypical megakaryocytes from different hematological disorders, as well as mast cells in mastocytosis were also LAT-positive, but all neoplasms of B cell origin, Hodgkin's lymphomas, and several nonlymphoid malignancies were negative. These data indicate that the anti-LAT antibody may be of value to diagnostic histopathologists for the identification of T cell neoplasms.

Authors
Facchetti, F; Chan, JK; Zhang, W; Tironi, A; Chilosi, M; Parolini, S; Notarangelo, LD; Samelson, LE
MLA Citation
Facchetti, F, Chan, JK, Zhang, W, Tironi, A, Chilosi, M, Parolini, S, Notarangelo, LD, and Samelson, LE. "Linker for activation of T cells (LAT), a novel immunohistochemical marker for T cells, NK cells, mast cells, and megakaryocytes: evaluation in normal and pathological conditions." Am J Pathol 154.4 (April 1999): 1037-1046.
PMID
10233842
Source
pubmed
Published In
The American journal of pathology
Volume
154
Issue
4
Publish Date
1999
Start Page
1037
End Page
1046
DOI
10.1016/S0002-9440(10)65356-4

Cutting edge: a role for the adaptor protein LAT in human NK cell-mediated cytotoxicity.

Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcgammaRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-gamma. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and "natural cytotoxicity," thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.

Authors
Jevremovic, D; Billadeau, DD; Schoon, RA; Dick, CJ; Irvin, BJ; Zhang, W; Samelson, LE; Abraham, RT; Leibson, PJ
MLA Citation
Jevremovic, D, Billadeau, DD, Schoon, RA, Dick, CJ, Irvin, BJ, Zhang, W, Samelson, LE, Abraham, RT, and Leibson, PJ. "Cutting edge: a role for the adaptor protein LAT in human NK cell-mediated cytotoxicity." J Immunol 162.5 (March 1, 1999): 2453-2456.
PMID
10072481
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
162
Issue
5
Publish Date
1999
Start Page
2453
End Page
2456

Essential role of LAT in T cell development.

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement. Phosphorylated LAT binds many critical signaling molecules. The central role of this molecule in TCR-mediated signaling has been demonstrated by experiments in a LAT-deficient cell line. To probe the role of LAT in T cell development, the LAT gene was disrupted by targeting. LAT-deficient mice appeared healthy. Flow cytometric analysis revealed normal B cell populations but the absence of any mature peripheral T cells. Intrathymic development was blocked within the CD4- CD8- stage. No gross abnormality of NK or platelet function was observed. LAT is thus critical to both T cell activation and development.

Authors
Zhang, W; Sommers, CL; Burshtyn, DN; Stebbins, CC; DeJarnette, JB; Trible, RP; Grinberg, A; Tsay, HC; Jacobs, HM; Kessler, CM; Long, EO; Love, PE; Samelson, LE
MLA Citation
Zhang, W, Sommers, CL, Burshtyn, DN, Stebbins, CC, DeJarnette, JB, Trible, RP, Grinberg, A, Tsay, HC, Jacobs, HM, Kessler, CM, Long, EO, Love, PE, and Samelson, LE. "Essential role of LAT in T cell development." Immunity 10.3 (March 1999): 323-332.
PMID
10204488
Source
pubmed
Published In
Immunity
Volume
10
Issue
3
Publish Date
1999
Start Page
323
End Page
332

Studies on the adapter molecule LAT.

Authors
Samelson, LE; Bunnell, SC; Trible, RP; Yamazaki, T; Zhang, W
MLA Citation
Samelson, LE, Bunnell, SC, Trible, RP, Yamazaki, T, and Zhang, W. "Studies on the adapter molecule LAT." Cold Spring Harb Symp Quant Biol 64 (1999): 259-263. (Review)
PMID
11232294
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
64
Publish Date
1999
Start Page
259
End Page
263

Molecular basis of T cell inactivation by CTLA-4.

CTLA-4, a negative regulator of T cell function, was found to associate with the T cell receptor (TCR) complex zeta chain in primary T cells. The association of TCRzeta with CTLA-4, reconstituted in 293 transfectants, was enhanced by p56(lck)-induced tyrosine phosphorylation. Coexpression of the CTLA-4-associated tyrosine phosphatase, SHP-2, resulted in dephosphorylation of TCRzeta bound to CTLA-4 and abolished the p56(lck)-inducible TCRzeta-CTLA-4 interaction. Thus, CTLA-4 inhibits TCR signal transduction by binding to TCRzeta and inhibiting tyrosine phosphorylation after T cell activation. These findings have broad implications for the negative regulation of T cell function and T cell tolerance.

Authors
Lee, KM; Chuang, E; Griffin, M; Khattri, R; Hong, DK; Zhang, W; Straus, D; Samelson, LE; Thompson, CB; Bluestone, JA
MLA Citation
Lee, KM, Chuang, E, Griffin, M, Khattri, R, Hong, DK, Zhang, W, Straus, D, Samelson, LE, Thompson, CB, and Bluestone, JA. "Molecular basis of T cell inactivation by CTLA-4." Science 282.5397 (December 18, 1998): 2263-2266.
PMID
9856951
Source
pubmed
Published In
Science
Volume
282
Issue
5397
Publish Date
1998
Start Page
2263
End Page
2266

LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway.

In this study, we present the further characterization of a mutant Jurkat T cell line, J.CaM2, that is defective in TCR-mediated signal transduction. Although initial TCR-mediated signaling events such as the inducible tyrosine phosphorylation of the TCR-zeta chain and ZAP-70 are intact in J.CaM2, subsequent events, including increases in intracellular calcium, Ras activation, and IL-2 gene expression are defective. Subsequent analysis of J.CaM2 demonstrated a severe deficiency in pp36/LAT expression, a recently cloned adaptor protein implicated in TCR signaling. Importantly, reexpression of LAT in J.CaM2 restored all aspects of TCR signaling. These results demonstrate a necessary and exclusive role for LAT in T cell activation.

Authors
Finco, TS; Kadlecek, T; Zhang, W; Samelson, LE; Weiss, A
MLA Citation
Finco, TS, Kadlecek, T, Zhang, W, Samelson, LE, and Weiss, A. "LAT is required for TCR-mediated activation of PLCgamma1 and the Ras pathway." Immunity 9.5 (November 1998): 617-626.
PMID
9846483
Source
pubmed
Published In
Immunity
Volume
9
Issue
5
Publish Date
1998
Start Page
617
End Page
626

LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation.

The linker molecule LAT is a critical substrate of the tyrosine kinases activated upon TCR engagement. Phosphorylated LAT binds Grb2, PLC-gamma1, and other signaling molecules. We demonstrate that human LAT is palmitoylated and that palmitoylated LAT predominantly localizes into glycolipid-enriched microdomains (GEMs). Although the LAT transmembrane domain is sufficient for membrane localization, palmitoylation at C26 and C29 is essential for efficient partitioning into GEMs. LAT palmitoylation is necessary for its tyrosine phosphorylation. After T cell activation, most tyrosine-phosphorylated LAT molecules and a fraction of PLC-gamma1 and other signaling molecules are present in GEMs. LAT is central to T cell activation and is a novel linker molecule shown to require targeting to membrane microdomains for signaling.

Authors
Zhang, W; Trible, RP; Samelson, LE
MLA Citation
Zhang, W, Trible, RP, and Samelson, LE. "LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation." Immunity 9.2 (August 1998): 239-246.
PMID
9729044
Source
pubmed
Published In
Immunity
Volume
9
Issue
2
Publish Date
1998
Start Page
239
End Page
246

ZAP-70-dependent and -independent activation of Erk in Jurkat T cells. Differences in signaling induced by H2o2 and Cd3 cross-linking.

Oxidative stress in T cells induces signaling events similar to those initiated by T cell antigen receptor engagement, including tyrosine phosphorylation and activation of the critical protein-tyrosine kinase ZAP-70. Distal signaling events such as the activation of mitogen-activated protein kinases and downstream transcription factors are also initiated by oxidative stimuli. In this study P116, a ZAP-70-negative Jurkat T cell line, was used to investigate the role of ZAP-70 in mediating activation of Erk in response to H2O2. Consistent with the hypothesis that ZAP-70 is required for activation of Erk in response to an oxidative stimulus, Erk1 and Erk2 could be rapidly activated in Jurkat cells but not in P116 cells upon addition of H2O2. P116 cells became competent for H2O2-induced Erk activation upon stable transfection with wild-type ZAP-70. An in vivo ZAP-70 substrate, SLP-76, implicated in Erk activation, also became rapidly tyrosine-phosphorylated in Jurkat cells, but not in P116 cells, upon treatment with H2O2. Surprisingly, although ZAP-70 was required for H2O2-mediated Erk activation, Erk activation in response to T cell antigen receptor engagement did not require ZAP-70. In addition to demonstrating a requirement for ZAP-70 in H2O2-stimulated Erk activation, these results provide the first evidence for the existence of a ZAP-70-independent pathway for Erk activation in T cells.

Authors
Griffith, CE; Zhang, W; Wange, RL
MLA Citation
Griffith, CE, Zhang, W, and Wange, RL. "ZAP-70-dependent and -independent activation of Erk in Jurkat T cells. Differences in signaling induced by H2o2 and Cd3 cross-linking." J Biol Chem 273.17 (April 24, 1998): 10771-10776.
PMID
9553143
Source
pubmed
Published In
The Journal of biological chemistry
Volume
273
Issue
17
Publish Date
1998
Start Page
10771
End Page
10776

Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line.

T-cell antigen receptor (TCR) engagement activates multiple protein tyrosine kinases (PTKs), including the Src family member, Lck, and the Syk-related PTK, ZAP-70. Studies in ZAP-70-deficient humans have demonstrated that ZAP-70 plays crucial roles in T-cell activation and development. However, progress toward a detailed understanding of the regulation and function of ZAP-70 during TCR signaling has been hampered by the lack of a suitable T-cell model for biochemical and genetic analyses. In this report, we describe the isolation and phenotypic characterization of a Syk- and ZAP-70-negative somatic mutant derived from the Jurkat T-cell line. The P116 cell line displays severe defects in TCR-induced signaling functions, including protein tyrosine phosphorylation, intracellular Ca2+ mobilization, and interleukin-2 promoter-driven transcription. These signaling defects were fully reversed by reintroduction of catalytically active versions of either Syk or ZAP-70 into the P116 cells. However, in contrast to ZAP-70 expression, Syk expression triggered a significant degree of cellular activation in the absence of TCR ligation. Transfection experiments with ZAP-70-Syk chimeric proteins indicated that both the amino-terminal regulatory regions and the carboxy-terminal catalytic domains of Syk and ZAP-70 contribute to the distinctive functional properties of these PTKs. These studies underscore the crucial role of ZAP-70 in TCR signaling and offer a powerful genetic model for further analyses of ZAP-70 regulation and function in T cells.

Authors
Williams, BL; Schreiber, KL; Zhang, W; Wange, RL; Samelson, LE; Leibson, PJ; Abraham, RT
MLA Citation
Williams, BL, Schreiber, KL, Zhang, W, Wange, RL, Samelson, LE, Leibson, PJ, and Abraham, RT. "Genetic evidence for differential coupling of Syk family kinases to the T-cell receptor: reconstitution studies in a ZAP-70-deficient Jurkat T-cell line." Mol Cell Biol 18.3 (March 1998): 1388-1399.
PMID
9488454
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
18
Issue
3
Publish Date
1998
Start Page
1388
End Page
1399

LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation.

Despite extensive study, several of the major components involved in T cell receptor-mediated signaling remain unidentified. Here we report the cloning of the cDNA for a highly tyrosine-phosphorylated 36-38 kDa protein, previously characterized by its association with Grb2, phospholipase C-gamma1, and the p85 subunit of phosphoinositide 3-kinase. Deduced amino acid sequence identifies a novel integral membrane protein containing multiple potential tyrosine phosphorylation sites. We show that this protein is phosphorylated by ZAP-70/Syk protein tyrosine kinases leading to recruitment of multiple signaling molecules. Its function is demonstrated by inhibition of T cell activation following overexpression of a mutant form lacking critical tyrosine residues. Therefore, we propose to name the molecule LAT-linker for activation of T cells.

Authors
Zhang, W; Sloan-Lancaster, J; Kitchen, J; Trible, RP; Samelson, LE
MLA Citation
Zhang, W, Sloan-Lancaster, J, Kitchen, J, Trible, RP, and Samelson, LE. "LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation." Cell 92.1 (January 9, 1998): 83-92.
PMID
9489702
Source
pubmed
Published In
Cell
Volume
92
Issue
1
Publish Date
1998
Start Page
83
End Page
92

Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein.

To investigate the cellular dynamics of ZAP-70, we have studied the distribution and regulation of its intracellular location using a ZAP-70 green fluorescent protein chimera. Initial experiments in epithelial cells indicated that ZAP-70 is diffusely located throughout the quiescent cell, and accumulates at the plasma membrane upon cellular activation, a phenotype enhanced by the coexpression of Lck and the initiation of ZAP-70 kinase activity. Subsequent studies in T cells confirmed this phenotype. Intriguingly, a large amount of ZAP-70, both chimeric and endogenous, resides in the nucleus of quiescent and activated cells. Nuclear ZAP-70 becomes tyrosine phosphorylated upon stimulation via the T cell receptor, indicating that it may have an important biologic function.

Authors
Sloan-Lancaster, J; Zhang, W; Presley, J; Williams, BL; Abraham, RT; Lippincott-Schwartz, J; Samelson, LE
MLA Citation
Sloan-Lancaster, J, Zhang, W, Presley, J, Williams, BL, Abraham, RT, Lippincott-Schwartz, J, and Samelson, LE. "Regulation of ZAP-70 intracellular localization: visualization with the green fluorescent protein." J Exp Med 186.10 (November 17, 1997): 1713-1724.
PMID
9362531
Source
pubmed
Published In
The Journal of Experimental Medicine
Volume
186
Issue
10
Publish Date
1997
Start Page
1713
End Page
1724

ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis.

Activation-induced cell death (AICD) is initiated by the TCR-dependent up-regulation of Fas ligand (FasL) mRNA. The subsequently generated soluble or cell-associated FasL gene products bind Fas, leading to apoptosis of the T cells. Although TCR stimulation is essential to initiate AICD, little is known about which TCR-initiated second messengers are required for FasL expression. We provide evidence in this work that T cells lacking the tyrosine kinase ZAP-70 are unable to up-regulate FasL and undergo AICD. Transfection of wild-type ZAP-70 into the ZAP-70-deficient T cells restores their sensitivity to TCR-induced apoptosis, whereas transfection of catalytically inactive ZAP-70 does not. These results provide clear evidence that ZAP-70 tyrosine kinase is essential in up-regulating FasL for TCR-induced apoptosis.

Authors
Eischen, CM; Williams, BL; Zhang, W; Samelson, LE; Lynch, DH; Abraham, RT; Leibson, PJ
MLA Citation
Eischen, CM, Williams, BL, Zhang, W, Samelson, LE, Lynch, DH, Abraham, RT, and Leibson, PJ. "ZAP-70 tyrosine kinase is required for the up-regulation of Fas ligand in activation-induced T cell apoptosis." J Immunol 159.3 (August 1, 1997): 1135-1139.
PMID
9233606
Source
pubmed
Published In
Journal of immunology (Baltimore, Md. : 1950)
Volume
159
Issue
3
Publish Date
1997
Start Page
1135
End Page
1139

MHC class I-peptide interactions and TCR recognition.

The recent crystal structure determinations of MHC class I molecules with single bound peptides have allowed us to understand the guidelines that govern peptide binding in a given MHC allele. Evolution has provided for MHC class I molecules whose antigen binding clefts possess distinct physical and chemical properties as a result of the particular arrangement of variable residues lining the binding cleft. As a result, a given molecule binds a unique set of peptides, the position and identity of whose anchor residues are dictated by the features of the cleft. In addition to the interactions between the anchor residues and the cleft, the peptide is held in the groove by a highly conserved array of hydrogen bonds. A tantalizing application of our newfound understanding of peptide binding will be in the design of model peptides that either block or enhance immune response, in order to achieve effective treatments for a variety of disorders of the immune system.

Authors
Young, AC; Zhang, W; Sacchettini, JC; Nathenson, SG
MLA Citation
Young, AC, Zhang, W, Sacchettini, JC, and Nathenson, SG. "MHC class I-peptide interactions and TCR recognition." Cancer Surv 22 (1995): 17-36. (Review)
PMID
7536627
Source
pubmed
Published In
Cancer surveys
Volume
22
Publish Date
1995
Start Page
17
End Page
36

The three-dimensional structure of H-2Db at 2.4 A resolution: implications for antigen-determinant selection.

Solution at 2.4 A resolution of the structure of H-2Db with the influenza virus peptide NP366-374 (ASNEN-METM) and comparison with the H-2Kb-VSV (RGY-VYQGL) structure allow description of the molecular details of MHC class I peptide binding interactions for mice of the H-2b haplotype, revealing a strategy that maximizes the repertoire of peptides than can be presented. The H-2Db cleft has a mouse-specific hydrophobic ridge that causes a compensatory arch in the backbone of the peptide, exposing the arch residues to TCR contact and requiring the peptide to be at least 9 residues. This ridge occurs in about 40% of the known murine D and L allelic molecules, classifying them as a structural subgroup.

Authors
Young, AC; Zhang, W; Sacchettini, JC; Nathenson, SG
MLA Citation
Young, AC, Zhang, W, Sacchettini, JC, and Nathenson, SG. "The three-dimensional structure of H-2Db at 2.4 A resolution: implications for antigen-determinant selection." Cell 76.1 (January 14, 1994): 39-50.
PMID
7506996
Source
pubmed
Published In
Cell
Volume
76
Issue
1
Publish Date
1994
Start Page
39
End Page
50

Structural organization of the genes encoding human and murine FK-506-binding protein (FKBP) 13 and comparison to FKBP1

FK506-binding protein (FKBP) 12 and FKBP 13 are members of a family of proteins which bind the immunosuppressant drugs, FK506 and rapamycin. FKBP12 and FKBP13 are encoded by distinct genes, designated FKBP1 and FKBP2, respectively. The structure of human FKBP1 was previously characterized. We now report the genomic structure of the human and murine FKBP2 genes. Comparison of FKBP1 and FKBP2 reveals significant homology and correlation of intron positions in the C-terminal region, suggesting that these genes may have evolved from a common ancestral gene.

Authors
Hendrickson, BA; Zhang, W; Craig, RJ; Jin, Y-J; Bierer, BE; Burakoff, S; DiLella, AG
MLA Citation
Hendrickson, BA, Zhang, W, Craig, RJ, Jin, Y-J, Bierer, BE, Burakoff, S, and DiLella, AG. "Structural organization of the genes encoding human and murine FK-506-binding protein (FKBP) 13 and comparison to FKBP1." Gene 134.2 (1993): 271-275.
Source
scival
Published In
Gene
Volume
134
Issue
2
Publish Date
1993
Start Page
271
End Page
275
DOI
10.1016/0378-1119(93)90106-D

Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition.

To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.

Authors
Zhang, W; Young, AC; Imarai, M; Nathenson, SG; Sacchettini, JC
MLA Citation
Zhang, W, Young, AC, Imarai, M, Nathenson, SG, and Sacchettini, JC. "Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition." Proc Natl Acad Sci U S A 89.17 (September 1, 1992): 8403-8407.
PMID
1325657
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
89
Issue
17
Publish Date
1992
Start Page
8403
End Page
8407

Specificity of interaction between MHC (major histocompatibility complex) class I molecules, antigenic peptides, and the T cell receptor

Authors
Nathenson, SG; Bleek, GMV; Geliebter, J; Hasenkrug, K; Imarai, M; Joyce, S; Kesari, K; Kumar, A; McCue, B; Nakagawa, M; Palmieri, E; Sheil, J; Shibata, K; Sun, R; Zhang, W
MLA Citation
Nathenson, SG, Bleek, GMV, Geliebter, J, Hasenkrug, K, Imarai, M, Joyce, S, Kesari, K, Kumar, A, McCue, B, Nakagawa, M, Palmieri, E, Sheil, J, Shibata, K, Sun, R, and Zhang, W. "Specificity of interaction between MHC (major histocompatibility complex) class I molecules, antigenic peptides, and the T cell receptor." Developmental and Comparative Immunology 15.SUPPL. 1 (1991): S33-.
Source
scival
Published In
Developmental and Comparative Immunology
Volume
15
Issue
SUPPL. 1
Publish Date
1991
Start Page
S33
DOI
10.1016/0145-305X(91)90201-9
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