Tyrosine 319, a Newly Identified Phosphorylation Site of ZAP-70, Plays a Critical Role in T Cell Antigen Receptor Signaling

Bartolo, Vincenzo Di; Mège, Dominique; Germain, Valérie; Pelosi, Michele; Dufour, Evelyne; Michel, Frédérique; Magistrelli, Giovanni; Isacchi, Antonella; Acuto, Oreste

doi:10.1074/jbc.274.10.6285

Cited by 133 publications

(146 citation statements)

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“…HS1 is known to be tyrosine phosphorylated as a consequence of anti-CD3 stimulation, possibly through the action of Syk (26,27), but again the site of phosphorylation was not previously identified. Up-regulation of ZAP-70 catalytic activity results from phosphorylation of Tyr-493 by Lck and autophosphorylation of Tyr-319 after tandem interactions of the ZAP-70 SH2 domains with immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3 (21,28). Consistent with the reported sequential phosphorylation of ZAP-70 and CD3 , we observed maximal phosphorylation of Tyr-493 and Tyr-319 of ZAP-70 to occur 1 min after maximal CD3 phosphorylation (Fig.…”

Section: Discussionsupporting

confidence: 85%

Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

Salomon

Ficarro

Brill

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

271

229

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The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography͞ mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.M any cellular processes are directly controlled through the reversible phosphorylation of protein tyrosine residues. These regulatory functions are ultimately affected through the coordinated phosphorylation of numerous tyrosine residues across multiple proteins over time. Clearly, there are benefits to individually characterizing specific components of a particular pathway, such as identifying a site of phosphorylation on a given protein, the kinase responsible for the modification, or the identity of subsequently interacting proteins. Ultimately, though, a thorough understanding of these signaling pathways at the molecular level requires the wide-scale, simultaneous evaluation of these phosphorylation events as they occur over time.To date, two-dimensional gel electrophoresis (2D-GE) remains the most common methodology for assessing wide-scale changes in phosphorylation (1). However, this methodology is relatively slow, and suffers from a number of well documented operational limitations. For example, 2D-GE has been shown to be poorly suited for the direct detection and analysis of medium to low abundance proteins from whole cell lysates, a particular concern in the case of regulatory proteins such as kinases, which often exist at very low copy numbers per cell (2). Even with the improved dynamic range afforded by multiple 2D-GE runs of prefractionated samples, the individual gelisolated proteins still require further characterization by using methods such as two-dimensional tryptic phosphopeptide mapping (3), Edman degradation (4), or precursor ion scan...

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Section: Discussionsupporting

confidence: 85%

Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

Salomon

Ficarro

Brill

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

271

229

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“…Mouse 13B8.2 Ab inhibits soluble gp120 binding to soluble and membrane CD4, but fails to block either gp120-bearing virion binding to CD4 or HIV entry (54), thus suggesting that 13B8.2 and gp120 signaling could be closely related but not equivalent. This argument is correlated with the fact that gp120, like rIgG 1 13B8.2, induces CD4 raft compartmentalization, as demonstrated by us and others (55), but preferentially triggers phosphorylation of signalactivating Zap70 Tyr 319 and Tyr 493 residues (41,42,56), rather than phosphorylating the signal-inhibiting Zap70 Tyr 292 residue (57), which we observed upon rIgG 1 13B8.2 binding. rIgG 1 13B8.2 drives the gp120-induced phosphorylation pattern toward a Zap70-negative regulation pathway, explaining why anti-CD4 13B8.2 Ab treatment induces inhibition of NF-B activation and transcription inhibition of the viral genome, whereas gp120 binding leads to NF-B activation and productive transcription of the viral genome in the case of HIV infection (23).…”

Section: Discussionsupporting

confidence: 82%

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

Chentouf

Ghannam

Bès

et al. 2007

The Journal of Immunology

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The biological effects of rIgG1 13B8.2, directed against the CDR3-like loop on the D1 domain of CD4, are partly due to signals that prevent NF-κB nuclear translocation, but the precise mechanisms of action, particularly at the level of membrane proximal signaling, remain obscure. We support the hypothesis that rIgG1 13B8.2 acts by interfering with the spatiotemporal distribution of signaling or receptor molecules inside membrane rafts. Upon cross-linking of Jurkat T lymphocytes, rIgG1 13B8.2 was found to induce an accumulation/retention of the CD4 molecule inside polyoxyethylene-20 ether Brij 98 detergent-resistant membranes at 37°C, together with recruitment of TCR, CD3ζ, p56 Lck, Lyn, and Syk p70 kinases, linker for activation of T cells, and Csk-binding protein/phosphoprotein associated with glycosphingolipid adaptor proteins, and protein kinase Cθ, but excluded Zap70 and its downstream targets Src homology 2-domain-containing leukocyte protein of 76 kDa, phospholipase Cγ1, and p95vav. Analysis of key upstream events such as Zap70 phosphorylation showed that modulation of Tyr292 and Tyr319 phosphorylation occurred concomitantly with 13B8.2-induced Zap70 exclusion from the membrane rafts. 13B8.2-induced differential raft partitioning was epitope, cholesterol, and actin dependent but did not require Ab hyper-cross-linking. Fluorescence confocal imaging confirmed the spatiotemporal segregation of the CD4 complex inside rafts and concomitant Zap70 exclusion, which occurred within 10–30 s following rIgG1 13B8.2 ligation, reached a plateau at 1 min, and persisted until the end of the 1-h experiment. The differential spatiotemporal partitioning between the CD4 receptor and the Zap70-signaling kinase inside membrane rafts interrupts the proximal signal cross-talk leading to subsequent NF-κB nuclear translocation and explains how baculovirus-expressed CD4-CDR3-like-specific rIgG1 13B8.2 acts to induce its biological effects.

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“…That ZAP-70 is directly linked to these disease states shows the need for very tight regulation of ZAP-70 and suggests ZAP-70 as a therapeutic target for pharmacologic intervention. Thus, a detailed understanding at the molecular level of the regulation of catalytic activity of ZAP-70 is of considerable interest.Several tyrosine phosphorylation sites in ZAP-70 have been identified that serve a role in its regulation (12)(13)(14)(15)(16)(17)(18). Two of these tyrosine residues, Tyr-492 and Tyr-493 in human ZAP-70, are positioned in the activation loop of the catalytic domain, and their phosphorylation either by Lck (13,15) or by ZAP-70 itself in trans (17) contributes to the activation of ZAP-70 by stabilizing the open and extended conformation of the activation loop.…”

mentioning

confidence: 99%

“…Two of these tyrosine residues, Tyr-492 and Tyr-493 in human ZAP-70, are positioned in the activation loop of the catalytic domain, and their phosphorylation either by Lck (13,15) or by ZAP-70 itself in trans (17) contributes to the activation of ZAP-70 by stabilizing the open and extended conformation of the activation loop. Two regulatory tyrosine residues of the SH2-kinase linker, Tyr-315 and Try319, are known to become phosphorylated (16,17,19) when ZAP-70 is recruited to the TCR and have been implicated in an autoinhibitory mechanism controlling the catalytic activity of 16,17,[19][20][21][22].…”

mentioning

confidence: 99%

Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response

Deindl

Kadlecek

Cao

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The delivery of signals from the activated T cell antigen receptor (TCR) inside the cell relies on the protein tyrosine kinase ZAP-70 (-associated protein of 70 kDa). A recent crystal structure of inactive full-length ZAP-70 suggests that a central interface formed by the docking of the two SH2 domains of ZAP-70 onto the kinase domain is crucial for suppressing catalytic activity. Here we validate the significance of this autoinhibitory interface for the regulation of ZAP-70 catalytic activity and the T cell response. For this purpose, we perform in vitro catalytic activity assays and binding experiments using ZAP-70 proteins purified from insect cells to examine activation of ZAP-70. Furthermore, we use cell lines stably expressing wild-type or mutant ZAP-70 to monitor proximal events in T cell signaling, including TCR-induced phosphorylation of ZAP-70 substrates, activation of the MAP kinase pathway, and intracellular Ca 2؉ levels. Taken together, our results directly correlate the stability of the autoinhibitory interface with the activation of these key events in the T cell response.activation ͉ catalytic activity ͉ conformation ͉ ITAM T he protein tyrosine kinase ZAP-70 (-associated protein of 70 kDa) (1) participates in the initial events of T cell antigen receptor (TCR) signaling and thus is of critical importance for the adaptive immune response (2-5). On activation of the TCR, 2 tyrosines in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 or cytoplasmic tails of the TCR are phosphorylated by a Src family kinase member. Through the binding of 2 SH2 (Src homology 2) domains at the N terminus of ZAP-70 to these ITAM phosphotyrosines, ZAP-70 is then specifically recruited to a stimulated TCR. These 2 SH2 domains of ZAP-70 are tightly associated as a tandem unit with a precise structural organization that enables high-affinity ITAM peptide binding (6). In the very closely related Syk (spleen tyrosine) kinase, the destabilization of this structural organization by mutation results in both uncoupling of the 2 SH2 units and a dramatic reduction in ITAM binding affinity (7). Recruitment of ZAP-70 to the TCR results in activation of the catalytic domain located at the C terminus of ZAP-70 and signal propagation by phosphorylation of its downstream target molecules, including the ZAP-70 substrates LAT (linker for the activation of T cells) and SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kDa). Both LAT and SLP-76 are crucial adaptor proteins that, once phosphorylated, provide a platform for the assembly of signaling molecules (8, 9). Ultimately, these signaling events produce effector secretory responses, modulate transcription, and trigger T cell proliferation and differentiation.ZAP-70 plays critical roles in pre-TCR and TCR signaling, thymocyte development, and peripheral T cell function. The loss of ZAP-70 catalytic activity results in profound immunodeficiency, and reduced activity of ZAP-70 may result in autoimmunity (4, 10). Furthermore, the expression of ZAP-70 in B c...

show abstract

Tyrosine 319, a Newly Identified Phosphorylation Site of ZAP-70, Plays a Critical Role in T Cell Antigen Receptor Signaling

Cited by 133 publications

References 50 publications

Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry

Recombinant Anti-CD4 Antibody 13B8.2 Blocks Membrane-Proximal Events by Excluding the Zap70 Molecule and Downstream Targets SLP-76, PLCγ1, and Vav-1 from the CD4-Segregated Brij 98 Detergent-Resistant Raft Domains

Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response

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