Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine kinase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, presumably by two mechanisms: an autophosphorylation and a trans-phosphorylation by the Src-family PTK Lck. These events have been implicated in both positive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr315 and Tyr319 in the interdomain B of ZAP-70 are autophosphorylated in vitro and become phosphorylated in vivo upon TCR triggering. Moreover, by mutational analysis, we demonstrate that phosphorylation of Tyr319 is required for the positive regulation of ZAP-70 function. Indeed, overexpression in Jurkat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr319 was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired anti-TCR-induced activation of the nuclear factor of activated T cells and interleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr315 had little or no effect. The inhibitory effect of ZAP-70-Y319F correlated with a substantial loss of its activation-induced tyrosine phosphorylation and up-regulation of catalytic activity, as well as with a decreased in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-76 and LAT. Collectively, our data reveal the pivotal role of Tyr319 phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.     

Interdomain B in ZAP-70 Regulates but Is Not Required for ZAP-70 Signaling Function in Lymphocytes

The protein tyrosine kinase ZAP-70 plays an important role in T-cell activation and development. After T-cell receptor stimulation, ZAP-70 associates with the receptor and is phosphorylated on many tyrosines, including Y292, Y315, and Y319 within interdomain B. Previously, we demonstrated that Y292 negatively regulates ZAP-70 function and that Y315 positively regulates ZAP-70 function by interacting with Vav. Recent studies have suggested that Y319 also positively regulate ZAP-70 function. Paradoxically, removal of interdomain B (to create the construct designated Δ), containing the Y292, Y315, and Y319 sites, did not eliminate the ability of ZAP-70 to induce multiple gene reporters in Syk-deficient DT-40 B cells and ZAP-70/Syk-deficient Jurkat cells. Here we show that Δ still utilizes the same pathways as wild-type ZAP-70 to mediate NF-AT induction. This is manifested by the ability of Δ to restore induction of calcium fluxes and mitogen-activated protein kinase activation and by the ability of dominant negative Ras and FK506 to block the induction of NF-AT activity mediated by Δ. Biochemically we show that the stimulated tyrosine phosphorylation of Vav, Shc, and ZAP-70 itself is diminished, whereas that of Slp-76 is increased in cells reconstituted with Δ. Deletion of interdomain B did not affect the ability of ZAP-70 to bind to the receptor. The in vitro kinase activity of ZAP-70 lacking interdomain B was markedly reduced, but the kinase activity was still required for the protein’s in vivo activity. Based on these data, we concluded that interdomain B regulates but is not required for ZAP-70 signaling function leading to cellular responses.     

Role of the Src homology 2 domains and interdomain regions in ZAP-70 phosphorylation and enzymatic activity.

The protein tyrosine kinase ZAP-70, which mediates T-cell antigen receptor (TCR) signalling, contains three distinct functional modules, two tandemly arranged SH2 domains, a kinase domain and a linker region (interdomain B) that connects them. ZAP-70 enzymatic activation is strictly dependent on the binding, via its SH2 domains, to the triggered TCR and on tyrosine phosphorylation. Here we utilized recombinant ZAP-70 and carried out a mutational analysis to understand the structural requirements for its activation. We show that deletion of both SH2 domains corresponding to the first 254 residues moderately increases ZAP-70 enzymatic activity on an exogenous substrate in vitro, results in increased tyrosine phosphorylation and produces subtle conformational changes, as judged by altered SDS/PAGE migration. Mutation of Tyr292, 315 and 319 to Phe in the interdomain B region, which constitute the major phosphorylation sites both in vitro and in vivo, did not affect ZAP-70 enzymatic activity. Moreover, deletion analysis of the interdomain B region established residues 320-619 as a minimal region endowed with full kinase activity. We propose that binding of ZAP-70 to the TCR promotes, through conformational changes, its extensive phosphorylation on tyrosine. However, Tyr292, 315 and 319 do not affect ZAP-70 enzymatic activity and may influence ZAP-70 signalling only indirectly by mediating its association with intracellular transducers.     

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.     

Activation of Zap-70 tyrosine kinase due to a structural rearrangement induced by tyrosine phosphorylation and/or ITAM binding

The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR zeta chain through its two src-homology 2 (SH2) domains results in its activation coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely understood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activation in a spectrophotometric substrate phosphorylation assay. Under these conditions, we observed a lag phase of several minutes before full ZAP-70 activation, which was not observed using a truncated form lacking the first 254 residues, suggesting that it might be due to an intramolecular interaction involving the interdomain A and SH2 region. Accordingly, the lag phase could be reproduced by testing the truncated form in the presence of recombinant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished the lag phase and resulted in a more active enzyme. The same results were obtained using a ZAP-70 mutant lacking the interdomain B tyrosines. These findings are consistent with a mechanism in which ZAP-70 phosphorylation/autophosphorylation on tyrosine(s) other than 292, 315, and 319, as well as engagement of the SH2 domains by the phosphorylated TCR, can induce a conformational change leading to accelerated enzyme kinetics and higher catalytic efficiency.     

Cutting edge: T cell migration regulated by CXCR4 chemokine receptor signaling to ZAP-70 tyrosine kinase

Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. The signaling pathways by which chemokine receptors regulate lymphocyte migration remain incompletely characterized. We demonstrate that Jurkat T cells lacking the ZAP-70 tyrosine kinase exhibit reduced migration in response to the CXCR4 ligand CXCL12 when compared with wild-type Jurkat T cells. Expression of wild-type, but not kinase-inactive, ZAP-70 resulted in enhanced migration of ZAP-70-deficient Jurkat T cells. The tyrosine residue at position 292 in the interdomain B region of ZAP-70 exerts a negative regulatory effect on ZAP-70-dependent migration. Stimulation of Jurkat T cells with CXCL12 also resulted in ZAP-70-dependent tyrosine phosphorylation of the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) adapter protein. Although CXCL12-dependent migration of SLP-76-deficient Jurkat T cells was impaired, re-expression of SLP-76 did not enhance migration. These results suggest a novel function for ZAP-70, but not SLP-76, in CXCR4 chemokine receptor signaling in human T cells.     

Phosphorylation of Tyr342 in the linker region of Syk is critical for Fc epsilon RI signaling in mast cells

The linker region of Syk and ZAP70 tyrosine kinases plays an important role in regulating their function. There are three conserved tyrosines in this linker region; Tyr317 of Syk and its equivalent residue in ZAP70 were previously shown to negatively regulate the function of Syk and ZAP70. Here we studied the roles of the other two tyrosines, Tyr342 and Tyr346 of Syk, in Fc epsilon RI-mediated signaling. Antigen stimulation resulted in Tyr342 phosphorylation in mast cells. Syk with Y342F mutation failed to reconstitute Fc epsilon RI-initiated histamine release. In the Syk Y342F-expressing cells there was dramatically impaired receptor-induced phosphorylation of multiple signaling molecules, including LAT, SLP-76, phospholipase C-gamma2, but not Vav. Compared to wild-type Syk, Y342F Syk had decreased binding to phosphorylated immunoreceptor tyrosine-based activation motifs and reduced kinase activity. Surprisingly, mutation of Tyr346 had much less effect on Fc epsilon RI-dependent mast cell degranulation. An anti-Syk-phospho-346 tyrosine antibody indicated that antigen stimulation induced only a very minor increase in the phosphorylation of this tyrosine. Therefore, Tyr342, but not Tyr346, is critical for regulating Syk in mast cells and the function of these tyrosines in immune receptor signaling appears to be different from what has been previously reported for the equivalent residues of ZAP70.     

Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.     

Zap-70-independent Ca(2+) mobilization and Erk activation in Jurkat T cells in response to T-cell antigen receptor ligation

The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-gamma1 (PLCgamma1) and increased the intracellular Ca(2+) concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCgamma1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.     

CTLA-4 suppresses proximal TCR signaling in resting human CD4(+) T cells by inhibiting ZAP-70 Tyr(319) phosphorylation: a potential role for tyrosine phosphatases

The balance between positive and negative signals plays a key role in determining T cell function. CTL-associated Ag-4 is a surface receptor that can inhibit T cell responses induced upon stimulation of the TCR and its CD28 coreceptor. Little is known regarding the signaling mechanisms elicited by CTLA-4. In this study we analyzed CTLA-4-mediated inhibition of TCR signaling in primary resting human CD4(+) T cells displaying low, but detectable, CTLA-4 cell surface expression. CTLA-4 coligation with the TCR resulted in reduced downstream protein tyrosine phosphorylation of signaling effectors and a striking inhibition of extracellular signal-regulated kinase 1/2 activation. Analysis of proximal TCR signaling revealed that TCR zeta-chain phosphorylation and subsequent zeta-associated protein of 70 kDa (ZAP-70) tyrosine kinase recruitment were not significantly affected by CTLA-4 engagement. However, the association of p56(lck) with ZAP-70 was inhibited following CTLA-4 ligation, correlating with reduced actions of p56(lck) in the ZAP-70 immunocomplex. Moreover, CTLA-4 ligation caused the selective inhibition of CD3-mediated phosphorylation of the positive regulatory ZAP-70 Y319 site. In addition, we demonstrate protein tyrosine phosphatase activity associated with the phosphorylated CTLA-4 cytoplasmic tail. The major phosphatase activity was attributed to Src homology protein 2 domain-containing tyrosine phosphatase 1, a protein tyrosine phosphatase that has been shown to be a negative regulator of multiple signaling pathways in hemopoietic cells. Collectively, our findings suggest that CTLA-4 can act early during the immune response to regulate the threshold of T cell activation.     

Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivo substrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.     

T cell activation-induced CrkII binding to the Zap70 protein tyrosine kinase is mediated by Lck-dependent phosphorylation of Zap70 tyrosine 315

The Zap70 protein tyrosine kinase controls TCR-linked signal transduction pathways and is critical for T cell development and responsiveness. Following engagement of TCR, the Zap70 undergoes phosphorylation on multiple tyrosine residues that are implicated in the regulation of its catalytic activity and interaction with signaling effector molecules downstream of the TCR. We have shown previously that the CT10 regulator of kinase II (CrkII) adapter protein interacts with tyrosine-phosphorylated Zap70 in TCR-engaged T cells, and now extend these studies to show that Tyr315 in the Zap70 interdomain B region is the site of interaction with CrkII. A point mutation of Tyr315 (Y315F) eliminated the CrkII-Zap70 interaction capacity. Phosphorylation of Tyr315 and Zap70 association with CrkII were both dependent upon the Lck protein tyrosine kinase. Previous studies demonstrated the Tyr315 is the Vav-Src homology 2 (SH2) binding site, and that replacement of Tyr315 by Phe impaired the function of Zap70 in TCR signaling. However, fluorescence polarization-based binding studies revealed that the CrkII-SH2 and the Vav-SH2 bind a phosphorylated Tyr315-Zap70-derived peptide with affinities of a similar order of magnitude (Kd of 2.5 and 1.02 microM, respectively). The results suggest therefore that the biological functions attributed to the association of Zap70 with Vav following T cell activation may equally reflect the association of Zap70 with CrkII, and further support a regulatory role for CrkII in the TCR-linked signal transduction pathway.     

T-cell antigen receptor ligation induces tyrosine phosphorylation of phospholipase C-gamma 1

Ligand-mediated perturbation of the T-cell antigen receptor (TCR) triggers a rapid increase in phosphoinositide-specific phospholipase C (PLC) activity in resting T-cells. Although the mechanism by which TCR ligation regulates PLC activity is unknown, recent studies suggest that coupling of this receptor complex to PLC activity is dependent on an intermediate protein tyrosine phosphorylation event(s). In the present study, we demonstrate that antibody-mediated TCR cross-linkage results in the tyrosine phosphorylation of PLC-gamma 1. Stimulation of the TCR for 30 s induced a 4-5-fold increase in the level of PLC activity recovered in anti-phosphotyrosine (Tyr(P)) antibody immunoprecipitates from stimulated Jurkat cells. The appearance of PLC activity in the immunoprecipitates preceded the onset of phosphoinositide hydrolysis in vivo, which began 30-60 s after TCR ligation. Furthermore, the TCR-mediated increase in anti-Tyr(P) antibody-bound PLC activity was inhibited by staurosporine at drug concentrations identical with those required for in vivo inhibition of TCR-dependent phosphoinositide breakdown. Immunoblot analyses demonstrated that TCR ligation dramatically increased the level of tyrosine-phosphorylated PLC-gamma 1 present in anti-Tyr(P) antibody immunoprecipitates from stimulated Jurkat cells. These results strongly suggest that the TCR complex expressed by Jurkat cells is functionally coupled to the phosphoinositide-dependent signaling pathway through the tyrosine phosphorylation of PLC-gamma 1.     

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.     

Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.