T cell receptor–dependent S-acylation of ZAP-70 controls activation of T cells

Abstract

ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity. ZAP-70 is a tyrosine kinase essential for T cell immune responses. Upon engagement of the T cell receptor (TCR), ZAP-70 is recruited to the specialized plasma membrane domains, becomes activated, and is released to phosphorylate its laterally segregated targets. A shift in ZAP-70 distribution at the plasma membrane is recognized as a critical step in TCR signal transduction and amplification. However, the molecular mechanism supporting stimulation-dependent plasma membrane compartmentalization of ZAP-70 remains poorly understood. In this study, we identified previously uncharacterized lipidation (S-acylation) of ZAP-70 using Acyl-Biotin Exchange assay, a technique that selectively captures S-acylated proteins. We found that this posttranslational modification of ZAP-70 is dispensable for its enzymatic activity. However, the lipidation-deficient mutant of ZAP-70 failed to propagate the TCR pathway suggesting that S-acylation is essential for ZAP-70 interaction with its protein substrates. The kinetics of ZAP-70 S-acylation were consistent with TCR signaling events indicating that agonist-induced S-acylation is a part of the signaling mechanism controlling T cell activation and function. Taken together, our results suggest that TCR-induced S-acylation of ZAP-70 can serve as a critical regulator of T cell-mediated immunity. ZAP-70 (“CD3ζ-chain-associated protein kinase 70”) is one of the first proteins activated upon T cell receptor (TCR) engagement by the peptide/major histocompatibility complex on the surface the antigen-presenting cell (1Brownlie R.J. Zamoyska R. T cell receptor signalling networks: Branched, diversified and bounded.Nat. Rev. Immunol. 2013; 13: 257-269Crossref PubMed Scopus (276) Google Scholar). Stimulation of the TCR leads to activation of the Src-family kinase Lck, which then phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) of TCR-associated CD3ζ-chain (1Brownlie R.J. Zamoyska R. T cell receptor signalling networks: Branched, diversified and bounded.Nat. Rev. Immunol. 2013; 13: 257-269Crossref PubMed Scopus (276) Google Scholar, 2Salmond R.J. Filby A. Qureshi I. Caserta S. Zamoyska R. T-cell receptor proximal signaling via the Src-family kinases, Lck and Fyn, influences T-cell activation, differentiation, and tolerance.Immunol. Rev. 2009; 228: 9-22Crossref PubMed Scopus (245) Google Scholar). Double phosphorylation of ITAMs creates a high-affinity docking site for Src homology 2 (SH2) domains of ZAP-70 resulting in its recruitment to the TCR–CD3 complex. Binding to ITAMs triggers conformational changes that make ZAP-70 more accessible to phosphorylation by Lck and subsequently leads to ZAP-70 autophosphorylation and full activation (3Wang H. Kadlecek T.A. Au-Yeung B.B. Goodfellow H.E.S. Hsu L.Y. Freedman T.S. Weiss A. ZAP-70: An essential kinase in T-cell signaling.Cold Spring Harb. Perspect. Biol. 2010; 2: 1-17Crossref Scopus (203) Google Scholar, 4Hsu L.Y. Cheng D.A. Chen Y. Liang H.E. Weiss A. Destabilizing the autoinhibitory conformation of Zap70 induces up-regulation of inhibitory receptors and T cell unresponsiveness.J. Exp. Med. 2017; 214: 833-849Crossref PubMed Scopus (6) Google Scholar, 5Au-Yeung B.B. Shah N.H. Shen L. Weiss A. ZAP-70 in signaling, Biology, and disease.Annu. Rev. Immunol. 2018; 36: 127-156Crossref PubMed Scopus (32) Google Scholar). Activated ZAP-70 is released from ITAMs and proceeds to target its effectors, primarily membrane-bound scaffolding proteins LAT and SLP-76 (6Su X. Ditlev J.A. Hui E. Xing W. Banjade S. Okrut J. King D.S. Taunton J. Rosen M.K. Vale R.D. Phase separation of signaling molecules promotes T cell receptor signal transduction.Science. 2016; 352: 595-599Crossref PubMed Scopus (379) Google Scholar). Phosphorylation of LAT and SLP-76 nucleates the assembly of the multiprotein signaling complex, which further propagates the downstream TCR signaling cascade resulting in activation of the phospholipase PLC-γ1, cytoplasmic calcium release from endoplasmic reticulum, and, ultimately, transcriptional responses associated with T cell activation and clonal expansion (6Su X. Ditlev J.A. Hui E. Xing W. Banjade S. Okrut J. King D.S. Taunton J. Rosen M.K. Vale R.D. Phase separation of signaling molecules promotes T cell receptor signal transduction.Science. 2016; 352: 595-599Crossref PubMed Scopus (379) Google Scholar, 7Stathopulos P.B. Zheng L. Li G.-Y. Plevin M.J. Ikura M. Structural and mechanistic insights into STIM1-mediated initiation of store-operated calcium entry.Cell. 2008; 135: 110-122Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 8Smith-Garvin J.E. Koretzky G.A. Jordan M.S. T cell activation.Annu. Rev. Immunol. 2009; 27: 591-619Crossref PubMed Scopus (1186) Google Scholar, 9Katz Z.B. Novotná L. Blount A. Lillemeier B.F. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.Nat. Immunol. 2017; 18: 86-95Crossref PubMed Scopus (43) Google Scholar). Thus, the signaling function of the ZAP-70 kinase relies on two temporally and spatially segregated events. During the first step, the phosphorylated TCR–CD3 complex amplifies the initial antigenic signal through repeated cycles of ZAP-70 recruitment, activation, and release (9Katz Z.B. Novotná L. Blount A. Lillemeier B.F. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.Nat. Immunol. 2017; 18: 86-95Crossref PubMed Scopus (43) Google Scholar). Through the next step, activated ZAP-70 translocates into distinct plasma membrane domains where it can access its protein substrates (9Katz Z.B. Novotná L. Blount A. Lillemeier B.F. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.Nat. Immunol. 2017; 18: 86-95Crossref PubMed Scopus (43) Google Scholar, 10Lillemeier B.F. Mörtelmaier M.A. Forstner M.B. Huppa J.B. Groves J.T. Davis M.M. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.Nat. Immunol. 2010; 11: 90-96Crossref PubMed Scopus (441) Google Scholar, 11Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (534) Google Scholar). This compartmentalization of early phosphorylation events not only ensures rapid TCR signal amplification and distribution but can also help to avoid premature or unspecific T cell activation. It is not clear, however, what mechanism prevents activated and decoupled ZAP-70 from being dispersed back into the cytoplasm and promotes its prompt translocation to the laterally segregated downstream targets. It has been proposed that both plasma membrane anchoring and lateral distribution of proteins within the plasma membrane can be mediated by S-acylation—reversible lipidation of cysteine thiols with long-chain fatty acids (12Chamberlain L.H. Shipston M.J. The physiology of protein S-acylation.Physiol. Rev. 2015; 95: 341-376Crossref PubMed Scopus (149) Google Scholar, 13Levental I. Grzybek M. Simons K. Raft domains of variable properties and compositions in plasma membrane vesicles.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 11411-11416Crossref PubMed Scopus (156) Google Scholar). In particular, this posttranslational modification (also referred to as palmitoylation) was found to be essential for biological activity of two immediate ZAP-70 effectors, Lck and LAT (14Kabouridis P.S. Magee A.I. Ley S.C. S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes.EMBO J. 1997; 16: 4983-4998Crossref PubMed Scopus (308) Google Scholar, 15Levental I. Lingwood D. Grzybek M. Coskun U. Simons K. Palmitoylation regulates raft affinity for the majority of integral raft proteins.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 22050-22054Crossref PubMed Scopus (344) Google Scholar). It has been shown that, although the acylation-deficient mutant of Lck was still catalytically active, it was unable to phosphorylate ITAMs and failed to activate ZAP-70, thus leading to abrogated TCR signaling (14Kabouridis P.S. Magee A.I. Ley S.C. S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes.EMBO J. 1997; 16: 4983-4998Crossref PubMed Scopus (308) Google Scholar, 16Kosugi A. Hayashi F. Liddicoat D.R. Yasuda K. Saitoh S. Hamaoka T. A pivotal role of cysteine 3 of Lck tyrosine kinase for localization to glycolipid-enriched microdomains and T cell activation.Culture. 2001; 76: 133-138Google Scholar, 17Yurchak L.K. Sefton B.M. Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase.Microbiology. 1995; 15: 6914-6922Google Scholar). Furthermore, our that initiation of the receptor signaling pathway in T relies on rapid agonist-induced changes in Lck S-acylation and of this modification T to D. and of the tyrosine kinase Lck Natl. Acad. Sci. U. S. A. 2015; PubMed Scopus Google Scholar). S-acylation of LAT was found to be essential for its to the TCR signaling and of downstream signaling phosphorylation of PLC-γ1, calcium and activation of I. Lingwood D. Grzybek M. Coskun U. Simons K. Palmitoylation regulates raft affinity for the majority of integral raft proteins.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 22050-22054Crossref PubMed Scopus (344) Google Scholar, M. H. M. Hayashi K. Y. R. L. Y. M. A. S. J. T. activation and localization of LAT in T as a of a Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. L. W. A. plasma membrane but signaling by for activation of T Immunol. 2009; PubMed Scopus Google Scholar, J. Weiss A. T.S. of LAT in glycolipid-enriched microdomains is required for T cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. of membrane protein LAT in T cell receptor signaling Biol. PubMed Scopus Google Scholar). In this study, we previously S-acylation of We found that this posttranslational modification is TCR and identified the cysteine in as the site of ZAP-70 of this cysteine not the of the kinase but to interaction of ZAP-70 with its kinase Lck, resulting in ZAP-70 phosphorylation However, the acylation-deficient mutant of ZAP-70 failed to phosphorylate its downstream LAT and of the TCR signaling our a previously uncharacterized posttranslational modification of ZAP-70 that as a critical regulator of its signaling function in T cell activation. We first identified ZAP-70 as S-acylated protein in our initial at the of proteins in the proximal TCR signaling T cell protein we the Acyl-Biotin Exchange assay, a that for rapid and of S-acylated proteins and of protein 36: PubMed Google Scholar). in proteins in T cell were with by of with The were then and using and the proteins were by This in with a of the proximal TCR signaling to previously uncharacterized S-acylation of TCR signaling protein phospholipase PLC-γ1, and tyrosine kinase ZAP-70 in both cell and T A and that this posttranslational modification is more and to a role of S-acylation in the of the ZAP-70 signaling function. next was to the site of ZAP-70 the cysteine we ZAP-70 protein using J. L. X. Y. X. An for 2008; PubMed Scopus Google Scholar). on the two cysteine at and of being to the of we that in the part of the protein is the site of S-acylation The was further by a that a of this was associated with in a T. I. H. S. I. M. can the of with J. 2009; PubMed Scopus Google Scholar). S-acylation of we T to T cell ZAP-70 either or of in and the assay, we found that the of the cysteine by or in of ZAP-70 S-acylation and and indicating that is required for this posttranslational S-acylation of ZAP-70 was in T the of early TCR signaling, we that of the TCR can rapid changes in ZAP-70 S-acylation this we T with to activate the TCR signaling pathway and the kinetics of ZAP-70 lipidation using the We found that T cell in a but in S-acylation of ZAP-70, at 2 TCR engagement and proximal signaling Lck, kinetics with more S-acylation to indicating distinct enzymatic of its In to in S-acylation of ZAP-70 on in cytoplasmic we a using T in calcium release of phospholipase to T-cell of a T-cell Biol. PubMed Scopus Google Scholar). We found that of calcium signaling not of ZAP-70 but the TCR-induced changes in ZAP-70 lipidation were in this cell we found that the in ZAP-70 S-acylation upon TCR the phosphorylation of ZAP-70 and TCR signaling proteins suggesting that to protein S-acylation can be by TCR engagement and calcium A of ZAP-70 cysteine in to was associated with a of in a however, the molecular mechanism this of The within the part of the ZAP-70 kinase suggesting that of a in the of the ZAP-70 its kinase function. this we in kinase to the can the of ZAP-70 to phosphorylate its protein the phosphorylation ZAP-70 was from either or ZAP-70 and with SLP-76 phosphorylation of we found that the not of ZAP-70 kinase activity as from phosphorylation of SLP-76 at and Thus, that the acylation-deficient mutant of ZAP-70 is catalytically the acylation-deficient mutant of ZAP-70 the to phosphorylate its we that of protein lipidation still ZAP-70 activation by its recruitment from the to the plasma membrane and interaction with the of S-acylation on plasma membrane localization of ZAP-70, we We with ZAP-70 and the of proteins in the of and T We found that both and ZAP-70 localization by laterally at the plasma membrane suggesting that S-acylation is not required for ZAP-70 translocation from the to the plasma further the plasma membrane of the acylation-deficient mutant of ZAP-70, we the with our the ZAP-70 mutant a with the membrane in both and activated The of the mutant of ZAP-70 at the plasma membrane that the acylation-deficient ZAP-70 can still with its S-acylation of ZAP-70 influences the interaction with its we Lck from or ZAP-70 in the Lck 2 of the TCR by but the proteins upon cell It is that we that of S-acylation in a more interaction ZAP-70 and shown in we found of Lck and ZAP-70 in both and activated cell TCR-induced recruitment of ZAP-70 to the plasma membrane and to Lck phosphorylation of the ZAP-70 tyrosine and within the and the tyrosine and in the activation of the T. Kadlecek T. a Weiss A. in ZAP-70: with receptor tyrosine Biol. 2005; PubMed Scopus Google Scholar). more interaction of ZAP-70 with Lck its we to ZAP-70 and the phosphorylation of and by with more of ZAP-70 with Lck, phosphorylation of the acylation-deficient ZAP-70 was by with and and we also that, being phosphorylated at both and the acylation-deficient mutant of ZAP-70 failed to propagate the TCR signaling pathway as from the phosphorylation of proximal TCR signaling proteins and suggest that S-acylation of ZAP-70 its into the plasma membrane domains where it can with its downstream targets. further the of ZAP-70 to TCR signal we with ZAP-70 with and early signaling events leading to T cell activation. shown in and of ZAP-70 phosphorylation of ZAP-70 LAT and leading to activation of downstream of the proximal TCR and In we found that the acylation-deficient mutant of ZAP-70 failed to phosphorylate its resulting in of the TCR signaling cascade and of the ZAP-70 not activation of Src-family Lck and of TCR signaling was also in acylation-deficient mutant of ZAP-70 of the T cell signaling events on calcium release from the M. A. signaling in Immunol. 2008; PubMed Scopus (276) Google Scholar, X. J. D. T-cell receptor complex is essential for signal Natl. Acad. Sci. U. S. A. 2010; 107: PubMed Scopus Google Scholar). with activation of PLC-γ1, we not TCR-induced cytoplasmic calcium in either or mutant of ZAP-70 and and on we that S-acylation of ZAP-70 is essential for the activation of the proximal TCR signaling We next to of the early TCR signaling events in acylation-deficient ZAP-70 leads to T cell activation. the T cell transcriptional we ZAP-70 with for and to of a essential for T cell activation and We found that, in to ZAP-70, of ZAP-70 in in of in to TCR our that of ZAP-70 S-acylation to of and cell activation on the surface of we of and in the acylation-deficient mutant of ZAP-70 and The in T cell activation in was by a of and indicating that the signaling downstream of ZAP-70 were not that S-acylation of ZAP-70 is essential for T cell activation. A cysteine of ZAP-70 was to be associated with in a T. I. H. S. I. M. can the of with J. 2009; PubMed Scopus Google Scholar). However, the molecular mechanism this of In this study, we identified as the site of a posttranslational modification as protein and of the that the creates the acylation-deficient of ZAP-70 that is unable to early TCR signal transduction events resulting in T cell activation. S-acylation of ZAP-70 was in T however, we found that engagement of the TCR triggers changes in ZAP-70 lipidation We a rapid in ZAP-70 S-acylation 2 of TCR by a to This rapid and of ZAP-70 S-acylation the phosphorylation kinetics of proximal TCR signaling ZAP-70 Thus, suggest that S-acylation of ZAP-70 a role during initiation of early TCR signaling events and that the controlling ZAP-70 S-acylation are part of the proximal TCR signaling the of ZAP-70 we to the acylation-deficient ZAP-70, is to activation of the TCR signaling However, we were that of the the protein resulting in ZAP-70 to or premature the within the part of the kinase a more by the of ZAP-70 to its protein substrates. We both by ZAP-70 in and in kinase in which SLP-76 protein was to ZAP-70 This that ZAP-70 the to phosphorylate its indicating that this not in protein This was further by our that the mutant can still be to the plasma membrane and phosphorylated by Lck of the cysteine in the by on ZAP-70 signaling function as from the of mutant to T cell activation in ZAP-70 Furthermore, we found that of by also in a ZAP-70 of cytoplasmic calcium TCR and of TCR signaling and Thus, suggest that the of the mutant in our was by of S-acylation kinase activity. We found that the S-acylation of ZAP-70 is critical for its signaling function in the proximal TCR being catalytically active, the acylation-deficient mutant of ZAP-70 the TCR signaling cascade by to phosphorylate its LAT and thus calcium responses and downstream signaling and T cell activation. The acylation-deficient ZAP-70 was unable to phosphorylate its substrates is not our initial of S-acylation not the interaction ZAP-70 and its kinase Lck, but resulting in a phosphorylation at and and, of activated ZAP-70 A by that activated ZAP-70 is by Lck at the TCR–CD3 complex during events that to amplification of the initial TCR signal (9Katz Z.B. Novotná L. Blount A. Lillemeier B.F. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.Nat. Immunol. 2017; 18: 86-95Crossref PubMed Scopus (43) Google Scholar). Upon release from the the activated ZAP-70 kinase is at the plasma membrane and translocates into the spatially segregated domains its substrates (9Katz Z.B. Novotná L. Blount A. Lillemeier B.F. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.Nat. Immunol. 2017; 18: 86-95Crossref PubMed Scopus (43) Google Scholar, 10Lillemeier B.F. Mörtelmaier M.A. Forstner M.B. Huppa J.B. Groves J.T. Davis M.M. TCR and Lat are expressed on separate protein islands on T cell membranes and concatenate during activation.Nat. Immunol. 2010; 11: 90-96Crossref PubMed Scopus (441) Google Scholar, 11Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (534) Google Scholar). S-acylation has been proposed to serve as a molecular for protein lateral and into plasma membrane domains I. Grzybek M. Simons K. protein with membrane 2010; PubMed Scopus Google Scholar). TCR-induced S-acylation can as a mechanism supporting membrane of activated ZAP-70 and its to LAT and In this the acylation-deficient ZAP-70 mutant is recruited and phosphorylated at the TCR complex but of lipidation prevents it from its substrates and that can of protein distribution with S-acylation regulates ZAP-70 lateral and with its substrates in T by our is the of for ZAP-70 S-acylation and upon T cell In protein S-acylation is by a of protein A. and the role of proteins in T cell activation and Immunol. 2011; PubMed Scopus Google Scholar, D.A. A. R.J. by a of protein Full Text Full Text PDF PubMed Scopus Google Scholar). to is the role of in the of the immune responses. we identified as a controlling T cell activation and of T into T and Y. R. D. of T cell receptor signaling by protein Biol. PubMed Scopus Google Scholar). of S-acylation of Lck, Fyn, and suggesting that this be in the of proximal TCR However, the of ZAP-70 to be of the the fatty and the cysteine more a protein and 2 were the only to of proteins J.A. A cytoplasmic that from protein and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. G.A. 2 the of 2010; PubMed Scopus Google Scholar). the was to a of Y. A. T. Kobayashi K. M. of 2016; 36: PubMed Scopus Google Scholar, E. proteins are protein that and 2015; Scopus Google Scholar). However, the for and the role of in the of the immune remains to be In our a previously uncharacterized posttranslational modification of ZAP-70 with long-chain fatty as and of this modification S-acylation of ZAP-70 as a signal transduction mechanism supporting the function of ZAP-70 in T immunity. the proteins were from Lck ZAP-70 LAT SLP-76 and were from The were for and The were A 2 and and were from were in with 2 to and cell were in T were from was into using was by to ZAP-70, ZAP-70 and ZAP-70 and ZAP-70 in the ZAP-70 or ZAP-70 was into the from using the assembly with the and as the in and were at for by and to membranes were with in in and with at by were then with or in for at in membranes were on the and were in the using the ZAP-70, ZAP-70, ZAP-70, ZAP-70 or ZAP-70 were generated by of the with of and of into at in from M.L. D. J. M.M. K. S. E. and of TCR signaling is by phosphorylation of ZAP-70 and of the Natl. Acad. Sci. 2018; PubMed Scopus Google The the was and and were at of was in A of of was to the at and the was at at for were in S-acylation was using the from R. M.A. of protein by 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J.A. D. of protein in Full Text Full Text PDF PubMed Scopus Google Scholar, R. of protein S-acylation using Exp. PubMed Scopus Google Scholar). We for were in of in 2 and cell was to by and to the at a of of with to a This was by the at for to a at the and The was and the protein was in of by at in a the were by of in to to a of was for at with at in a was by of and protein were in and A as R. of protein S-acylation using Exp. 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A. 2010; 107: PubMed Scopus Google and in with a with were on a with a were 3 with a were for 2 to a and then the were with of to initiate TCR signaling and for were from of the and A of were in The were then with ZAP-70 or ZAP-70 at two of and using a were in for and then in and in and to and in with a with were on a with a with were and for 3 with were for were with a in and The were at for at to cell and The was and at for at The resulting was in with and 2 The was as the or and by A of or ZAP-70 or ZAP-70 were in of of was as ZAP-70 were with of and at This was by with of A for at The were then with the and with the kinase and and in of the kinase and SLP-76 were then to the and at for was to the The was for phosphorylation of SLP-76 by with and in kinase was as a to the are in the and the The of in to this We of of and of at for and We of at and of for with and We of at for critical of the R. T. and A. M. A and the R. T. and Y. 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