T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.

Center for Cancer Research Development Proteomics Core Facility Rhode Island Hospital Providence RI USA

Department of Chemistry Brown University Providence RI USA

Department of Molecular Biology Cell Biology and Biochemistry Brown University Providence RI USA

Departments of Molecular and Cell Biology University of California Berkeley Berkeley CA USA

Division of Biology and Medicine Alpert Medical School Brown University Providence RI USA

Division of Rheumatology Rosalind Russell and Ephraim P Engleman Arthritis Research Center Department of Medicine University of California San Francisco San Francisco CA USA

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

The Howard Hughes Medical Institute University of California Berkeley Berkeley CA USA

The Howard Hughes Medical Institute University of California San Francisco San Francisco CA USA

See more in PubMed

van der Merwe PA, Dushek O. Mechanisms for T cell receptor triggering. Nat Rev Immunol. 2011;11:47–55. PubMed

Chakraborty AK, Weiss A. Insights into the initiation of TCR signaling. Nat Immunol. 2014;15:798–807. PubMed PMC

Malissen B, Bongrand P. Early T cell activation: integrating biochemical, structural, and biophysical cues. Annu Rev Immunol. 2015;33:539–561. PubMed

Hartmann J, Schussler-Lenz M, Bondanza A, Buchholz CJ. Clinical development of CAR T cells-challenges and opportunities in translating innovative treatment concepts. EMBO Mol Med. 2017;9:1183–1197. PubMed PMC

Nika K, et al. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity. 2010;32:766–777. PubMed PMC

Thill PA, Weiss A, Chakraborty AK. Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor Signaling In Vivo. Mol Cell Biol. 2016;36:2396–2402. PubMed PMC

Courtney AH, et al. A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45. Mol Cell. 2017;67:498–511.e496. PubMed PMC

Stepanek O, et al. Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance. Cell. 2014;159:333–345. PubMed PMC

van Oers NS, Killeen N, Weiss A. Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes. J Exp Med. 1996;183:1053–1062. PubMed PMC

Hatada MH, et al. Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor. Nature. 1995;377:32–38. PubMed

Love PE, Hayes SM. ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb Perspect Biol. 2010;2:a002485. PubMed PMC

Deindl S, et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell. 2007;129:735–746. PubMed

Yan Q, et al. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Mol Cell Biol. 2013;33:2188–2201. PubMed PMC

Shah NH, et al. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor. Elife. 2016;5 doi: 10.7554/eLife.20105. PubMed DOI PMC

Pelosi M, et al. Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck. J Biol Chem. 1999;274:14229–14237. PubMed

Wang H, et al. ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb Perspect Biol. 2010;2:a002279. PubMed PMC

Mukherjee S, et al. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases. Sci Signal. 2013;6:ra1. PubMed PMC

Balagopalan L, Coussens NP, Sherman E, Samelson LE, Sommers CL. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb Perspect Biol. 2010;2:a005512. PubMed PMC

Katz ZB, Novotna L, Blount A, Lillemeier BF. A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli. Nat Immunol. 2017;18:86–95. PubMed PMC

Luis BS, Carpino N. Insights into the suppressor of T-cell receptor (TCR) signaling-1 (Sts-1)-mediated regulation of TCR signaling through the use of novel substrate-trapping Sts-1 phosphatase variants. FEBS J. 2014;281:696–707. PubMed PMC

Yang M, et al. K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8(+) T cell activation. Nat Immunol. 2015;16:1253–1262. PubMed

Mayer BJ. The discovery of modular binding domains: building blocks of cell signalling. Nat Rev Mol Cell Biol. 2015;16:691–698. PubMed

Dinkel H, et al. ELM 2016–data update and new functionality of the eukaryotic linear motif resource. Nucleic Acids Res. 2016;44:D294–300. PubMed PMC

Michie AM, Zuniga-Pflucker JC. Regulation of thymocyte differentiation: pre-TCR signals and beta-selection. Semin Immunol. 2002;14:311–323. PubMed

Moran AE, Hogquist KA. T-cell receptor affinity in thymic development. Immunology. 2012;135:261–267. PubMed PMC

Zhang K, Zhou B, Wang Y, Rao L, Zhang L. The TLR4 gene polymorphisms and susceptibility to cancer: a systematic review and meta-analysis. Eur J Cancer. 2013;49:946–954. PubMed

Shen S, Zhu M, Lau J, Chuck M, Zhang W. The essential role of LAT in thymocyte development during transition from the double-positive to single-positive stage. J Immunol. 2009;182:5596–5604. PubMed PMC

Rudd ML, Tua-Smith A, Straus DB. Lck SH3 domain function is required for T-cell receptor signals regulating thymocyte development. Mol Cell Biol. 2006;26:7892–7900. PubMed PMC

Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene. 2004;23:7990–8000. PubMed

Purbhoo MA, et al. The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor zeta chain. J Biol Chem. 2001;276:32786–32792. PubMed

McCoy ME, Finkelman FD, Straus DB. Th2-specific immunity and function of peripheral T cells is regulated by the p56Lck Src homology 3 domain. J Immunol. 2010;185:3285–3294. PubMed

Paster W, et al. A THEMIS:SHP1 complex promotes T-cell survival. EMBO J. 2015;34:393–409. PubMed PMC

Aguado E, et al. Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science. 2002;296:2036–2040. PubMed

Sommers CL, et al. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science. 2002;296:2040–2043. PubMed

Miyaji M, et al. Genetic evidence for the role of Erk activation in a lymphoproliferative disease of mice. Proc Natl Acad Sci U S A. 2009;106:14502–14507. PubMed PMC

Kortum RL, et al. A phospholipase C-gamma1-independent, RasGRP1-ERK-dependent pathway drives lymphoproliferative disease in linker for activation of T cells-Y136F mutant mice. J Immunol. 2013;190:147–158. PubMed PMC

Ravichandran KS, et al. Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation. Science. 1993;262:902–905. PubMed

Ravichandran KS, Lorenz U, Shoelson SE, Burakoff SJ. Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol Cell Biol. 1995;15:593–600. PubMed PMC

Ravichandran KS, Lorenz U, Shoelson SE, Burakoff SJ. Interaction of Shc with Grb2 regulates the Grb2 association with mSOS. Ann N Y Acad Sci. 1995;766:202–203. PubMed

Zhou MM, et al. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature. 1995;378:584–592. PubMed

Ravichandran KS, et al. Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo. Mol Cell Biol. 1997;17:5540–5549. PubMed PMC

Pratt JC, et al. Requirement for Shc in TCR-mediated activation of a T cell hybridoma. J Immunol. 1999;163:2586–2591. PubMed

Arbulo-Echevarria MM, et al. A Stretch of Negatively Charged Amino Acids of Linker for Activation of T-Cell Adaptor Has a Dual Role in T-Cell Antigen Receptor Intracellular Signaling. Front Immunol. 2018;9:115. doi: 10.3389/fimmu.2018.00115. PubMed DOI PMC

Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–1190. PubMed PMC

Yu K, Salomon AR. PeptideDepot: flexible relational database for visual analysis of quantitative proteomic data and integration of existing protein information. Proteomics. 2009;9:5350–5358. PubMed PMC

Yu K, Salomon AR. HTAPP: high-throughput autonomous proteomic pipeline. Proteomics. 2010;10:2113–2122. PubMed PMC

Ahsan N, Belmont J, Chen Z, Clifton JG, Salomon AR. Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction. J Proteomics. 2017;165:69–74. PubMed PMC

Elias JE, Gygi SP. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods. 2007;4:207–214. PubMed

Demirkan G, Yu K, Boylan JM, Salomon AR, Gruppuso PA. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1) PLoS One. 2011;6:e21729. PubMed PMC

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