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.
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van der Merwe, P. A. & Dushek, O. Mechanisms for T cell receptor triggering. Nat. Rev. Immunol. 11, 47–55 (2011).
Chakraborty, A. K. & Weiss, A. Insights into the initiation of TCR signaling. Nat. Immunol. 15, 798–807 (2014).
Malissen, B. & Bongrand, P. Early T cell activation: integrating biochemical, structural, and biophysical cues. Annu. Rev. Immunol. 33, 539–561 (2015).
Hartmann, J., Schüßler-Lenz, M., Bondanza, A. & Buchholz, C. J. Clinical development of CAR T cells: challenges and opportunities in translating innovative treatment concepts. EMBO Mol. Med. 9, 1183–1197 (2017).
Nika, K. et al. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity 32, 766–777 (2010).
Thill, P. A., Weiss, A. & Chakraborty, A. K. 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. 36, 2396–2402 (2016).
Courtney, A. H. et al. A Phosphosite within the SH2 domain of Lck regulates its activation by CD45. Mol Cell 67, 498–511.e496 (2017).
Stepanek, O. et al. Coreceptor scanning by the T cell receptor provides a mechanism for T cell tolerance. Cell 159, 333–345 (2014).
van Oers, N. S., Killeen, N. & Weiss, A. Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes. J. Exp. Med. 183, 1053–1062 (1996).
Hatada, M. H. et al. Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor. Nature 377, 32–38 (1995).
Love, P. E. & Hayes, S. M. ITAM-mediated signaling by the T-cell antigen receptor. Cold Spring Harb. Perspect. Biol. 2, a002485 (2010).
Deindl, S. et al. Structural basis for the inhibition of tyrosine kinase activity of ZAP-70. Cell 129, 735–746 (2007).
Yan, Q. et al. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Mol. Cell. Biol. 33, 2188–2201 (2013).
Shah, N. H. et al. An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor. eLife 5, e20105 (2016).
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. 274, 14229–14237 (1999).
Wang, H. et al. ZAP-70: an essential kinase in T-cell signaling. Cold Spring Harb. Perspect. Biol. 2, a002279 (2010).
Mukherjee, S. et al. Monovalent and multivalent ligation of the B cell receptor exhibit differential dependence upon Syk and Src family kinases. Sci. Signal. 6, ra1 (2013).
Balagopalan, L., Coussens, N. P., Sherman, E., Samelson, L. E. & Sommers, C. L. The LAT story: a tale of cooperativity, coordination, and choreography. Cold Spring Harb. Perspect. Biol. 2, a005512 (2010).
Katz, 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. 18, 86–95 (2017).
Luis, B. S. & 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. 281, 696–707 (2014).
Yang, M. et al. K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8+ T cell activation. Nat. Immunol. 16, 1253–1262 (2015).
Mayer, B. J. The discovery of modular binding domains: building blocks of cell signalling. Nat. Rev. Mol. Cell Biol. 16, 691–698 (2015).
Dinkel, H. et al. ELM 2016: data update and new functionality of the eukaryotic linear motif resource. Nucleic Acids Res. 44, D294–D300 (2016). D1.
Michie, A. M. & Zúñiga-Pflücker, J. C. Regulation of thymocyte differentiation: pre-TCR signals and beta-selection. Semin. Immunol. 14, 311–323 (2002).
Moran, A. E. & Hogquist, K. A. T-cell receptor affinity in thymic development. Immunology 135, 261–267 (2012).
Zhang, W. et al. Essential role of LAT in T cell development. Immunity 10, 323–332 (1999).
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. 182, 5596–5604 (2009).
Rudd, M. L., Tua-Smith, A. & Straus, D. B. Lck SH3 domain function is required for T-cell receptor signals regulating thymocyte development. Mol. Cell. Biol. 26, 7892–7900 (2006).
Palacios, E. H. & Weiss, A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23, 7990–8000 (2004).
Purbhoo, M. A. 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. 276, 32786–32792 (2001).
McCoy, M. E., Finkelman, F. D. & Straus, D. B. Th2-specific immunity and function of peripheral T cells is regulated by the p56Lck Src homology 3 domain. J. Immunol. 185, 3285–3294 (2010).
Paster, W. et al. A THEMIS:SHP1 complex promotes T-cell survival. EMBO J. 34, 393–409 (2015).
Aguado, E. et al. Induction of T helper type 2 immunity by a point mutation in the LAT adaptor. Science 296, 2036–2040 (2002).
Sommers, C. L. et al. A LAT mutation that inhibits T cell development yet induces lymphoproliferation. Science 296, 2040–2043 (2002).
Miyaji, M. et al. Genetic evidence for the role of Erk activation in a lymphoproliferative disease of mice. Proc. Natl. Acad. Sci. USA 106, 14502–14507 (2009).
Kortum, R. L. et al. A phospholipase C-γ1-independent, RasGRP1-ERK-dependent pathway drives lymphoproliferative disease in linker for activation of T cells–Y136F mutant mice. J. Immunol. 190, 147–158 (2013).
Ravichandran, K. S. et al. Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation. Science 262, 902–905 (1993).
Ravichandran, K. S., Lorenz, U., Shoelson, S. E. & Burakoff, S. J. Interaction of Shc with Grb2 regulates association of Grb2 with mSOS. Mol. Cell. Biol. 15, 593–600 (1995).
Ravichandran, K. S., Lorenz, U., Shoelson, S. E. & Burakoff, S. J. Interaction of Shc with Grb2 regulates the Grb2 association with mSOS. Ann. NY Acad. Sci. 766, 202–203 (1995).
Zhou, M. M. et al. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378, 584–592 (1995).
Ravichandran, K. S. et al. Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo. Mol. Cell. Biol. 17, 5540–5549 (1997).
Pratt, J. C. et al. Requirement for Shc in TCR-mediated activation of a T cell hybridoma. J. Immunol. 163, 2586–2591 (1999).
Arbulo-Echevarria, M. M. 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. 9, 115 (2018).
Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Yu, K. & Salomon, A. R. PeptideDepot: flexible relational database for visual analysis of quantitative proteomic data and integration of existing protein information. Proteomics 9, 5350–5358 (2009).
Yu, K. & Salomon, A. R. HTAPP: high-throughput autonomous proteomic pipeline. Proteomics 10, 2113–2122 (2010).
Ahsan, N., Belmont, J., Chen, Z., Clifton, J. G. & Salomon, A. R. Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction. J. Proteomics 165, 69–74 (2017).
Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).
Demirkan, G., Yu, K., Boylan, J. M., Salomon, A. R. & Gruppuso, P. A. Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1). PLoS One 6, e21729 (2011).
We thank L. Samelson and C. Sommers (NIH) for sharing the LAT-deficient mouse line; T. Kadlecek for generating the J.Lck mutant Jurkat cell clone; A. Roque for animal husbandry; W. Paster (Medical University Vienna) for sharing the CD8 expression vector; T. Brdicka (IMG, Prague) for sharing the Lck-encoding DNA sequence; the UCSF Parnassus Flow Cytometry Core for maintaining FACSAria instruments and services; the NIH Tetramer Core Facility for providing the H-2Kb OVA tetramers and H-2Ab OVA tetramers; and D. L. Donermeyer, B. B. Au-Yeung, H. Wang, and C. Morley for critical reading of the manuscript and providing comments. This work was supported by the Jane Coffin Childs Fund 61-1560 (to W.-L.L.); the Damon Runyon Cancer Research Foundation 2198-14 (to N.H.S.); the Czech Science Foundation GJ16-09208Y (to O.S.); the Howard Hughes Medical Institute (to A.W. and J.K.); NIH, NIAID P01 AI091580-06 (to A.W., J.K., and A.R.S.); R01 AI083636 and P30 GM110759 (to A.R.S.); and DRC Center Grant P30 DK063720 (UCSF Parnassus Flow Cytometry Core).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 The proline-rich motif in the membrane-proximal segment of LAT is highly evolutionarily conserved.
a, The amino acid sequences of 42 mammalian LATs. b, The frequency of individual amino acids in human LAT or the whole human proteome. c, Relative frequency of individual amino acids of human LAT normalized to the whole human proteome. d, Relative frequency of proline residues of key adaptor or kinase proteins (normalized to the whole human proteome) in the antigen receptor signaling pathway
Supplementary Figure 2 The proline-rich motif in the membrane-proximal segment of LAT promotes TCR signaling.
(a) Immunoblot analysis of screening LAT mutant Jurkat variants that lack the proline-rich region, probed with anti-C terminal LAT antibody or anti-tubulin antibody (loading control). Numbers to the right of cropped blots indicate molecular masses (kDa). Data are representative of at least two independent experiments. (b) Characterization of CRISPR/Cas9-induced amino acid changes of mutant LAT in J.dPro or J.Het cells. Each line represents the amino acid sequences encoded by one allele of DNA. “-” indicates the amino acid deletion. “n” indicates the insertion of asparagine resulted from the CRISPR/Cas9-induced nucleotide insertions. Wild type human LAT sequences are shown at the top. (c) CRISPR/Cas9 generated LAT-deficient J.LAT cells, or Jurkat cells were loaded with Indo-1 AM, stimulated with 0.5 μg/ml anti-CD3 and the changes in relative calcium-sensitive fluorescence ratios over time are shown. The center of measure indicates mean ± s.d. n = 3 technical replicates. Data are representative of two independent experiments. (d) Immunoblot analyses of Jurkat, J.LAT cells, or LAT-deficient JCaM2 were left unstimulated or stimulated with 0.5 μg/ml anti-CD3 for 2 min. Numbers to the right of cropped blots indicate molecular masses (kDa). Data are representative of at least three independent experiments with similar results
Immunoblot analyses of J.LAT cells reconstituted with WT or AIARSA mutant LAT were either unstimulated or stimulated with anti-CD3 (concentration as indicated above blots) for 2 min (a), or with 0.5 μg/ml anti-CD3 over time (stimulation time as indicated, b). Numbers to the right of cropped blots indicate molecular masses (kDa). Data are representative of four experiments
Absolute number analysis of bone marrow chimeric studies shown in Fig. 3. (a) Bar graph showing absolute number of CD45.2+ mCherry+ DN, DP, CD4SP, and CD8SP thymocytes as in Fig. 3d. (b) Absolute number analysis of CD45.2+ mCherry+ DN1, DN2, DN3 and DN4 cells as in Fig. 3f. (c) Absolute number analysis of post positive selection CD45.2+ mCherry+ DP3 cells as in Fig. 3h. (a, b, c) Each symbol represents an individual mouse. All bar graphs indicate mean±s.d. n = 4 independent animals. All experiments were repeated twice with four independent animals. *P = 0.0286; ns, not significant, two-tailed Mann-Whitney test
Supplementary Figure 5 Peripheral T cells ectopically expressing the mutant AIARSA LAT exhibit an increased memory-cell-like phenotype.
Flow cytometry of splenocytes from lethally irradiated bone marrow BoyJ chimeras reconstituted with CD45.2+ B6 hematopoietic stem cells transduced with lentivirus expressing WT LAT-P2A-mCherry (WT; n = 4 recipients) or mutant AIARSA LAT-P2A-mCherry (AIARSA; n = 4 recipients), as described in Fig. 3. (a) Flow cytometric analysis of CD45.2+ mCherry+ splenocytes. Numbers in outlined areas indicate percent cells in each quadrant. Bar graph depicts the frequency or the absolute number of CD4+ and CD8+ splenocytes among CD45.2+mCherry+ cells (mean±s.d.). n = 4 independent animals. ns, not significant; two-tailed Mann-Whitney test. (b) Surface expression of TCR or CD5 in CD45.2+mCherry+ CD4+ or CD8+ T cells. (c and d) Flow cytometric analysis of CD62L versus CD44 expression of CD45.2+ mCherry+ CD4+ (c) or CD8+ (d) T cells. Bar graphs present the frequencies or the absolute number of naive cell populations (CD62Lhi CD44lo), or memory cell populations (CD44hiCD62Llo or CD44hiCD62Lhi). Bar indicates mean±s.d. n = 4 independent animals. *P = 0.0286; ns, not significant, two-tailed Mann-Whitney test. (a, b, c, d) Data are representative of two independent experiments
LAT deficient J.LAT cells were reconstituted with WT or AIARSA mutant LAT, fused with a Myc-tag at the C-terminus. Cells were stimulated with 0.5 μg/ml anti-CD3 for 0, 2, or 10 min. Samples were immunoprecipitated (IP) with anti-Myc antibody and subjected to mass spectrometry analysis. (a) Ratio fold-change in Lck peptides identified by mass spectrometry analysis was illustrated as heat map among each sample. White block indicates peptide was not identified as the peak area was not observed. (b) Immunoblot analysis of samples IP’d with anti-Myc or whole cell lysates for Lck (LAT PIPRSP motif dependent), Grb2 (LAT PIPRSP motif independent) or LAT. Data are representative of two independent experiments with similar results. c, Illustration of the proposed model whereby Lck serves as a kinase and an adaptor protein. New data suggest Lck interacts via its SH3 domain with the LAT PIPRSP motif and also interacts via its SH2 domain with Zap70 phospho-Y319, as well as associating via its unique domain with coreceptor CD4 or CD8 cytoplasmic segment. Arrows indicate Lck’s ability to phosphorylate CD3 or Zap70 (left) or Zap70’s ability to phosphorylate LAT (right)
Supplementary Figure 7 Illustration of a HEK293-cell-based cellular system to examine the adaptor function of Lck and its role in enhancing the phosphorylation of LAT by Zap70.
a, Scheme of mutant Lck, Zap70 and LAT variants that were used in experiments in Fig. 5. b, Illustration of experimental design using HEK293 cell based system to study Lck’s roles as an adaptor protein in Zap70-mediated phosphorylation of LAT
Supplementary Figure 8 The coexpression of human CD8 may assist mouse OT-I TCR in binding the mouse MHC-I H-2Kb.
a, CD69 upregulation assay of human CD8+ OT-I TCR+ Jurkat cells cultured with OVA peptide-pulsed antigen presenting T2-Kb cells over a wide range of OVA peptide concentration. J.OT-I.hCD8.Lck-FLAG cells were retrovirally transduced to express human CD8, whereas J.OT-I.Lck-FLAG cells did not express human CD8. Each symbol represents the mean±s.e.m. of percent of CD69+ cells. n = 4 independent experiments at OVA concentration of 10, 10-7, 10-8 μM; n = 6 independent experiments at OVA concentration between 1 and 10-6 μM. Data are representative of six independent experiments. b, The flow cytometry analysis of MHC class I H-2Kb OVA tetramers staining of J.OT-I.hCD8.Lck-FLAG cells or J.OT-I.Lck-FLAG cells. The unstained sample of J.OT-I.hCD8.Lck-FLAG cells was used as the no stained control. Experiments were repeated independently twice with similar results
Supplementary Figures 1–8
The 42 mammalian sequences of LAT
All of the interacting proteins identified by mass spectrometry analysis from immunoprecipitation samples from WT LAT or AIARSA mutant LAT over time
SH3 domain-containing proteins identified by mass spectrometry analysis from immunoprecipitation samples from WT LAT or AIARSA mutant LAT over time
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Lo, W., Shah, N.H., Ahsan, N. et al. Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT. Nat Immunol 19, 733–741 (2018). https://doi.org/10.1038/s41590-018-0131-1
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