Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT

Abstract

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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Fig. 1: A conserved proline-rich region in the membrane-proximal region of LAT is important for TCR signaling.
Fig. 2: A PIPRSP motif in LAT promotes the phosphorylation of LAT.
Fig. 3: Ectopic expression of LAT containing the PIPRSP-mutant motif AIARSA impairs thymocyte beta and positive selection in vivo.
Fig. 4: Mutation of the PIPRSP motif in LAT impedes TCR signal transduction of DP thymocytes.
Fig. 5: Interaction of the Lck SH3 domain and the LAT PIPRSP motif enhances Zap70-dependent phosphorylation of LAT.
Fig. 6: TCR engagement triggers the association of LAT with TCR signaling complexes.

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Acknowledgements

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).

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Contributions

W.-L.L., N.H.S., and A.W. designed the experiments, W.-L.L., N.H.S., N.A., and V.H. conducted the experiments; W.-L.L., N.H.S., N.A., A.R.S., J.K., and A.W. analyzed data and provided intellectual input; V.H. and O.S. provided advice and reagents. W.-L.L., N.H.S., N.A., V.H., O.S., A.R.S., and A.W. wrote the manuscript.

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Correspondence to Arthur Weiss.

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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

Supplementary Figure 3 The PIPRSP motif in LAT promotes the phosphorylation of LAT and PLCγ1.

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

Supplementary Figure 4 Mutant AIARSA LAT impedes thymic development.

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

Supplementary Figure 6 TCR stimulation induces the interaction of Lck with the LAT PIPRSP motif.

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 information

Supplementary Text and Figures

Supplementary Figures 1–8

Reporting Summary

Supplementary Table 1

The 42 mammalian sequences of LAT

Supplementary Table 2

All of the interacting proteins identified by mass spectrometry analysis from immunoprecipitation samples from WT LAT or AIARSA mutant LAT over time

Supplementary Table 3

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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