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Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling

Nature Immunology volume 15, pages 186–194 (2014)Cite this article

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Abstract

Signaling via the T cell antigen receptor (TCR) is initiated by Src-family kinases (SFKs). To understand how the kinase Csk, a negative regulator of SFKs, controls the basal state and the initiation of TCR signaling, we generated mice that express a Csk variant sensitive to an analog of the common kinase inhibitor PP1 (CskAS). Inhibition of CskAS in thymocytes, without engagement of the TCR, induced potent activation of SFKs and proximal TCR signaling up to phospholipase C-γ1 (PLC-γ1). Unexpectedly, increases in inositol phosphates, intracellular calcium and phosphorylation of the kinase Erk were impaired. Altering the actin cytoskeleton pharmacologically or providing costimulation via CD28 'rescued' those defects. Thus, Csk has a critical role in preventing TCR signaling. However, our studies also revealed a requirement for actin remodeling, initiated by costimulation, for full TCR signaling.

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Figure 1: Inhibition of CskAS in primary mouse T cells induces hyperactivation of SFKs and phosphorylation of TCR-proximal signaling molecules.
Figure 2: Increases in intracellular Ca2+ and Erk phosphorylation are impaired following inhibition of CskAS.
Figure 3: Increases in intracellular Ca2+ and Erk phosphorylation are restored by alteration of the actin cytoskeleton following inhibition of CskAS.
Figure 4: Mature DCs restore the Ca2+ increase and Erk phosphorylation following inhibition of CskAS.
Figure 5: Restoration of full TCR signaling after inhibition of CskAS requires both MHC and the CD28 ligands CD80 and CD86 on DCs.
Figure 6: CD28 signaling induces actin remodeling and restores robust Erk phosphorylation following inhibition of CskAS.

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Acknowledgements

We thank J.A. Bluestone (University of California, San Francisco) for CD80-CD86–deficient mice; J.B. Bolen (Moderna Therapeutics) for the anti-Lck hybridoma; H. Wang (University of California, San Francisco) for anti-ζ; A. Roque and Z. Wang for assisting with animal husbandry and cell sorting, respectively; N. Killeen and Z. Yang for technical assistance with BAC transgenesis; J.S. Shin for technical assistance with isolation of thymic DCs; H. Liu and the University of California, San Francisco Helen Diller Family Comprehensive Cancer Center Mass Spectrometry Core Facility for technical and analytical expertise; and A. DeFranco, C. Lowell and H. Wang for critical reading of the manuscript and discussions. Supported by the Agency for Science, Technology and Research in Singapore (Y.X.T.), the Cancer Research Institute (B.N.M.) and the US National Institutes of Health (PO1 AI091580 to A.W. and 5R01EB001987 to K.M.S.).

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Authors and Affiliations

  1. Division of Rheumatology, Department of Medicine, Rosalind Russell–Ephraim P. Engleman Medical Research Center for Arthritis, University of California, San Francisco, California, USA

    Ying Xim Tan, Boryana N Manz, Tanya S Freedman & Arthur Weiss

  2. Department of Chemistry, University of Southern California, California, USA

    Chao Zhang

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, USA

    Kevan M Shokat

  4. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Kevan M Shokat & Arthur Weiss

  5. Department of Microbiology and Immunology, University of California, San Francisco, California, USA

    Arthur Weiss

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Contributions

Y.X.T. and A.W. designed the research and wrote the manuscript; Y.X.T. did the research and analyzed the data; B.N.M. helped design, do and analyze the immunofluorescence experiments; T.S.F. designed, did and analyzed the data from the in vitro kinase assays; C.Z. and K.M.S. designed the CskAS allele and provided 3-IB-PP1; and all authors commented on the manuscript.

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Correspondence to Arthur Weiss.

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Integrated supplementary information

Supplementary Figure 1 The CskAS protein is active and can be specifically inhibited by the PP1 analog 3-IB-PP1.

(a) Substituting glycine for threonine at the gatekeeper position in the active site of Csk to generate analog sensitive Csk (CskAS) impairs but does not abrogate kinase activity in purified CskAS, as monitored by decreasing absorbance in an assay coupling ATP hydrolysis to NADH oxidation. (b) CskAS phosphorylates peptide at a rate in between that of CskWT and the kinase-impaired variant CskK222R. Error bars represent 95% confidence intervals (CI), n=4. The Csk variants are significantly different from each other (p<0.0001) in a one-way ANOVA test (or p=0.0041 (CskAS, CskK222R) in a two-tailed t test). (c) A bulky analog of kinase inhibitor PP1, 3-IB-PP1, specifically targets CskAS over CskWT. Data points are independent samples from four (CskWT) or five (CskAS) separate experiments, and lines represent fit dose-response curves. (d) 3-IB-PP1 has a 27-fold lower IC50 for CskAS than for CskWT. Error bars represent 95% CI of a global fit.

Supplementary Figure 2 T cells develop normally and respond normally to TCR stimulation in CskAS BAC transgenic mice.

(a) Immunoblot and densitometric analysis of Csk expression in wild-type (WT) and CskAS(AS) thymocytes. (b) Thymocytes or splenocytes from wild-type (WT) and CskAS(AS) thymi were surface stained for the indicated markers and analyzed by flow cytometry. Data are representative of five littermate pairs. (c) Mean thymic cellularity of ten wild-type (WT) and ten CskAS(AS) littermate thymi with means and s.e.m indicated. (d) Thymocytes from wild-type (WT:thin lines) or CskAS (AS:thick lines) mice loaded with Indo-1AM dye were stimulated with high dose (red lines: 20μg/mL) or low dose (blue lines: 5μg/mL) anti-CD3ɛ. Ratiometric assessment of intracellular calcium of CD4+CD8+ thymocytes over time is shown. Data are representative of three independent experiments.

Supplementary Figure 3 Inhibition of CskAS induces tyrosine phosphorylation but not CD69 upregulation in primary mouse T cells.

(271) Lighter exposure of pTyr blot from Figure 1d with quantification of p21 normalized to total ζ immunoprecipitated. (b) Immunoblot analysis of phosphorylated Akt or actin (loading control) in wild-type (WT) or CskAS (AS) thymocytes treated for 3min with DMSO, 10 μM 3-IB-PP1 or 20 μg/Ml anti-CD3ɛ. (c) Thymocytes from wild-type (WT) or CskAS (AS) mice were treated with DMSO, 10 μM 3-IB-PP1 or 10 μg/ml platebound anti-CD3ɛ for 12 hours, stained for cell surface CD69 and analyzed by flow cytometry, gated on CD4+CD8+ thymocytes. (d) Immunoblot analysis of phosphorylated tyrosine (p-Tyr) and Erk2 (loading control) in wild-type (WT) or CskAS (AS) purified peripheral CD4+ T cells treated for 3min as in b. (e) Immunoblot analysis of total and phosphorylated (p-) Zap70, Lat and PLC-γ1 in wild-type (WT) or CskAS (AS) purified peripheral CD4+ T cells treated for 3min as in b. Data are representative of two (d) or three (a-c and e) independent experiments.

Supplementary Figure 4 Simultaneous alteration of the actin cytoskeleton enhances Erk phosphorylation in thymocytes after inhibition of CskAS.

Phosphorylated Erk in wild-type (WT) or CskAS (AS) thymocytes stimulated for 2 min as indicated (10 μM 3-IB-PP1; CytoD, 10 μM cytochalasin D; LatA, 0.5 μM latrunculin A; Jpk, 1 μM jasplakinolide; 20 μg/mL anti-CD3ɛ), gated on CD4+CD8+ thymocytes. Data are representative of two independent experiments. Numbers above bracketed lines indicate percent cells with phosphorylated Erk.

Supplementary Figure 5 Thymic DCs, but not naive splenic DCs, ICAM-1 deficient DCs or chemokines secreted by mature splenic DCs, enhance Erk phosphorylation in thymocytes after CskAS inhibition.

Phosphorylated Erk in CskAS thymocytes (a, b and d) or in wild-type (WT) and CskAS (AS) thymocytes (c) pelleted with or without DCs at a 1:1 ratio and stimulated with vehicle (DMSO), 10 μM 3-IB-PP1, 50 ng/mL phorbol myristate acetate (PMA) (a-d) and 10 ng/mL stromal cell-derived factor (SDF-1) (d) for 3min, gated on CD4+CD8+ thymocytes. Numbers above bracketed lines indicate percent cells with phosphorylated Erk. (a) Naïve DCs were enriched from wild-type splenocytes and used immediately. (b) Thymic DCs were sorted from wild-type thymi as described in methods. (c) DCs were enriched from wild-type (WT) or ICAM-1 deficient (ICAM-1 KO) splenocytes and activated by overnight culture. (d) Thymocytes from CskAS mice were treated with DMSO or 50 ng/mL pertussis toxin (PTx) for 1h before mixing with DCs enriched from wild-type splenocytes and activated by overnight culture. Data are representative of two (a, d) or three (b, c) independent experiments.

Supplementary Figure 6 Model for TCR signal initiation and propagation in thymocytes.

(a) Perturbing the Csk-CD45 equilibrium regulating Lck by inhibiting CskAS results in potent Lck activation, thereby initiating phosphorylation of CD3 and ζ ITAMs and Zap70, and eventually activation of PLC-γ1 bound to the Lat signalosome. However, in the absence of actin turnover, active PLC-γ1 cannot access plasma membrane PtdIns(4,5)P2 to hydrolyze it because the dense cortical actin meshwork and its associated proteins may act as a barrier or because PLC-γ1 fails to be recruited to the actin cytoskeleton. (b) Inducing remodeling of the cortical actin cytoskeleton with cytochalasin D allows active PLC-γ1 to access and hydrolyze PtdIns(4,5)P2 to generate diacylglycerol (DAG) and Ins(1,4,5)P3. (c) During a thymocyte-APC interaction, TCR engagement by pMHC recruits CD4 or CD8 coreceptor bound active Lck. Association of CD80 or CD86 engaged CD28 with Lck allows for activation of local actin cytoskeletal remodeling. (d) This enables active PLC-γ1 to hydrolyze PtdIns(4,5)P2.

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Tan, Y., Manz, B., Freedman, T. et al. Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling. Nat Immunol 15, 186–194 (2014). https://doi.org/10.1038/ni.2772

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