Research ArticleImmunology

Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling

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Science Signaling  16 Apr 2019:
Vol. 12, Issue 577, eaar3349
DOI: 10.1126/scisignal.aar3349
  • Fig. 1 Deficient activation of T cells from PKCθ knockout mice expressing the pTyr nonbinding PKCθ mutant HR2A.

    (A) Alignment of sequences surrounding the pTyr-binding site in the C2 domains of nPKC subfamily members. Residues that form the pTyr-binding pocket are in gray, and Ala mutations of His63 and Arg68 in the HR2A mutant are shown. (B and C) Immunoblot for PKCθ in sorted (GFP+) primary CD4+ T cells that were transduced with control retrovirus (RV) encoding GFP alone (vector) or GFP and either PKCθWT or PKCθHR2A. Actin is a loading control. Results representative of five independent experiments are shown in (B). Quantified band intensity ratio values from (C) are means ± SEM pooled from five independent experiments. (D to F) Flow cytometry analysis of the surface abundance of the T cell activation markers CD25 and CD69 (D), proliferation (E), or IL-2 abundance (F) of CD4+ T cells that were either left unstimulated (No stim) or restimulated with mAbs specific for CD3 and CD28 (α-CD3 + CD28). Numbers above bracketed lines indicate the percentage of positive (D and F) or proliferating (E) cells. Mean fluorescence intensities (MFIs) of CD25 and CD69 are shown (D). Representative plots from five independent experiments are shown. (G) Abundance of IL-2 in supernatants of sorted CD4+ T cells transduced with RV encoding PKCθWT, PKCθHR2A and left unstimulated (0), restimulated with CD3 only (α-CD3) or CD3 and CD28 mAbs (α-CD3 + CD28). Data are means ± SEM of nine biological replicates pooled from three independent experiments. (H and I) In vitro kinase assay with Myc-tagged PKCθWT, PKCθHR2A, or the kinase-inactive mutant PKCθK409R immunoprecipitated (IP) from transfected human embryonic kidney (HEK) 293 (HEK293) cells. Immunoprecipitated proteins were mixed with radiolabeled ATP and myelin basic protein (MBP), and the reactions were separated on a gel for autoradiography and for immunoblotting for Myc and MBP. (H) is the representative blot, and (I) shows band densitometry ratio (means ± SEM) pooled from three independent experiments. ns, not significant; *P < 0.05; **P < 0.01.

  • Fig. 2 Deficient activation of CD4+ T cells from BM chimeric mice reconstituted with PKCθ knockout BM cells expressing PKCθHR2A.

    (A to D) Flow cytometry analysis of the surface abundance of CD25 and CD69 (A and B) and the production of IL-2 (C and D) by transduced (GFP+) or nontransduced (GFP) naïve CD4+ T cells from BM chimeric mice reconstituted with PKCθ knockout BM cells expressing EV (vector), PKCθWT, or PKCθHR2A. Cells were left unstimulated or restimulated with CD3 and CD28 mAbs. Numbers above bracketed lines (A) and in the gated boxes (C) indicate the percentage of positive cells from a single representative mouse in each group. MFI values of CD25 and CD69 (A) are shown, and averaged data pooled from six mice per group are shown in (B) and (D). *P < 0.05; **P < 0.01; ***P < 0.001. Results are representative of four independent experiments. (E and F) Flow cytometry analysis of the proliferation (E) and the amounts of intracellular IFN-γ (TH1) or IL-4 (TH2) cytokines (F) in sorted (GFP+) CD4+ cells as in (A) to (D). Numbers in (E) and (F) indicate the percentage of proliferating cells or the frequency of IFN-γ– or IL-4–containing cells among gated CD4+ cells, respectively. Results are representative of six mice per group and three independent experiments.

  • Fig. 3 The PKCθ C2 domain interacts with the kinase Zap70.

    (A) Purified recombinant GST-tagged WT or HR2A mutant forms of the PKCθ C2 domain, GST-C2 and GST-C2-HR2A, respectively, were used for pull-down assays of lysates from unstimulated or PV-stimulated Jurkat T cells. Bound material was separated by SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblotted for pTyr and GST. The boxed area was subjected to LC-MS/MS analysis. Data are representative of three independent experiments. (B) Identity of some of the peptides resolved by LC-MS/MS from PV-stimulated Jurkat T cells. (C) Representative pull-down experiment using purified recombinant GST-C2 and GST-C2-HR2A to pull down proteins from lysates of unstimulated or PV-stimulated primary CD4+ T cells from PKCθ knockout mice. Pulled-down material (top three panels) or WCLs (bottom two panels) were analyzed by immunoblotting for the indicated proteins. Data are representative of three independent experiments. (D to G) Primary B6 (D and E) or OT-II (F and G) CD4+ T cells were left unstimulated or stimulated for the indicated times with BM-derived dendritic cells (APC) pulsed with SEE or OVA peptide, respectively. PKCθ immunoprecipitates (IP) or WCLs were immunoblotted for the indicated proteins. Representative immunoblots of three independent experiments are shown (D and F) alongside quantification of Zap70 by densitometry (E and G) pooled from three independent experiments. *P < 0.05; **P < 0.01. IgG, immunoglobulin G.

  • Fig. 4 Identification and characterization of Zap70 pTyr residues required for binding to the PKCθ C2 domain.

    (A) Purified recombinant GST (control) or GST-C2WT was used to pull down proteins in lysates of PV-stimulated 293T cells expressing WT Zap70 or the indicated Zap70 mutants. Pulled-down material and WCLs were separated by SDS-PAGE, stained with Ponceau S, and immunoblotted for Zap70. This experiment is representative of three independent experiments. (B) Binding of recombinant GST and GST-C2WT to the indicated immobilized synthetic Zap70 peptides was assessed by an ELISA. Non, non–peptide-coated control wells. (C) Binding of the indicated recombinant proteins to wells coated with the pTyr126 peptide determined by ELISA. Pooled data from four independent experiments are shown in (B) and (C); **P < 0.01. (D and E) GST pull-down with lysates of WT (JE6.1) or Lck-deficient (Jcam1.6) Jurkat cells that were left unstimulated or stimulated with PV or CD3 and CD28 mAbs as indicated. C2WT-bound proteins or WCLs were immunoblotted for the indicated proteins. Data are representative of three independent experiments. Pooled data from three independent experiments as (D) were quantitated by densitometry and shown in (E). **P < 0.01; ***P < 0.001.

  • Fig. 5 Binding of pTyr by PKCθ is required for early TCR signaling.

    GFP+ primary CD4+ T cells were isolated from BM chimeric mice reconstituted with PKCθ knockout BM cells transduced with RV encoding GFP and either PKCθWT or PKCθHR2A (A to D) or EV (empty vector) (C and D). The cells were left unstimulated (unstim.) or stimulated (stim.) with mAbs against CD3 and CD28 for 5 min before cell lysates were subjected to SDS-PAGE and immunoblotted for the indicated proteins. p-Lck and p-TCRζ were determined by the dominant lanes of molecular mass around 55 kDa (Lck) and 23 kDa (TCRζ) from the total pTyr blot (4G10), respectively. Representative raw data are shown in (A) and (C), and pooled data from three independent experiments were quantitated by densitometry in (B) and (D). *P < 0.05; **P < 0.01. (E) Cells isolated and stimulated as in (A) and (C) were fixed and stained intracellularly with an APC-conjugated Ab against phosphorylated Itk (p-Itk). Plot is representative of three independent experiments.

  • Fig. 6 pTyr binding by PKCθ is required for PKCθ IS localization and association with CD28.

    (A) Confocal imaging of Prkcq−/− OT-II CD4+ T cells that were transduced with an RV encoding GFP-tagged WT or HR2A mutant PKCθ (green), mixed with APCs labeled with the cell-tracking dye CMAC (blue), and pulsed with OVA peptide (+OVA). Fixed conjugates were stained for Zap70 and a secondary fluorescent Ab (AF555). A representative cell is shown. Scale bar, 10 μm. (B) Quantification of PKCθ localization within (IS) or outside (No IS) the IS in the T cell–APC conjugates analyzed in (A). Analysis was performed only on conjugates that displayed IS-localized Zap70 and detectable PKCθ. n = ~120. (C to E) GFP+ CD4+ T cells transduced in vitro (C and D) or derived from BM chimeras reconstituted in vivo (E) with pMIG vectors expressing PKCθWT, PKCθHR2A, or PKCθ-δV3 were stimulated with mAbs against CD3 and CD28 for 5 min. CD28 and PKCθ interaction was detected by coimmunoprecipitation (co-IP). Representative blots are shown above the corresponding band densitometry quantifications pooled from three independent experiments. **P < 0.01; ***P < 0.001.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/577/eaar3349/DC1

    Fig. S1. Normal T cell development in PKCθHR2A BM chimeric mice.

    Fig. S2. Impaired activation of peripheral T cells expressing PKCθHR2A.

    Fig. S3. Defective early TCR signaling in primary CD4+ T cells from Prkcq−/− B6 mice.

    Fig. S4. Effect of PKCθHR2A on Zap70 recruitment to TCRζ and on formation of the PKCθ-CARMA1 complex.

    Table S1. Abs and other reagents used in this study.

  • This PDF file includes:

    • Fig. S1. Normal T cell development in PKCθHR2A BM chimeric mice.
    • Fig. S2. Impaired activation of peripheral T cells expressing PKCθHR2A.
    • Fig. S3. Defective early TCR signaling in primary CD4+ T cells from Prkcq−/− B6 mice.
    • Fig. S4. Effect of PKCθHR2A on Zap70 recruitment to TCRζ and on formation of the PKCθ-CARMA1 complex.
    • Table S1. Abs and other reagents used in this study.

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