pubmed.ncbi.nlm.nih.gov

A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28 - PubMed

  • ️Sat Jan 01 2011

A motif in the V3 domain of the kinase PKC-θ determines its localization in the immunological synapse and functions in T cells via association with CD28

Kok-Fai Kong et al. Nat Immunol. 2011.

Abstract

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. Here we found that the V3 domain of PKC-θ was necessary and sufficient for localization to the immunological synapse mediated by association with the coreceptor CD28 and dependent on the kinase Lck. We identified a conserved proline-rich motif in V3 required for association with CD28 and immunological synapse localization. We found association with CD28 to be essential for PKC-θ-mediated downstream signaling and the differentiation of T helper type 2 cells (T(H)2 cells) and interleukin 17-producing helper T cells (T(H)17 cells) but not of T helper type 1 cells (T(H)1 cells). Ectopic expression of V3 sequestered PKC-θ from the immunological synapse and interfered with its functions. Our results identify a unique mode of CD28 signaling, establish a molecular basis for the immunological synapse localization of PKC-θ and indicate V3-based 'decoys' may be therapeutic modalities for T cell-mediated inflammatory diseases.

PubMed Disclaimer

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors have no competing financial interests.

Figures

Figure 1
Figure 1

Requirement of PKC-θ-V3 for IS-cSMAC localization and signaling. (a) Immunofluorescence imaging of Prkcq−/ − OT-II Tg CD4+ T cells infected with retrovirus expressing GFP-tagged wild-type (WT) PKC-θ, PKC-θ-ΔV3 or PKC-θ+δV3 (green) and mixed (1:1 ratio) with CMAC (blue)-labeled APC, pre-incubated with or without Ova peptide. Fixed conjugates were stained with anti-talin plus a secondary Alexa 647-coupled antibody (red). (b) Quantitation of PKC-θ IS-cSMAC localization analyzed in ~40 T-APC conjugates as described in a. Only conjugates that had reorganized their talin and that had detectable PKC-θ were analyzed. ** p < .05. (c) Normalized luciferase (Luc) activity in MCC-specific T hybridoma cells cotransfected with empty vector or the indicated Xpress-tagged PKC-θ vectors, together with CD28-response element (RE/AP)-Luc reporter and a β-Gal reporter. Cells were incubated with I-Ek- and B7-1-expressing DCEK fibroblasts in the absence or presence of MCC peptide for 6 h. Transfected PKC-θ expression revealed by anti-Xpress immunoblotting is shown at the bottom. ** p < .05. Data are from three experiments. (d–e) CD69 (d) or CD25 and PKC-θ. (e) expression in GFP+ CD4+ T cells sorted from Rag1−/ − mice reconstituted with Prkcq−/ − BM cells transduced with empty vector, WT PKC-θ, or PKC-θ+δV3 and left unstimulated or stimulated overnight with anti-CD3 plus anti-CD28 mAbs. Data are representative of two experiments. (f–g) IL-2 production (f) and cell proliferation (g) in GFP+ CD4+ T cells isolated as in d and stimulated or not with anti-CD3 plus anti-CD28 mAbs for 48 h. Data are from two experiments.

Figure 2
Figure 2

The PKCθ V3 domain is required and sufficient for CD28 interaction. (a) Association of PKC-θ-V3 with CD28 in Prkcq−/ − CD4+ T cells infected with retrovirus expressing empty pMIG vector, or with the indicated PKCθ or PKCδ vectors. Cells were harvested and restimulated with CD3+CD28 mAbs for 5 min. Cell lysates were immunoprecipitated with anti-CD28 mAb, resolved by SDS-PAGE, and immunoblotted with the indicated Abs. (b) Jurkat T cells cotransfected with Myc-tagged PKCθ-V3 plus CFP-tagged CD28 were mixed with SEE-pulsed Raji B cells at a 1:1 ratio. Conjugates were fixed and stained with a rabbit anti-Myc Ab plus a secondary Alexa 488-coupled antibody. Data are from four experiments.

Figure 3
Figure 3

A PR motif in the V3 domain of PKCθ determines its IS localization and interacts with CD28. (a) PKCθ localization in CD4+ T cells from OT-II Tg mice infected with retrovirus expressing GFP-tagged WT PKCθ, PKCδ with an inserted PR motif (PKCδ+θPR), or WT PKCδ (green). Cells were stimulated, and conjugates were fixed, stained and analyzed as in Fig. 1a. (b) Quantitative analysis of the results shown in (a) was performed as in Fig. 1a. ** p < .05. (c) PKC-CD28 association in Prkcq−/ − CD4+ T cells infected with retrovirus expressing WT PKCθ, PKCδ+θPR, or WT PKCδ. Cells were harvested, restimulated with CD3+CD28 mAbs for 5 min (left panel) or left unstimulated (right panel). Asterisk in the right panel indicates the position of the immunoprecipitating antibody heavy chain. (d) Normalized Luc activity in MCC-specific T hybridoma cells cotransfected with the same vectors as in (a) together with an RE/AP and a β-Gal reporter plasmids. Cells were stimulated as in Fig. 1c, and Normalized Luc activity was determined in triplicates. ** p < .05. Data are from two experiments.

Figure 4
Figure 4

Importance of the PxxP motif in the V3 domain of PKCθ for IS localization and CD28 interaction. (a) Quantitative PKC-θ localization analysis using Prkcq−/ − OT-II CD4+ T cells infected with retrovirus expressing GFP-tagged PKCθ, or PKCθ-GFP fusion vectors containing the indicated proline mutations. Representative images are shown in Supplementary Fig. 5. ** p < .05. (b) PKC-θ-CD28 association in PKCθ −/ − CD4+ T cells infected with retrovirus expressing WT or proline-mutated PKC-θ. Cells were harvested and restimulated with CD3+CD28 mAbs for 5 min (c) MCC-specific T hybridoma cells were cotransfected with empty pEF vector or the indicated PKCθ mutants together with RE/APβ-Gal reporter plasmids. Cells were stimulated, and normalized Luc activity was determined as in Fig. 1c. ** p < .05. (d–f) T cell activation in PKC-θ-reconstituted BM chimeras. Prkcq−/ − BM cells transduced with retrovirus expressing empty pMIG vector, or the indicated PKCθ vectors were used to reconstitute Rag−/ − mice. Sorted GFP+ CD4+ T cells were left unstimulated or stimulated overnight with anti-CD3 plus anti-CD28 mAbs to determine the expression of CD69 or CD25 (d); or for 48 h to determine the production of IL-2 (f), and proliferation (g). Data are from two experiments.

Figure 5
Figure 5

Interaction between CD28 and the V3 of PKCθ is Lck-dependent. PKC-θ-Lck-CD28 association in Lck-deficient (JCam1.6) Jurkat cells cotransfected with Myc-tagged PKCθ-V3 plus WT Lck or its indicated mutants, Transfected cells were stimulated with anti-CD3 and anti-CD28 for 5 min, immunoprecipitated with an anti-Myc Ab, and immunoblotted for Lck and endogenous CD28. Data are from three experiments.

Figure 6
Figure 6

The V3 domain interferes with PKCθ-mediated signaling and T cell differentiation. (a) PKC-θ and V3 localization in OT-II CD4+ T cells infected with retrovirus expressing Myc-tagged non-mutated or proline-mutated V3 vectors. Infected cells (green) were harvested, stimulated, and fixed. Conjugates were stained with anti-Myc plus a secondary Alexa 555-coupled antibody (orange), and anti-PKCθ plus a secondary Alexa 647-coupled antibody (red). Cells were analyzed as in Fig. 1a. (b) Quantitative analysis of the results shown in (a) as in Fig. 1b. ** p < .05. (c) Reporter gene activation in MCC- specific T hybridoma cells cotransfected with indicated PKC-θ and/or V3 vectors together with RE/AP- Luc and β-Gal reporter plasmids. Cells were stimulated and analyzed as in Fig. 1c. (d) TH differentiation in naïve CD4+ T cells from B6 mice stimulated with anti-CD3 plus anti-CD28 mAbs, cultured under TH1-, TH2- or TH17-polarizing conditions, and retrovirally transduced with empty pMIG vector, or with the indicated PKC-θ V3 vectors. Cytokine-producing cells were analyzed by intracellular staining 8 h after restimulation. Right panels represent cumulative data showing percentage of cytokine-producing cells. Data are from six experiments.

Figure 7
Figure 7

V3 inhibits TH2-, but not TH1-mediated, lung inflammation. TH difefrentiation of anti-CD3- plus anti-CD28-stimulated OT-II CD4+ T cells cultured under TH2 (a–c) or TH1 (d, e) -polarizing conditions and retrovitrally transduced with the same PKCθ V3 vectors as in Fig. 6. Sorted GFP+ populations were adoptively transferred into naïve B6 mice, which were challenged with Ova. Total mononuclear cell infiltration in BAL fluid (a, d) and cytokine expression (b, c, e) were analyzed 1 d later. Data are from two experiments.

Comment in

  • PKC-θ: hitting the bull's eye.

    Dustin ML. Dustin ML. Nat Immunol. 2011 Oct 19;12(11):1031-2. doi: 10.1038/ni.2141. Nat Immunol. 2011. PMID: 22012436 No abstract available.

Similar articles

Cited by

References

    1. Dustin ML. The cellular context of T cell signaling. Immunity. 2009;30:482–492. - PMC - PubMed
    1. Dustin ML, Chakraborty AK, Shaw AS. Understanding the structure and function of the immunological synapse. Cold Spring Harb Perspect Biol. 2010;2:a002311. - PMC - PubMed
    1. Monks CR, Kupfer H, Tamir I, Barlow A, Kupfer A. Selective modulation of protein kinase C-θ during T-cell activation. Nature. 1997;385:83–86. - PubMed
    1. Monks CR, Freiberg BA, Kupfer H, Sciaky N, Kupfer A. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature. 1998;395:82–86. - PubMed
    1. Pfeifhofer C, et al. Protein kinase C θ affects Ca2+ mobilization and NFAT cell activation in primary mouse T cells. J Exp Med. 2003;197:1525–1535. - PMC - PubMed

Publication types

MeSH terms

Substances