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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.

Kong KF, Yokosuka T, Canonigo-Balancio AJ, Isakov N, Saito T, Altman A - Nat. Immunol. (2011)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

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.

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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.
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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.

Mentions: To determine if the V3 domain is required for the IS localization of PKC-θ, we constructed a V3 deletion mutant (PKC-θ-ΔV3). When retrovirally transduced into Prkcq−/ − TCR-transgenic (Tg) ovalbumin (Ova)-specific OT-II CD4+ T cells, wild-type PKC-θ localized in the center of IS, i.e., the cSMAC, following stimulation with Ova peptide-pulsed APCs (Fig. 1a,b), as evident from its central localization relative to that of talin, a known pSMAC marker4. In contrast, PKC-θ-ΔV3 did not translocate to the IS and instead remained largely cytosolic. Since V3 domain deletion could cause a gross conformational change, we next generated an exchange mutant, in which the native V3 domain of PKC-θ was replaced with the corresponding domain of PKC-δ (PKC-θ+δV3). Similar to PKC-θ-ΔV3, this mutant also failed to translocate to the IS/cSMAC (Fig. 1a,b). The peripheral IS localization of talin in T cells expressing both mutants indicates that the organization of a mature IS was not grossly impaired in the absence of wild-type PKC-θ. These data suggest that the unique V3 domain of PKC-θ is required for its selective IS/cSMAC localization.


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.

Kong KF, Yokosuka T, Canonigo-Balancio AJ, Isakov N, Saito T, Altman A - Nat. Immunol. (2011)

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.
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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.
Mentions: To determine if the V3 domain is required for the IS localization of PKC-θ, we constructed a V3 deletion mutant (PKC-θ-ΔV3). When retrovirally transduced into Prkcq−/ − TCR-transgenic (Tg) ovalbumin (Ova)-specific OT-II CD4+ T cells, wild-type PKC-θ localized in the center of IS, i.e., the cSMAC, following stimulation with Ova peptide-pulsed APCs (Fig. 1a,b), as evident from its central localization relative to that of talin, a known pSMAC marker4. In contrast, PKC-θ-ΔV3 did not translocate to the IS and instead remained largely cytosolic. Since V3 domain deletion could cause a gross conformational change, we next generated an exchange mutant, in which the native V3 domain of PKC-θ was replaced with the corresponding domain of PKC-δ (PKC-θ+δV3). Similar to PKC-θ-ΔV3, this mutant also failed to translocate to the IS/cSMAC (Fig. 1a,b). The peripheral IS localization of talin in T cells expressing both mutants indicates that the organization of a mature IS was not grossly impaired in the absence of wild-type PKC-θ. These data suggest that the unique V3 domain of PKC-θ is required for its selective IS/cSMAC localization.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

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.

Show MeSH
Related in: MedlinePlus