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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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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.
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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.

Mentions: The quality of T cell activation appears to correlate with the clustering of PKC-θ in the IS4,24. However, direct evidence that the IS/cSMAC localization of PKC-θ is essential for its downstream functions is missing. Therefore, we investigated whether loss or replacement of the PKC-θ V3 domain impairs the activation of key transcription factors that are essential for productive T cell activation and are known targets of PKC-θ5,8–12, i.e. NF-κB, AP-1 and NFAT (Fig. 1c). Stimulation of empty vector-transfected cells with peptide-APCs resulted consistently in minimal reporter gene stimulation (see also Figs. 3d, 4c, 6c), most likely reflecting the relatively weak stimulus provided by peptide-APC stimulation as compared to the more standard use of saturating anti-CD3-CD28 antibody concentrations. T cells transfected with wild-type PKC-θ showed a significant increase in the basal activities of a CD28 response element (RE/AP; Fig. 1c) and NFAT (Supplementary Fig. 2), which was further increased by peptide-APC stimulation. However, the activation of these reporter genes was completely abrogated in T cells transfected with PKC-θ-ΔV3 or PKC-θ+δV3 (Fig. 1c and Supplementary Fig. 2).


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: The quality of T cell activation appears to correlate with the clustering of PKC-θ in the IS4,24. However, direct evidence that the IS/cSMAC localization of PKC-θ is essential for its downstream functions is missing. Therefore, we investigated whether loss or replacement of the PKC-θ V3 domain impairs the activation of key transcription factors that are essential for productive T cell activation and are known targets of PKC-θ5,8–12, i.e. NF-κB, AP-1 and NFAT (Fig. 1c). Stimulation of empty vector-transfected cells with peptide-APCs resulted consistently in minimal reporter gene stimulation (see also Figs. 3d, 4c, 6c), most likely reflecting the relatively weak stimulus provided by peptide-APC stimulation as compared to the more standard use of saturating anti-CD3-CD28 antibody concentrations. T cells transfected with wild-type PKC-θ showed a significant increase in the basal activities of a CD28 response element (RE/AP; Fig. 1c) and NFAT (Supplementary Fig. 2), which was further increased by peptide-APC stimulation. However, the activation of these reporter genes was completely abrogated in T cells transfected with PKC-θ-ΔV3 or PKC-θ+δV3 (Fig. 1c and Supplementary Fig. 2).

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