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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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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.
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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.

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)

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.
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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.
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