Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3197934&req=5

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.

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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3197934&req=5

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.
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