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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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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.
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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.

Mentions: We hypothesized that the importance of the PKC-θ V3 domain for the enzyme’s IS localization and function reflects a critical association of the V3 domain with a ligand that recruits it to the IS. Given the reported colocalization of PKC-θ and CD28 in the IS25,26 and the phorbol ester-induced association between the two26, we examined whether anti-CD3-CD28 costimulation will cause PKC-θ to associate with CD28. Indeed, PKC-θ coimmunoprecipitated with CD28 from anti-CD3-CD28-stimulated T cells (Supplementary Fig. 3a). Maximal association was observed after 5 min of costimulation, and it declined thereafter but was still seen up to 30 min of stimulation. To identify the PKC-θ region required for this interaction, we transfected Jurkat T cells with a series of PKC-θ deletion mutants (Supplementary Fig. 3b). Upon anti-CD3-CD28 costimulation, wild-type PKC-θ as well as a deletion mutant of the N-terminal C2 domain (ΔC2), previously shown to negatively regulate the activation of PKC-θ27, coimmunoprecipitated with CD28 (Supplementary Fig. 3c). However, deletion of the V3 domain abolished this interaction. Deletion of both the C2 and C1a domains of PKC-θ (ΔC2+C1a) reduced, but did not abolish the interaction. We repeated this analysis in primary Prkcq−/ − CD4+ T cells, which were transduced with different PKC-θ- or PKC-δ-expressing retroviruses. The ΔV3 mutant (Fig. 2a, left panel) as well as the PKC-θ+δV3 mutant or wild-type PKC-δ (Fig. 2a, middle panel) did not coimmunoprecipitate with CD28. Thus, the V3 domain of PKC-θ is required for the inducible interaction with CD28. Moreover, the V3 domain was sufficient for this interaction since retrovirus-transduced Myc-tagged V3 coimmunoprecipitated with endogenous CD28 from Prkcq−/ − primary CD4+ T cells (Fig. 2a, right panel). The V3 domain also colocalized with CFP-tagged CD28 in the IS of cotransfected Jurkat T cells (Fig. 2b). Therefore, the PKC-θ V3 domain is necessary and sufficient for the CD3-CD28-induced interaction of PKC-θ with CD28.


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 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.
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Related In: Results  -  Collection

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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.
Mentions: We hypothesized that the importance of the PKC-θ V3 domain for the enzyme’s IS localization and function reflects a critical association of the V3 domain with a ligand that recruits it to the IS. Given the reported colocalization of PKC-θ and CD28 in the IS25,26 and the phorbol ester-induced association between the two26, we examined whether anti-CD3-CD28 costimulation will cause PKC-θ to associate with CD28. Indeed, PKC-θ coimmunoprecipitated with CD28 from anti-CD3-CD28-stimulated T cells (Supplementary Fig. 3a). Maximal association was observed after 5 min of costimulation, and it declined thereafter but was still seen up to 30 min of stimulation. To identify the PKC-θ region required for this interaction, we transfected Jurkat T cells with a series of PKC-θ deletion mutants (Supplementary Fig. 3b). Upon anti-CD3-CD28 costimulation, wild-type PKC-θ as well as a deletion mutant of the N-terminal C2 domain (ΔC2), previously shown to negatively regulate the activation of PKC-θ27, coimmunoprecipitated with CD28 (Supplementary Fig. 3c). However, deletion of the V3 domain abolished this interaction. Deletion of both the C2 and C1a domains of PKC-θ (ΔC2+C1a) reduced, but did not abolish the interaction. We repeated this analysis in primary Prkcq−/ − CD4+ T cells, which were transduced with different PKC-θ- or PKC-δ-expressing retroviruses. The ΔV3 mutant (Fig. 2a, left panel) as well as the PKC-θ+δV3 mutant or wild-type PKC-δ (Fig. 2a, middle panel) did not coimmunoprecipitate with CD28. Thus, the V3 domain of PKC-θ is required for the inducible interaction with CD28. Moreover, the V3 domain was sufficient for this interaction since retrovirus-transduced Myc-tagged V3 coimmunoprecipitated with endogenous CD28 from Prkcq−/ − primary CD4+ T cells (Fig. 2a, right panel). The V3 domain also colocalized with CFP-tagged CD28 in the IS of cotransfected Jurkat T cells (Fig. 2b). Therefore, the PKC-θ V3 domain is necessary and sufficient for the CD3-CD28-induced interaction of PKC-θ with CD28.

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