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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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Interaction between CD28 and the V3 of PKCθ is Lck-dependent. PKC-θ-Lck-CD28 association in Lck-deficient (JCam1.6) Jurkat cells cotransfected with Myc-tagged PKCθ-V3 plus WT Lck or its indicated mutants, Transfected cells were stimulated with anti-CD3 and anti-CD28 for 5 min, immunoprecipitated with an anti-Myc Ab, and immunoblotted for Lck and endogenous CD28. Data are from three experiments.
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Figure 5: Interaction between CD28 and the V3 of PKCθ is Lck-dependent. PKC-θ-Lck-CD28 association in Lck-deficient (JCam1.6) Jurkat cells cotransfected with Myc-tagged PKCθ-V3 plus WT Lck or its indicated mutants, Transfected cells were stimulated with anti-CD3 and anti-CD28 for 5 min, immunoprecipitated with an anti-Myc Ab, and immunoblotted for Lck and endogenous CD28. Data are from three experiments.

Mentions: The identification of the PxxP motif, a potential SH3-binding motif, in the PKC-θ V3 domain as being essential for CD28 association was intriguing because our mapping analysis of the CD28 cytoplasmic tail revealed that a C-terminal PR motif in murine CD28, i.e. a P206YAP209 motif, was required for the CD28-PKC-θ interaction (data not shown). This is the same CD28 motif required for PKC-θ-CD28 colocalization in the cSMAC, for IL-2 mRNA stabilization, and for lipid raft reorganization26,29–31, as well as for PKC-θ-dependent TH2- and TH17-mediated inflammatory responses13–17,21. Since direct association between the PR motifs in PKC-θ and CD28, respectively, is unlikely, we surmised that this interaction requires an intermediary molecule, with Lck kinase representing a strong candidate. Indeed, we found that the interaction between CD28 and V3 was absent in Lck-deficient Jurkat (JCam1.6) cells and was restored upon transfection with wild-type Lck, which was also included in the V3-CD28 complex (Fig. 5). The V3 domain associated with SH2-inactivated (R154K) Lck, but CD28 was not present in this complex. When JCam1.6 cells were reconstituted with SH3-inactivated (W97A) Lck, PKC-θ-V3 failed to precipitate both CD28 and Lck (Fig. 5). These findings suggest that Lck mediates the interaction between PKC-θ and CD28, with its SH3 domain binding the PR motif in PKC-θ-V3 and its SH2 domain binding phosphorylated Tyr-207 in the CD28 P206YAP209 motif30,32. This mode of a tripartite interaction (Supplementary Fig. 8) is consistent with findings that the SH2 domain of Lck has a much higher affinity than its SH3 domain for the phosphorylated CD28 PYAP motif33 and, conversely, that in stimulated T cells, the Lck SH3 domain is considerably more effective than the SH2 domain in binding PKC-θ34.


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)

Interaction between CD28 and the V3 of PKCθ is Lck-dependent. PKC-θ-Lck-CD28 association in Lck-deficient (JCam1.6) Jurkat cells cotransfected with Myc-tagged PKCθ-V3 plus WT Lck or its indicated mutants, Transfected cells were stimulated with anti-CD3 and anti-CD28 for 5 min, immunoprecipitated with an anti-Myc Ab, and immunoblotted for Lck and endogenous CD28. Data are from three experiments.
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Related In: Results  -  Collection

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Figure 5: Interaction between CD28 and the V3 of PKCθ is Lck-dependent. PKC-θ-Lck-CD28 association in Lck-deficient (JCam1.6) Jurkat cells cotransfected with Myc-tagged PKCθ-V3 plus WT Lck or its indicated mutants, Transfected cells were stimulated with anti-CD3 and anti-CD28 for 5 min, immunoprecipitated with an anti-Myc Ab, and immunoblotted for Lck and endogenous CD28. Data are from three experiments.
Mentions: The identification of the PxxP motif, a potential SH3-binding motif, in the PKC-θ V3 domain as being essential for CD28 association was intriguing because our mapping analysis of the CD28 cytoplasmic tail revealed that a C-terminal PR motif in murine CD28, i.e. a P206YAP209 motif, was required for the CD28-PKC-θ interaction (data not shown). This is the same CD28 motif required for PKC-θ-CD28 colocalization in the cSMAC, for IL-2 mRNA stabilization, and for lipid raft reorganization26,29–31, as well as for PKC-θ-dependent TH2- and TH17-mediated inflammatory responses13–17,21. Since direct association between the PR motifs in PKC-θ and CD28, respectively, is unlikely, we surmised that this interaction requires an intermediary molecule, with Lck kinase representing a strong candidate. Indeed, we found that the interaction between CD28 and V3 was absent in Lck-deficient Jurkat (JCam1.6) cells and was restored upon transfection with wild-type Lck, which was also included in the V3-CD28 complex (Fig. 5). The V3 domain associated with SH2-inactivated (R154K) Lck, but CD28 was not present in this complex. When JCam1.6 cells were reconstituted with SH3-inactivated (W97A) Lck, PKC-θ-V3 failed to precipitate both CD28 and Lck (Fig. 5). These findings suggest that Lck mediates the interaction between PKC-θ and CD28, with its SH3 domain binding the PR motif in PKC-θ-V3 and its SH2 domain binding phosphorylated Tyr-207 in the CD28 P206YAP209 motif30,32. This mode of a tripartite interaction (Supplementary Fig. 8) is consistent with findings that the SH2 domain of Lck has a much higher affinity than its SH3 domain for the phosphorylated CD28 PYAP motif33 and, conversely, that in stimulated T cells, the Lck SH3 domain is considerably more effective than the SH2 domain in binding PKC-θ34.

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