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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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V3 inhibits TH2-, but not TH1-mediated, lung inflammation. TH difefrentiation of anti-CD3- plus anti-CD28-stimulated OT-II CD4+ T cells cultured under TH2 (a–c) or TH1 (d, e) -polarizing conditions and retrovitrally transduced with the same PKCθ V3 vectors as in Fig. 6. Sorted GFP+ populations were adoptively transferred into naïve B6 mice, which were challenged with Ova. Total mononuclear cell infiltration in BAL fluid (a, d) and cytokine expression (b, c, e) were analyzed 1 d later. Data are from two experiments.
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Figure 7: V3 inhibits TH2-, but not TH1-mediated, lung inflammation. TH difefrentiation of anti-CD3- plus anti-CD28-stimulated OT-II CD4+ T cells cultured under TH2 (a–c) or TH1 (d, e) -polarizing conditions and retrovitrally transduced with the same PKCθ V3 vectors as in Fig. 6. Sorted GFP+ populations were adoptively transferred into naïve B6 mice, which were challenged with Ova. Total mononuclear cell infiltration in BAL fluid (a, d) and cytokine expression (b, c, e) were analyzed 1 d later. Data are from two experiments.

Mentions: We further analyzed the effect of PKC-θ-V3 in vivo in an airway inflammation model using a T cell adoptive transfer system. Mice receiving OT-II TH2 cells transduced with an empty vector developed an inflammatory response by comparison with PBS-challenged control mice, as evidenced by increased number of infiltrating leukocytes in the bronchoalveolar lavages (BAL) fluid (Fig. 7a) and augmented levels of signature TH2 cytokines, IL-4 (Fig. 7b) and IL-5 (Fig. 7c). Introduction of V3 into the transferred TH2 cells ameliorated the disease by decreasing the levels of infiltrating cells and TH2 cytokines to basal levels (Fig. 7a,b,c). However, expression of the PR motif-deleted V3 domain failed to inhibit the inflammatory response. The 4PA mutant partially rescued the inhibition, most likely due to the fact that surrounding amino acid residues in addition to the critical Pro residues also contribute to the regulatory function of the V3 domain.


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)

V3 inhibits TH2-, but not TH1-mediated, lung inflammation. TH difefrentiation of anti-CD3- plus anti-CD28-stimulated OT-II CD4+ T cells cultured under TH2 (a–c) or TH1 (d, e) -polarizing conditions and retrovitrally transduced with the same PKCθ V3 vectors as in Fig. 6. Sorted GFP+ populations were adoptively transferred into naïve B6 mice, which were challenged with Ova. Total mononuclear cell infiltration in BAL fluid (a, d) and cytokine expression (b, c, e) were analyzed 1 d later. Data are from two experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: V3 inhibits TH2-, but not TH1-mediated, lung inflammation. TH difefrentiation of anti-CD3- plus anti-CD28-stimulated OT-II CD4+ T cells cultured under TH2 (a–c) or TH1 (d, e) -polarizing conditions and retrovitrally transduced with the same PKCθ V3 vectors as in Fig. 6. Sorted GFP+ populations were adoptively transferred into naïve B6 mice, which were challenged with Ova. Total mononuclear cell infiltration in BAL fluid (a, d) and cytokine expression (b, c, e) were analyzed 1 d later. Data are from two experiments.
Mentions: We further analyzed the effect of PKC-θ-V3 in vivo in an airway inflammation model using a T cell adoptive transfer system. Mice receiving OT-II TH2 cells transduced with an empty vector developed an inflammatory response by comparison with PBS-challenged control mice, as evidenced by increased number of infiltrating leukocytes in the bronchoalveolar lavages (BAL) fluid (Fig. 7a) and augmented levels of signature TH2 cytokines, IL-4 (Fig. 7b) and IL-5 (Fig. 7c). Introduction of V3 into the transferred TH2 cells ameliorated the disease by decreasing the levels of infiltrating cells and TH2 cytokines to basal levels (Fig. 7a,b,c). However, expression of the PR motif-deleted V3 domain failed to inhibit the inflammatory response. The 4PA mutant partially rescued the inhibition, most likely due to the fact that surrounding amino acid residues in addition to the critical Pro residues also contribute to the regulatory function of the V3 domain.

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