Direct interaction between PKCθ and ZAP70 kinases is required for T cell activation (IRM7P.716)

Abstract

Abstract PKCθ plays a key role in T cell activation, but the mechanisms that control its activation are not fully understood. We previously reported that the PKCθ N-terminal C2 region is a novel regulatory phosphotyrosine (pTyr)-binding domain. However, the physiological pTyr-containing C2-binding ligand has remained unknown. Using a GST-PKCθ-C2 recombinant fusion protein in pull-down assays, we detected a ~75-kDa pTyr-containing protein that associated with wild-type PKCθ-C2, but not with a pTyr non-binding PKCθ-C2 mutant after TCR/CD28 costimulation. Mass spectrometry analysis identified this protein as ZAP70 kinase. The stimulation-induced association of ZAP70 with full-length PKCθ or PKCθ-C2 in T cells was confirmed by co-immunoprecipitation assays, and was mediated by residues pY315 and pY319 in interdomain B of ZAP70. This transient association upon T cell activation required Lck kinase, followed by rapid complex dissociation that led to ZAP70 phosphorylation on Y493, resulting in full activation of ZAP70 and downstream signals. Mutation of two PKCθ-C2 residues essential for pTyr binding abolished PKCθ’s ability to rescue TCR- and CD28-induced IL-2 production, proliferation and Th2 differentiation in PKCθ-deficient T cells, without affecting T cell development or Th1 differentiation. These data suggest anchoring of PKCθ-C2 domain to phosphorylated interdomain B of ZAP70 promotes the activation of both kinases, thereby provided a novel pharmaceutical target for immunosuppression.