Critical role of novel Thr‐219 autophosphorylation for the cellular function of PKCθ in T lymphocytes

Phosphopeptide mapping identified a major autophosphorylation site, phospho (p)Thr-219, between the tandem C1 domains of the regulatory fragment in protein kinase C (PKC)θ. Confirmation of this identification was derived using (p)Thr-219 antisera that reacted with endogenous PKCθ in primary CD3+ T cells after stimulation with phorbol ester, anti-CD3 or vanadate. The T219A mutation abrogated the capacity of PKCθ to mediate NF-κB, NF-AT and interleukin-2 promoter transactivation, and reduced PKCθ's ability in Jurkat T cells to phosphorylate endogenous cellular substrates. In particular, the T219A mutation impaired crosstalk of PKCθ with Akt/PKBα in NF-κB activation. Yet, this novel (p)Thr-219 site did not affect catalytic activity or second-messenger lipid-binding activity in vitro. Instead, the T219A mutation prevented proper recruitment of PKCθ in activated T cells. The PKCθT219A mutant defects were largely rescued by addition of a myristoylation signal to force its proper membrane localization. We conclude that autophosphorylation of PKCθ at Thr-219 plays an important role in the correct targeting and cellular function of PKCθ upon antigen receptor ligation.