Characterization of protein kinase C θ activation loop autophosphorylation and the kinase domain catalytic mechanism

Protein kinase C theta (PKC theta), a member of the Ca2+-independent novel subfamily of PKCs, is required for T-cell receptor (TCR) signaling and IL2 production. PKC theta-deficient mice have impaired Th2 responses in a murine ova-induced asthma model, while Th1 responses are normal. As an essential component of the TCR signaling complex, PKC theta is a unique T-cell therapeutic target in the specific treatment of T-cell-mediated diseases. We report here the PKC theta autophosphorylation characteristics and elucidation of the catalytic mechanism of the PKC theta kinase domain using steady-state kinetics. Key phosphorylated residues of the active PKC theta kinase domain expressed in Escherichia coli were characterized, and mutational analysis of the kinase domain was performed to establish the autophosphorylation and kinase activity relationships. Initial velocity, product inhibition, and dead-end inhibition studies provided assignments of the kinetic mechanism of PCK theta(362-706) as ordered, wherein ATP binds kinase first and ADP is released last. Effects of solvent viscosity and ATP gamma S on PKC theta catalysis demonstrated product release is partially rate limiting. Our studies provide important mechanistic insights into kinase activity and phosphorylation-mediated regulation of the novel PKC isoform, PKC theta. These results should aid the design and discovery of PKC theta antagonists as therapeutics for modulating T-cell-mediated immune and respiratory diseases.