Identification of the phosphorylated region responsible for the permissive activation of protein kinase C.

Protein kinase C (PKC) appears to require phosphorylation in order to function as an effector-dependent kinase (Pears, C., Stabel, S., Cazaubon, S., and Parker, P. J. (1992) Biochem. J. 283, 515-518). By site-directed mutagenesis of the PKC alpha cDNA, it has been shown that a region including 3 threonine residues (Thr-494, Thr-495, and Thr-497), present in the catalytic domain, is involved in controlling PKC activity. Substitution of these 3 threonine residues by alanine residues leads to the expression, in COS-1 cells, of an unphosphorylated protein with an apparent molecular mass of 76 kDa, similar to that determined for the primary translation product. The biochemical characterization of this PKC alpha mutant reveals that it is a functional phorbol ester-binding protein but retains no kinase activity. Coexpression of this PKC alpha mutant and wild type PKC beta demonstrates that the mutant has a dominant effect upon PKC beta phosphorylation. The location of this region and its phosphorylation in relation to PKC function are discussed.