Department of Chemistry, Indiana University, Bloomington 47405.
Increasing evidence has implicated a post-translational phosphorylation in the production of a catalytically competent protein kinase C. Here we present structural and biochemical evidence that Thr500 of protein kinase C-beta II is the residue phosphorylated by another kinase. Modeling studies indicate that this residue is part of a "lip" structure at the entrance of the catalytic site; phosphorylation on this lip, or "activation loop," is central to the regulation of three kinases whose structures have been elucidated (Taylor, S. S., and Radzio-Andzelm, E. (1994) Structure 2, 345-355). Biochemical data reveal that mutation of Thr500 to an acidic residue (Glu) results in expression of catalytically active protein kinase C in COS cells. In contrast, mutation of this residue to a neutral, non-phosphorylatable residue (Val) results in expression of inactive enzyme. Thus, negative charge at position 500 is required for catalytically competent protein kinase C-beta II. These data suggest that signal processing by protein kinase C cannot occur until the enzyme is first phosphorylated by a protein kinase C kinase.