Adenylyl cyclase type II is stimulated by PKC via C-terminal phosphorylation

Adenylyl cyclases of the type II family differ from other subforms in that they are conditionally stimulated via alpha(s)/beta gamma subunits and regulated by PKC mediated phosphorylation. AC II, stably expressed in HEK 239 cells, was incubated with the PKC activator tetradecanoylphorbol acetate (TPA). Using cells metabolically labeled with [P-32]phosphate, TPA caused concerted stimulation of basal and forskolin activated adenylyl cyclase together with incorporation of [P-32]phosphate into AC II protein. Enhanced phosphorylation was also indicated by a monoclonal anti-phosphothreonine antibody. Assignment of TPA-induced [P-32]phosphate-incorporation to specific sites was achieved by a combination of chemical and immunochemical methods. Three out of five [P-32]labeled peptides that were generated by fragmentation with N-chlorosuccinimide were also recognized by the monoclonal antibody BBC-4 [S. Mollner, T. Pfeuffer, Eur. J. Biochem. 171 (1988) 265-271] directed against an epitope 8 kDa from the extreme C-terminus. These findings suggested Ser-871 (consensus sequence ARSLK) and Thr-1057 (CTCR) as acceptor candidates of phorbolester induced phosphoryl transfer. (C) 1997 Elsevier Science B.V.