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 subunit s and regulated by PKC mediated phosphorylation. AC II, stably express ed in HEK 239 cells, was incubated with the PKC activator tetradecanoy lphorbol acetate (TPA). Using cells metabolically labeled with [P-32]p hosphate, TPA caused concerted stimulation of basal and forskolin acti vated adenylyl cyclase together with incorporation of [P-32]phosphate into AC II protein. Enhanced phosphorylation was also indicated by a m onoclonal anti-phosphothreonine antibody. Assignment of TPA-induced [P -32]phosphate-incorporation to specific sites was achieved by a combin ation of chemical and immunochemical methods. Three out of five [P-32] labeled peptides that were generated by fragmentation with N-chlorosuc cinimide were also recognized by the monoclonal antibody BBC-4 [S. Mol lner, T. Pfeuffer, Eur. J. Biochem. 171 (1988) 265-271] directed again st an epitope 8 kDa from the extreme C-terminus. These findings sugges ted Ser-871 (consensus sequence ARSLK) and Thr-1057 (CTCR) as acceptor candidates of phorbolester induced phosphoryl transfer. (C) 1997 Else vier Science B.V.