PHORBOL ESTER-INDUCED SENSITIZATION OF ADENYLYL-CYCLASE TYPE-II IS RELATED TO PHOSPHORYLATION OF THREONINE-1057

Following up the results from previous studies on chemical fragmentati on of TPA-treated, [P-32]-phosphate labeled adenylyl cyclase type II ( AC II) (Bol, G. F., Hulster, A., and Pfeuffer, T. in press) we have re placed serine 871 or threonine 1057 by alanine using site directed mut agenesis. Both mutants had unimpaired catalytic activity, however enha ncement by phorbolester TPA was reduced by 60-80% in the T1057A mutant , but not in the S871A mutant. The stimulation of adenylyl cyclase typ e II by beta gamma subunits of heterotrimeric G-proteins and that by P KC have been previously shown to be mutually exclusive (Zimmermann and Taussig (1996), J. Biol. Chem. 271, 27161-27166). This is in line wit h the present findings that AC II expressed in COS-1 cells was only ba rely stimulated (10%) by coexpressed beta gamma-subunits in presence o f TPA. Mutation of threonine 1057 to alanine however caused partial re gain of beta gamma-stimulation in the presence of TPA by 40%, as compa red to that of WT adenylyl cyclase type II which was 70% in the absenc e of TPA, These data strongly implicate the importance of threonine 10 57 as phosphate acceptor following PKC-mediated sensitisation of adeny lyl cyclase type II. (C) 1997 Academic Press.