IDENTIFYING THE MOLECULAR SWITCHES THAT DETERMINE WHETHER (RP)-CAMPS FUNCTIONS AS AN ANTAGONIST OR AN AGONIST IN THE ACTIVATION OF CAMP-DEPENDENT PROTEIN KINASE-I

Previous investigations revealed that under physiological conditions in the presence of MgATP the phosphorothioate analogue of cAMP, (R(p))-cAMPS, is a competitive inhibitor and antagonist for cAMP for cAMP-dependent protein kinases I and II [DeWit et al., (1984) Eur. J. Biochem. 142, 255-2601. For the type I holoenzyme, the antagonist properties of (R(p))-cAMPS are shown here to be absolutely dependent on MgATP. In the absence of MgATP, (R(p))-cAMPS serves as a weak agonist with a K(a) of 7.9-mu-M. The high-affinity binding of MgATP imposes a barrier on cAMP-induced activation of the holoenzyme - a barrier that both cAMP and (S(p))-cAMPS, but not (R(p))-cAMPS, can overcome. In the absence of MgATP, this barrier no longer exists, and (R(p))-cAMPS functions as an agonist. The holoenzyme also was formed with mutant regulatory subunits. Replacing the essential arginine, predicted to bind the exocyclic oxygens of cAMP, in site A with lysine abolishes high-affinity binding of cAMP to site A. The holoenzyme formed with this mutant R-subunit is activated by (R(p))-cAMPS in both the presence and absence of MgATP. These results suggest that the stereospecific requirements for holoenzyme activation involve this guanidinium side chain. Mutations that eliminate the high-affinity binding of MgATP, such as the introduction of an autophosphorylation site in the autoinhibitory domain, also generate a holoenzyme that can be activated by (R(p))-cAMPS. In the case of the type II holoenzyme, (R(p))-cAMPS is an antagonist in both the presence and absence of MgATP, emphasizing distinct roles for MgATP in these two forms of cAMP-dependent protein kinase.