title: 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.
abstract: Previous investigations revealed that under physiological conditions in the presence of MgATP the phosphorothioate analogue of cAMP, (Rp)-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, 255-260]. For the type I holoenzyme, the antagonist properties of (Rp)-cAMPS are shown here to be absolutely dependent on MgATP. In the absence of MgATP, (Rp)-cAMPS serves as a weak agonist with a Ka of 7.9 microM. The high-affinity binding of MgATP imposes a barrier on cAMP-induced activation of the homoenzyme--a barrier that both cAMP and (Sp)-cAMPS, but not (Rp)-cAMPS, can overcome. In the absence of MgATP, this barrier no longer exists, and (Rp)-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 (Rp)-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 (Rp)-cAMPS. In the case of the type II holoenzyme, (Rp)-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.
authors.affiliation: Department of Chemistry, University of California, San Diego, La Jolla 92093-0654.
authors.forename: W R
authors.initials: WR
authors.lastname: Dostmann
authors.forename: S S
authors.initials: SS
authors.lastname: Taylor
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D016296:Mutagenesis; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D013552:Swine; D013816:Thermodynamics; D013873:Thionucleotides / Q000494:pharmacology*
chemical_list: D047428:Protein Kinase Inhibitors; D013873:Thionucleotides; C016957:adenosine-3',5'-cyclic phosphorothioate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases