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

Biochemistry. 1991 Sep 3;30(35):8710-6. doi: 10.1021/bi00099a032.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / pharmacology
  • Animals
  • Binding, Competitive / drug effects
  • Cyclic AMP / analogs & derivatives*
  • Cyclic AMP / pharmacology
  • Enzyme Activation / drug effects
  • Mutagenesis
  • Protein Conformation
  • Protein Kinase Inhibitors
  • Protein Kinases / genetics*
  • Protein Kinases / metabolism
  • Swine
  • Thermodynamics
  • Thionucleotides / pharmacology*


  • Protein Kinase Inhibitors
  • Thionucleotides
  • adenosine-3',5'-cyclic phosphorothioate
  • Adenosine Triphosphate
  • Cyclic AMP
  • Protein Kinases