Using Markov State Models to Develop a Mechanistic Understanding of Protein Kinase A Regulatory Subunit RIα Activation in Response to cAMP Binding

J Biol Chem. 2014 Oct 24;289(43):30040-51. doi: 10.1074/jbc.M114.568907. Epub 2014 Sep 8.

Abstract

Protein kinase A (PKA) holoenzyme consists of two catalytic (C) subunits and a regulatory (R) subunit dimer (R(2)C(2)). The kinase is activated by the binding of cAMPs to the two cyclic nucleotide binding domains (CBDs), A and B, on each R-subunit. Despite extensive study, details of the allosteric mechanisms underlying the cooperativity of holoenzyme activation remain unclear. Several Markov state models of PKA-RIα were developed to test competing theories of activation for the R(2)C(2) complex. We found that CBD-B plays an essential role in R-C interaction and promotes the release of the first C-subunit prior to the binding to CBD-A. This favors a conformational selection mechanism for release of the first C-subunit of PKA. However, the release of the second C-subunit requires all four cAMP sites to be occupied. These analyses elucidate R-C heterodimer interactions in the cooperative activation of PKA and cAMP binding and represent a new mechanistic model of R(2)C(2) PKA-RIα activation.

Keywords: Allosteric Regulation; Computer Modeling; Cooperativity; Cyclic AMP (cAMP); Protein Kinase A (PKA).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cyclic AMP / metabolism*
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / chemistry
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / metabolism*
  • Enzyme Activation / drug effects
  • Markov Chains*
  • Models, Molecular*
  • Mutant Proteins / metabolism
  • Oligopeptides / pharmacology
  • Protein Multimerization / drug effects
  • Protein Structure, Tertiary
  • Reproducibility of Results

Substances

  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit
  • Mutant Proteins
  • Oligopeptides
  • kemptide
  • Cyclic AMP