Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells

J Biol Chem. 2018 Mar 23;293(12):4411-4421. doi: 10.1074/jbc.M117.809988. Epub 2018 Jan 29.


cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro, whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

Keywords: PKA Regulatory Subunit RI alpha (RI&α); phosphorylation; post-translational modification (PTM); protein kinase; protein kinase A (PKA); protein kinase G (PKG).

Publication types

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

MeSH terms

  • Binding Sites
  • Catalytic Domain
  • Cyclic AMP / metabolism*
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / genetics
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / metabolism*
  • Cyclic GMP-Dependent Protein Kinases / genetics
  • Cyclic GMP-Dependent Protein Kinases / metabolism*
  • HEK293 Cells
  • Humans
  • Phosphorylation
  • Protein Binding


  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit
  • Cyclic AMP
  • Cyclic GMP-Dependent Protein Kinases

Associated data

  • PDB/2qcs