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

ABSTRACT: 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 ? and ? isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RI? regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro, whereupon phosphorylated RI? 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 RI? phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RI? was validated as a target of PKGI? phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RI? (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 RI? 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 RI? 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 RI? creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

SUBMITTER: Haushalter KJ 

PROVIDER: S-EPMC5868259 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature