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 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.
Highlights
CAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells
To extend previous reports demonstrating that PKA RI␣ could be phosphorylated by PKGI␣, we performed in vitro phosphorylation reactions using purified recombinant bovine RI␣–PKAc holoenzyme and PKGI␣
These results are a bit unexpected, because cyclic nucleotide free and cAMPbound PKGI␣ should not be as active as the cGMP-bound kinase
Summary
Dept. of Chemistry & Biochemistry, University of California San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0654. The linker– hinge region of PKA regulatory subunit proteins contains the autoinhibitory motif that allows for selective inhibition of PKAc at the kinase active site; this inhibitor sequence (IS) differs significantly between RI and RII [11, 16]. Because of the pseudosubstrate nature of the IS in RI␣, the high affinity binding of this motif to PKAc (in complex with ATP and two magnesium ions, Mg2ATP) presents a kinetic barrier for activation, whereas this is not critical for RII subunits capable of phosphorylation by PKAc [16, 23]. The interaction of Ser101 with Gln and His facilitates a hydrogen-bond network near the activation-loop phosphorylation site Thr197 in PKAc. we can further postulate that phosphorylation of Ser101 will bring about a negative charge– charge repulsion effect caused by the juxtaposition of two phosphate groups within this binding interface. Finding evidence of direct PKG–PKA cross-talk in the context of cellular signaling would aid our understanding of how these two related pathways could regulate physiologically relevant systems where both proteins are expressed (i.e. smooth muscle and cardiac tissues) [25, 26]
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