Compensatory Regulation of RIα Protein Levels in Protein Kinase A Mutant Mice

Abstract

The cAMP-dependent protein kinase holoenzyme is assembled from regulatory (R) and catalytic (C) subunits that are expressed in tissue-specific patterns. Despite the dispersion of the R and C subunit genes to different chromosomal loci, mechanisms exist that coordinately regulate the intracellular levels of R and C protein such that cAMP-dependent regulation is preserved. We have created null mutations in the RI␤ and RII␤ regulatory subunit genes in mice, and find that both result in an increase in the level of RI␣ protein in tissues that normally express the ␤ isoforms. Examination of RI␣ mRNA levels and the rates of RI␣ protein synthesis in wild type and RII␤ mutant mice reveals that the mechanism of this biochemical compensation by RI␣ does not involve transcriptional or translational control. These in vivo findings are consistent with observations made in cell culture, where we demonstrate that the overexpression of C␣ in NIH 3T3 cells results in increased RI␣ protein without increases in the rate of RI␣ synthesis or the level of RI␣ mRNA. Pulse-chase experiments reveal a 4 -5-fold increase in the half-life of RI␣ protein as it becomes incorporated into the holoenzyme. Compensation by RI␣ stabilization may represent an important biological mechanism that safeguards cells from unregulated catalytic subunit activity.The cAMP-dependent protein kinase (PKA) 1 is a key regulatory enzyme responsible for the intracellular transduction of a variety of extracellular signals and for the maintenance of numerous aspects of cellular homeostasis (1). The holoenzyme is composed of a regulatory (R) subunit dimer complexed with two catalytic (C) subunits. Two molecules of cAMP bind to each R subunit causing release of enzymatically active C subunits, which then modify the activity of target proteins by reversible phosphorylation of serine or threonine residues located within an appropriate consensus sequence (2).Four R subunit isoforms and two C subunit isoforms of PKA have been characterized in the mouse (3). They are highly conserved among mammals, encoded by unique genes located on separate chromosomes, and show unique patterns of gene expression. The ␣-isoforms are expressed ubiquitously while ␤ isoforms show more restricted patterns of expression. RI␤ is induced relatively late in development and is highly expressed in neural tissues (4 -6). RII␤ is expressed during embryogenesis in mouse brain, spinal cord, and liver (7). In adult mice RII␤ protein is most abundant in brain and brown and white adipose tissue, with lower expression in testis and ovary (8). C␤ is most abundant in the brain, but lower levels of C␤ mRNA are found in all tissues examined (9).PKA holoenzymes can be separated by ion-exchange chromatography and analysis of a variety of mammalian tissues has revealed significant differences in the ratio of type I (RI-containing) to type II (RII-containing) holoenzyme (10). In rats and mice, brain and adipose tissue contain principally the type II holoenzyme, while heart and liver contain mainly type I. The rat...

Sodium/Calcium Exchanger (NCX1) Macromolecular Complex

“…RI subunits are dynamic and developmentally regulated (58). The association of RI with NCX1 complex suggests that the PKA modulation of the NCX1 is more dynamic than previously appreciated and that phosphorylation may be an important signal or regulator during development.…”

Deficiency of the RIIβ subunit of PKA affects locomotor activity and energy homeostasis in distinct neuronal populations

“…We have shown previously that RIIβ deficiency leads to a compensatory increase in type I R subunits but a dramatic decrease in total PKA activity in the striatum (7). This decrease in PKA activity is likely due to the release of free C subunit, which turns over rapidly when not complexed in a holoenzyme with R subunits (30). The RIIβ KO mice exhibit deficits in c-fos and dynorphin gene induction in D1R neurons and reduced induction of neurotensin and c-fos by D2R antagonists (7,11), suggesting that both the direct and indirect pathways are affected by the RIIβ mutation.…”

Mutually regulated expression of caspase-activated DNase and its inhibitor for apoptotic DNA fragmentation

“…For example, the cAMP-dependent protein kinase is composed of regulatory and catalytic subunits, and the regulatory subunit is rapidly degraded in cells when it is not associated with the catalytic subunit. 15,16 Since ICAD-L is complexed with CAD, 5 it is possible that the ICAD-L that is associated with CAD is more stable than is uncomplexed ICAD-L. This may explain the lack of effect of CAD on the expression of ICAD-S, which is not complexed with CAD.…”