The Major Catalytic Subunit Isoforms of cAMP-dependent Protein Kinase Have Distinct Biochemical Properties in Vitro and in Vivo

Abstract

Two isoforms of the catalytic subunit of cAMPdependent protein kinase, C␣ and C␤1, are known to be widely expressed in mammals. Although much is known about the structure and function of C␣, few studies have addressed the possibility of a distinct role for the C␤ proteins. The present study is a detailed comparison of the biochemical properties of these two isoforms, which were initially expressed in Escherichia coli and purified to homogeneity. C␤1 demonstrated higher K m values for some peptide substrates than did C␣, but C␤1 was insensitive to substrate inhibition, a phenomenon that was observed with C␣ at substrate concentrations above 100 M. C␣ and C␤1 displayed distinct IC 50 values for the ␣ and ␤ isoforms of the protein kinase inhibitor, protein kinase inhibitor (5-24) peptide, and the type II␣ regulatory subunit (RII␣). Of particular interest, purified type II holoenzyme containing C␤1 exhibited a 5-fold lower K a value for cAMP (13 nM) than did type II holoenzyme containing C␣ (63 nM). This latter result was extended to in vivo conditions by employing a transcriptional activation assay. In these experiments, luciferase reporter activity in COS-1 cells expressing RII␣ 2 C␤1 2 holoenzyme was half-maximal at 12-fold lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RII␣ 2 C␣ 2 holoenzyme. These results provide evidence that type II holoenzyme formed with C␤1 is preferentially activated by cAMP in vivo and suggest that activation of the holoenzyme is determined in part by interactions between the regulatory and catalytic subunits that have not been described previously.cAMP exerts its effects in mammalian cells primarily through the activation of cAMP-dependent protein kinase (cAK), 1 a tetrameric enzyme consisting of two catalytic (C) and two regulatory (R) subunits. Occupation of the cyclic nucleotide binding sites of the R subunits by cAMP results in relief of R subunit inhibition of the C subunits and dissociation of the holoenzyme complex. The dissociated, active C subunit can then affect the cell physiology via phosphorylation of a wide variety of protein substrates (1-3).One possible explanation for the diversity of cellular responses to cAMP is the presence of multiple isoforms of cAK. Three C and four R mammalian subunit isoforms have been described to date: C␣, C␤, C␥, RI␣, RI␤, RII␣, and RII␤. Splice variants of the prototypic C␤ protein (C␤1) also have been reported, giving rise to C␤2 and C␤3 isoforms (4, 5). C␣ and C␤1 have been cloned from numerous sources (6 -9), whereas C␥ has only been cloned from human testis and has not been reported in other species (10). The amino acid sequences of C␣ and C␤1 within a given species are 91% identical (7); however, the amino acid identity of C␣ proteins from different species is significantly greater (98 -100%) (7, 11), suggesting that each kinase plays a distinct role(s) in cellular regulation (7,12). Furthermore, C␣ is ubiquitously expressed in mammalian tissues, whereas C␤1...

Inhibition of Xenopus oocyte meiotic maturation by catalytically inactive protein kinase A

“…As the available anti-PKA antibodies cross-reacted with both XPKA␣ and XPKA␤, it was not possible to determine which one was the main isoform in oocytes. It should be noted, however, that overexpression of either XPKA␣ and XPKA␤ can block oocyte maturation and only subtle differences have been reported between mammalian PKA␣ and PKA␤ in substrate specificity and binding to the regulatory subunits (32).…”

cAMP-dependent Protein Kinase Types I and II Differentially Regulate cAMP Response Element-mediated Gene Expression

“…Specifically, RI␣-containing holoenzymes need a 5-fold higher concentration of cAMP for activation than RI␤-containing holoenzymes (22,23). Moreover, type II holoenzymes containing C␤ are also more sensitive to dissociation by cAMP than are the C␣-containing type II holoenzymes (24). Thus, the preferential combination of C␤-RI␤ in NG108-15 cells may confer a greater sensitivity to cAMP for this holoenzyme than for the C␣-RII␤ holoenzyme.…”

Cyclic AMP- and Cyclic GMP-dependent Protein Kinases Differ in Their Regulation of Cyclic AMP Response Element-dependent Gene Transcription

“…24 h after transfection, cells were incubated for an additional 24 h in Dulbecco's modified Eagle's medium (DMEM) with or without added cyclic nucleotides. Following treatment, cells were washed twice with ice-cold PBS, scraped into homogenization buffer, sonicated, and assayed for both luciferase and ␤-galactosidase activities as described (43).…”

“…Control cells were transfected with the HCG-luciferase reporter plasmid alone. Transfected cells were treated with or without 8-Br-cGMP (1 mM) for 20 h. CV-1 and HEK293 cells were chosen for this experiment because regulation of CRE-dependent gene transcription by cAK has been characterized by transfection experiments previously in these cell lines (36,43,47). In both CV-1 and HEK293 cells, transfection of wild type cGKI␤ only minimally stimulated luciferase gene transcription in the absence of cyclic nucleotide treatment (Fig.…”