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 cAMP-dependent protein kinase, Cα and Cβ1, are known to be widely expressed in mammals. Although much is known about the structure and function of Cα, few studies have addressed the possibility of a distinct role for the Cβ 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. Cβ1 demonstrated higher K(m) values for some peptide substrates than did Cα, but Cβ1 was insensitive to substrate inhibition, a phenomenon that was observed with Cα at substrate concentrations above 100 μM. Cα and Cβ1 displayed distinct IC50 values for the and β isoforms of the protein kinase inhibitor, protein kinase inhibitor (5-24) peptide, and the type IIα regulatory subunit (RIIα). Of particular interest, purified type II holoenzyme containing Cβ1 exhibited a 5-fold lower K(α) value for cAMP (13 nm) than did type II holoenzyme containing Cα (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 RIIα2Cβ12 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 RIIα2Cα2 holoenzyme. These results provide evidence that type II holoenzyme formed with Cβ1 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.