THE MAJOR CATALYTIC SUBUNIT ISOFORMS OF CAMP-DEPENDENT PROTEIN-KINASEHAVE DISTINCT BIOCHEMICAL-PROPERTIES IN-VITRO AND IN-VIVO

Two isoforms of the catalytic subunit of cAMP-dependent protein kinase , C alpha and C beta 1, are known to be widely expressed in mammals, A lthough much is known about the structure and function of C alpha, few studies have addressed the possibility of a distinct role for the C b eta proteins, The present study is a detailed comparison of the bioche mical properties of these two isoforms, which were initially expressed in Escherichia coil and purified to homogeneity, C beta 1 demonstrate d higher K-m values for some peptide substrates than did C alpha but C beta 1 was insensitive to substrate inhibition, a phenomenon that was observed with C alpha at substrate concentrations above 100 mu M. C a lpha and C beta l displayed distinct IC50 values for the a and beta is oforms of the protein kinase inhibitor, protein kinase inhibitor (5-24 ) peptide, and the type II alpha regulatory subunit (RII alpha), Of pa rticular interest, purified type II holoenzyme containing C beta 1 exh ibited a B-fold lower K-alpha value for cAMP (13 nM) than did type II holoenzyme containing C alpha (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-l cells ex pressing RII alpha(2)C beta 1(2) holoenzyme was half-maximal at 12-fol d lower concentrations of 8-(4-chlorophenylthio)-cAMP and 5-fold lower concentrations of forskolin than in COS-1 cells expressing RII alpha( 2)Ca(2) holoenzyme. These results provide evidence that type II holoen zyme formed with C beta 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.