Differential transcriptional regulation by the alpha- and gamma-catalytic subunit isoforms of cAMP-dependent protein kinase

摘要

The Cgamma and Calpha isoforms of the cAMP-dependent protein kinase (PKA) share 83% identity including all critical catalytic and substrate-binding residues defined to date. Compared to Calpha, Cgamma has a different substrate specificity and a selective pseudosubstrate specificity, exhibiting inhibition by regulatory subunits, but not by the protein kinase inhibitor. In these studies, Cgamma-mediated gene transcription regulation was compared with that of Calpha in four cell lines using transient transfection/dual luciferase assays. As compared to Cgamma, Calpha more efficiently activated a cAMP-response element (CRE)-regulated fragment of the human alpha-glycoprotein hormone promoter which was coupled to a firefly luciferase reporter gene (pGHalpha-fluc). This occurred in Cos7, Y1, and Kin8 adrenal cells by 23-, 6.5-, and 1.4-fold, respectively. In contrast, Cgamma, but not Calpha, activated the Sp1RE-regulated herpes simplex virus thymidine kinase promoter which was coupled to a Renilla luciferase reporter (pTK-rluc). In Sp1-deficient Sf9 cells, pGHalpha-fluc expression was maintained for both isoforms, but cotransfection with an Sp1 expression plasmid was necessary and sufficient for activation of pTK-rluc expression by Cgamma. In all cell lines, cotransfection with a PDK1 expression plasmid enhanced the transcriptional activation of both Calpha and Cgamma (1.5- to Mold), while a catalytically inactive PDK1 mutant (PDK.KD) did not. These results suggest that both Ca and Cy can activate CRE-responsive genes; however, Calpha does so with better efficiency than Cgamma. In contrast to Calpha, Cgamma activates transcription of genes containing pTK-like Sp1RE sites. Activation of different C subunit isoforms can provide a means to diversify cAMP-mediated transcription, possibly affecting cell phenotype. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 61]