Differential transcriptional regulation by the alpha- and gamma-catalytic subunit isoforms of cAMP-dependent protein kinase.
The C gamma and C alpha isoforms of the cAMP-dependent protein kinase (PKA) share 83% identity including all critical catalytic and substrate-binding residues defined to date. Compared to C alpha, C gamma 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, C gamma-mediated gene transcription regulation was compared with that of C alpha in four cell lines using transient transfection/dual luciferase assays. As compared to C gamma, C alpha 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 (pGH alpha-fluc). This occurred in Cos7, Y1, and Kin8 adrenal cells by 23-, 6.5-, and 1.4-fold, respectively. In contrast, C gamma, but not C alpha, 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, pGH alpha-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 C gamma. In all cell lines, cotransfection with a PDK1 expression plasmid enhanced the transcriptional activation of both C alpha and C gamma (1.5- to 3-fold), while a catalytically inactive PDK1 mutant (PDK.KD) did not. These results suggest that both C alpha and C gamma can activate CRE-responsive genes; however, C alpha does so with better efficiency than C gamma. In contrast to C alpha, C gamma 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.
Center for Pediatric Research, Eastern Virginia Medical School, 855 West Brambleton Avenue, Norfolk, VA 23510, USA.
Rana C
RC
Morris
Gary Z
GZ
Morris
Weiqing
W
Zhang
Merica
M
Gellerman
Stephen J
SJ
Beebe
D064413:3-Phosphoinositide-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002478:Cells, Cultured; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D018139:Simplexvirus / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013379:Substrate Specificity; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D000509:alpha-Fetoproteins / Q000235:genetics
C097353:AFP protein, human; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D007527:Isoenzymes; C517834:PRKAR1A protein, human; C526224:Prkag1 protein, mouse; C517835:Prkar1a protein, mouse; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D000509:alpha-Fetoproteins; D008156:Luciferases; D011494:Protein Kinases; D013937:Thymidine Kinase; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; C526222:PRKAG1 protein, human; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C463959:protein kinase A Calpha; C463939:protein kinase A Cgamma; D055372:AMP-Activated Protein Kinases