Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells.
Adenylyl cyclase type 6 (AC6) activity is inhibited by protein kinase C (PKC) in vitro; however, in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. To investigate the effects of PKC activation on AC6 activity in intact cells, we constructed human embryonic kidney (HEK) 293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and cyclic AMP-dependent protein kinase (PKA) phosphorylation site, Ser674 (AC6-S674A). In contrast to in vitro observations, we observed a PKC-mediated enhancement of forskolin- and isoproterenol-stimulated cyclic AMP accumulation in HEK-AC6 cells. Phorbol 12-myristate 13-acetate also potentiated cyclic AMP accumulation in cells expressing endogenous AC6, including Chinese hamster ovary cells and differentiated Cath.a differentiated cells. In HEK-AC6-S674A cells, the potentiation of AC6 stimulation was significantly greater than in cells expressing AC6-WT. The positive effect of PKC activation on AC6 activity seemed to involve Raf1 kinase because the Raf1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) inhibited the PKC potentiation of AC6 activity. Furthermore, the forskolin-stimulated activity of a recombinant AC6 in which the putative Raf1 regulatory sites have been eliminated was not potentiated by activation of PKC. The ability of Raf1 to regulate AC6 may involve a direct interaction because AC6 and a constitutively active Raf1 construct were coimmunoprecipitated. In addition, we report that epidermal growth factor receptor activation also enhances AC6 signaling in a Raf1-dependent manner. These data suggest that Raf1 potentiates drug-stimulated cyclic AMP accumulation in cells expressing AC6 after activation of multiple signaling pathways.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-2091, USA.
Michael A
MA
Beazely
Jamie K
JK
Alan
Val J
VJ
Watts
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D017931:DNA Primers; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011493:Protein Kinase C / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D005576:Colforsin; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate