Received for publication April 16, 2004.
Revised October 5, 2004.
Accepted for publication October 5, 2004.

PKC and EGF stimulation of Raf1 potentiates AC6 activation in intact cells

Abstract

Adenylyl cyclase type 6 (AC6) activity is inhibited by PKC in vitro, however in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. In order to investigate the effects of PKC activation on AC6 activity in intact cells, we constructed HEK293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and 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. PMA also potentiated cyclic AMP accumulation in cells expressing endogenous AC6 including CHO cells and differentiated CAD 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 appeared to involve Raf1 kinase because the Raf1 inhibitor, GW5074, inhibited the PKC potentiation of AC6 activity. Further, 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 co-immunoprecipitated. In addition, we report that EGF 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.

Key words: NGF/EGF, Gs family, Adenylyl cyclases, Protein Kinase C, Raf family, Drug tolerance/dependence

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