Adenylyl cyclases (AC), a family of enzymes that catalyze the synthesis of cyclic AMP, are critical regulators of cellular functions. The activity of adenylyl cyclase is stimulated by a range of hormone receptors, primarily via interactions with G-proteins. However, recently we identified an alternate mechanism by which growth factors sensitize adenylyl cyclase activation. We suggested that this mechanism might involve a raf kinase-mediated serine phosphorylation of AC. However, the direct involvement of a specific form of raf kinase was yet to be demonstrated. Further, whether this mechanism is generalized to other isoforms of AC was unknown. In HEK 293 cells, we now demonstrate that in reconstitution studies, c-raf kinase can phosphorylate AC VI. Further, AC VI co-immunprecipitates with both c-raf and b-raf kinase. Raf kinase-dependent regulation of AC VI is dependent on the integrity of S750 in the fourth intracellular loop of the enzyme and S603/S608 in the C1b region of the molecule. To examine how generalized this effect is, we studied representative isoforms of the major subfamilies of AC viz, AC I, AC II and AC V. Raf kinase-dependent sensitization/ phosphorylation of adenylyl cyclases was common to AC VI, AC V and AC II isoforms but not AC I. In aggregate these studies indicate that raf kinase associates with adenylyl cyclases. Further, raf kinase regulation of AC is isoform-selective. These functional interactions between adenylyl cyclases and raf kinases suggest an important, but previously unrecognized protein-protein interaction between these two key regulatory enzymes.

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