Coordinated regulation of Rap1 and thyroid differentiation by cyclic AMP and protein kinase A

Originally identified as an antagonist of Ras action, Rap1 exhibits many Ra s-independent effects, including a role in signaling pathways initiated by cyclic AMP (cAMP). Since cAMP is a critical mediator of the effects of thyr otropin (TSH) on cell proliferation and differentiation, we examined the re gulation of Rap1 by TSH in a continuous line of rat thyroid-like cells. Bot h cAMP and protein kinase A (PRA) contribute to the regulation of Rap1 acti vity and signaling by TSH. TSH activates Rap1 through a cAMP-mediated and P KA-independent mechanism. TSH phosphorylates Rap1 in a PKA-dependent manner . Interference with PKA activity blocked phosphorylation but not the activa tion of Rap1. Rather, PKA inhibitors prolonged Rap1 activation, as did expr ession of a Rap1A mutant lacking a PKA phosphorylation site. These results indicate that PKA elicits negative feedback regulation on cAMP-stimulated R ap1 activity in some cells. The dual regulation of Rap1 by cAMP and PRA ext ends to downstream effecters. The ability of TSH to stimulate Akt phosphory lation was markedly enhanced by the expression of activated Rap1A and was r epressed in cells expressing a putative dominant-negative Rap1A mutant. Alt hough the expression of activated Rap1A was sufficient to stimulate wortman nin-sensitive Akt phosphorylation, TSH further increased Akt phosphorylatio n in a phosphatidylinositol 3-kinase- and PKA-dependent manner. The ability of TSH to phosphorylate Akt was impaired in cells expressing a Rap1A mutan t that could be activated but not phosphorylated. These findings indicate t hat dual signals, Rap1 activation and phosphorylation, contribute to TSH-st imulated Akt phosphorylation. Rap1 plays an essential role in cAMP-regulate d differentiation. TSH effects on thyroid-specific gene expression, but not its effects on proliferation, were markedly enhanced in cells expressing a ctivated Rap1A and repressed in cells expressing a dominant-negative Rap1A mutant. These findings reveal complex regulation of Rap1 by cAMP including PKA-independent activation and PKA-dependent negative feedback regulation. Both signals appear to be required for TSH signaling to Akt.