DIFFERENTIAL REGULATION OF RAF-1 AND B-RAF AND RAS-DEPENDENT ACTIVATION OF MITOGEN-ACTIVATED PROTEIN-KINASE BY CYCLIC-AMP IN PC12 CELLS

Growth factor stimulation of the mitogen-activated protein (MAP) kinas e pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve grow th factor-induced MAP kinase activation nor differentiation in PC12 ph eochromocytoma cells, Instead, in PC12 cells cAMP activates MAP kinase . Since one of the major differences between the Ras/Raf/MAP kinase ca scades of these cell types is the expression of B-Raf in PC12 cells, w e compared the effects of cAMP on Raf-1 and B-Raf, In PC12 cells maint ained in serum-containing medium, B-Raf was refractory to inhibition b y cAMP, whereas Raf-1 was effectively inhibited, In contrast, both B-R af and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The e ffect of cAMP is thus dependent upon growth conditions, with B-Raf bei ng resistant to cAMP inhibition in the presence of serum. These result s were extended by studies of Rat-1 fibroblasts into which B-Raf had b een introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effe ctively inhibited, In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth fact or-induced activation of MAP kinase and mitogenesis. These results ind icate that Raf-1 and B-Raf are differentially sensitive to inhibition by cAMP and that B-Raf expression can contribute to cell type-specific differences in the regulation of the MAP kinase pathway. In contrast to the situation in PC12 cells, cAMP by itself did not stimulate MAP k inase in B-Raf-expressing Rat-1 cells. The activation of MAP kinase by cAMP in PC12 cells was inhibited by the expression of a dominant nega tive ras mutant, indicating that cAMP acts on a target upstream of Ras . Thus, it appears that a signaling component upstream of Ras is also required for cAMP stimulation of MAP kinase in PC12 cells.