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) kinase pathway in fibroblasts is inhibited by cyclic AMP (cAMP) as a result of inhibition of Raf-1. In contrast, cAMP inhibits neither nerve growth factor-induced MAP kinase activation nor differentiation in PC12 pheochromocytoma cells. Instead, in PC12 cells cAMP activates MAP kinase. Since one of the major differences between the Ras/Raf/MAP kinase cascades of these cell types is the expression of B-Raf in PC12 cells, we compared the effects of cAMP on Raf-1 and B-Raf. In PC12 cells maintained in serum-containing medium, B-Raf was refractory to inhibition by cAMP, whereas Raf-1 was effectively inhibited. In contrast, both B-Raf and Raf-1 were inhibited by cAMP in serum-starved PC12 cells. The effect of cAMP is thus dependent upon growth conditions, with B-Raf being resistant to cAMP inhibition in the presence of serum. These results were extended by studies of Rat-1 fibroblasts into which B-Raf had been introduced by transfection. As in PC12 cells, B-Raf was resistant to inhibition by cAMP in the presence of serum, whereas Raf-1 was effectively inhibited. In addition, the expression of B-Raf rendered Rat-1 cells resistant to the inhibitory effects of cAMP on both growth factor-induced activation of MAP kinase and mitogenesis. These results indicate 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 kinase 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 negative Ras mutant, indicating that cAMP acts on a target upstream of Ras. Thus, it appears that a signaling component upstream of Ras is also require for cAMP stimulation of MAP kinase in PC12 cells.
Erhardt
P
P
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Troppmair
J
J
Rapp
U R
UR
Cooper
G M
GM
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000096:biosynthesis* / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts; D009414:Nerve Growth Factors / Q000494:pharmacology; D016716:PC12 Cells; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D018631:ras Proteins / Q000502:physiology*
D009414:Nerve Growth Factors; D011518:Proto-Oncogene Proteins; D004815:Epidermal Growth Factor; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins