REGULATION OF THE MAP KINASE CASCADE IN PC12 CELLS - B-RAF ACTIVATES MEK-1 (MAP KINASE OR ERK KINASE) AND IS INHIBITED BY CAMP

In PC12 cells, cAMP stimulates the MAP kinase pathway by an unknown me chanism. Firstly, we examined the role of calcium ion mobilization and of protein kinase C in cAMP-stimulated MAP kinase activation. We show that cAMP stimulates p44(mapk) independently of these events. Secondl y, we studied the role of B-Raf in this process. We observed that NGF, PMA and cAMP induce the phosphorylation of B-Raf as well as an upward shift in its electrophoretic mobility. We show that B-Raf is activate d following NGF and PMA treatment of PC12 cells, and that it can phosp horylate and activate MEK-1. However, cAMP inhibits B-Raf autokinase a ctivity as well as its ability to phosphorylate and activate MEK-1. Th is inhibition is likely to be due to a direct effect since me found th at PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf bind s to p21(ras), but more important, this binding to p21(ras) is virtual ly abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf ham pers its interaction with p21(ras), which is responsible for the PKA-m ediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP kinase through the activation of an u nidentified MEK kinase and/or the inhibition of a MEK phosphatase.