B-RAF-DEPENDENT REGULATION OF THE MEK-1 MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY IN PC12 CELLS AND REGULATION BY CYCLIC-AMP

Growth factor receptor tyrosine kinase regulation of the sequential ph osphorylation reactions leading to mitogen-activated protein (MAP) kin ase activation in PC12 cells has been investigated. In response to epi dermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kina se-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-spe cificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate ME K-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in re sponse to epidermal growth factor, nerve growth factor, or platelet-de rived growth factor was unaffected by protein kinase A activation. Eve n though elevated cAMP levels inhibited Raf activation, the growth fac tor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. T he results demonstrate that tyrosine kinase receptor activation of MEK -1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-l, whose activation is inhibited by protein kinase A, and MEK activators, whos e activation is independent of cAMP regulation.