ONCOGENES, GROWTH-FACTORS AND PHORBOL ESTERS REGULATE RAF-1 THROUGH COMMON MECHANISMS

We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Ra f-1. Specifically, we determined the role of Ras binding and the phosp horylation of serines 338/339, tyrosines 340/341 and the activation lo op (491-508) in response to these stimuli in COS-7 cells. An intact Ra s binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA, Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340 /341 phosphorylation sites also blocked Raf-1 activation by all stimul i tested. Loss of the serine 497 and serine 499 PKC alpha phosphorylat ion sites failed to significantly reduce Raf-1 activation by any stimu lus including PMA. Alanine substitution of all other potential phospho rylation sites within the Raf-1 activation loop had little or no effec t on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of different stimuli through a co mmon mechanism involving association with Ras-GTP and multiple phospho rylations of the amino-terminal region of the catalytic domain. Phosph orylation of the activation loop does not appear to be a significant m echanism of Raf-1 kinase regulation in COS-7 cells.