REVERSAL OF RAF-1 ACTIVATION BY PURIFIED AND MEMBRANE-ASSOCIATED PROTEIN PHOSPHATASES

The Raf-1 protein kinase participates in transduction of mitogenic sig nals, but its mechanisms of activation are incompletely understood. Tr eatment of human Raf-1 purified from insect Sf9 cells co-expressing c- H-Ras and Src(Y527F) (in which phenylalanine replaces tyrosine at resi due 527) with either serine-threonine or tyrosine phosphatases resulte d in enzymatic inactivation of Raf-1. Inactivation of purified Raf-1 w as blocked by addition of either the 14-3-3 zeta protein or heat shock protein 90, Loading of plasma membranes from transformed cells with g uanosine triphosphate (GTP) resulted in inactivation of endogenous or exogenous Raf-1; inactivation was blocked by inclusion of protein phos phatase inhibitors. These results suggest the existence of protein pho sphatases in the cell membrane that are regulated by GTP and are respo nsible for Raf-1 inactivation.