NEGATIVE MODULATION OF MEMBRANE LOCALIZATION OF THE RAF-1 PROTEIN-KINASE BY HYPERPHOSPHORYLATION

The serine/threonine-specific protein kinase Raf-l plays a key role in mitogenic signal transduction by coupling Pus to the mitogen-activate d protein (MAP) kinase cascade. Pus-mediated translocation to the plas ma membrane represents a crucial step in the process of serum-stimulat ed Raf-l kinase activation. The exact role of the multisite phosphoryl ation in Raf regulation, however, is not clear. We have previously rep orted that the mobility shift-associated hyperphosphorylation of Raf c orrelates with a reduction of serum-stimulated Raf kinase activity (Wa rtmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701), He re we show that incubation of serum-starved CHO cells with D609, a pur ported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-l that is due to hyperphosphorylat ion on sites identical to those observed following mitogen stimulation . Subcellular fractionation analyses revealed that D609-induced mobili ty shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-l. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibit or of activation of the MAP kinase kinase MEK, prevented D6O9-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels, Taken together, these data suggest that mobility s hift-associated hyperphosphorylation of Raf-l, by virtue of reducing t he amount of plasma membrane-bound Raf-l, represents a negative feedba ck mechanism contributing to the desensitization of the MAP kinase sig naling cascade.