ASSOCIATION OF MEK1 WITH P21(RAS)CENTER-DOT-GMPPNP IS DEPENDENT ON B-RAF

We have previously reported that immobilized p21(ras) forms a GMPPNP-d ependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK acti vity previously described to associate with immobilized p21(ras) . GMP PNP. The association between MEK1 and immobilized p21(ras) . GMPPNP in creased its specific activity towards p42(MAPK). We detected the speci fic association of B-Raf with immobilized p21(ras) . GMPPNP. In contra st to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Ra f significantly reduced, by 96%, the amount of MEK1 activity associate d with immobilized p21(ras) . GMPPNP. The decrease in MEK1 activity co rrelated with complete loss in the binding of both B-Raf and MEK1 prot eins with immobilized p21(ras) . GMPPNP. These data suggest that the p 21(ras) . GMPPNP-dependent activation of MEK1 in brain extracts is dep endent on the presence of the B-Raf protein kinase.