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-dependent 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 activity previously described to associate with immobilized p21(ras) . GMPPNP. The association between MEK1 and immobilized p21(ras) . GMPPNP increased its specific activity towards p42(MAPK). We detected the specific association of B-Raf with immobilized p21(ras) . GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21(ras) . GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21(ras) . GMPPNP. These data suggest that the p21(ras) . GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.