RAS AND RAF-1 FORM A SIGNALING COMPLEX WITH MEK-1 BUT NOT MEK-2

Recent studies have demonstrated the existence of a physical complex c ontaining p21(ras) (RAS), p74(raf-1) (RAF-1), and MEK-1. Although it i s clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the acti vation state of the cell and whether MEK-2 is also present in the comp lex. To analyze the regulation and specificity of this complex, we uti lized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvat ion or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate bu t bound only a small fraction of cytosolic MEK-1, and further MEK-1 co uld bind immobilized RAS only if exogenous RAF-1 was added to the lysa te. This indicates that binding of MEK-1 to RAS depends on the presenc e of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was n ot detected in the RAS signalling complex. A proline-rich region of ME K-1 containing a phosphorylation site appears to be essential for sign alling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed c ells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 acti vation.