BOTH P21(RAS) AND PP60(V-SRC) ARE REQUIRED, BUT NEITHER ALONE IS SUFFICIENT, TO ACTIVATE THE RAF-1 KINASE

The raf genes encode a family of cytoplasmic proteins with intrinsic p rotein-serine/threonine kinase activity. The c-raf gene is the cellula r homolog of v-raf, the transforming gene of murine sarcoma virus 3611 . The constitutive kinase activity of the v-Raf protein has been impli cated in transformation and mitogenesis. The activity of Raf-1, the pr otein product of the c-raf gene, is normally suppressed by a regulator y N-terminal domain. Activation of various tyrosine-kinase growth fact or receptors results in activation of Raf-1 and its hyperphosphorylati on. Further, Raf-1 has been shown to act either downstream or independ ently of the p21ras protein, as indicated by experiments involving mic roinjection of anti-Ras antibodies. To investigate the potential role of p21ras in the activation of Raf-1 by tyrosine kinases, we have used the baculovirus/Sf9 cell system to overproduce various wild-type and mutant forms of pp60src, p21ras, and Raf-1 proteins. We show that eith er pp60v-src or p21c-ras can independently activate the autokinase act ivity of Raf-1, but only to a limited extent. Surprisingly, both pp60v -src and p21c-ras are required to fully activate Raf-1. Analysis of th e Raf-1 autokinase activity in vitro shows that Raf-1 autophosphorylat ion sites are distributed equally on serine and threonine residues. Wh en Raf-1 is analyzed by immunoblotting, as previously reported for mam malian cell experiments, a marked increase in the apparent molecular w eight of Raf-1 is seen only when it is coexpressed with both pp60v-src and p21ras.