QUANTITATIVE-ANALYSIS OF THE COMPLEX BETWEEN P21(RAS) AND THE RAS-BINDING DOMAIN OF THE HUMAN RAF-1 PROTEIN-KINASE

The Ras-binding domain (RBD) of human Raf-1 was purified from Escheric hia coli, and its interaction with Ras was investigated. Its dissociat ion constant with p21(ras)guanyl-5'-yl imidodiphosphate was found to b e 18 nM, with a slight preference for H-ras over K- and N-ras. Oncogen ic forms bind with slightly lower affinity. The affinity of RED for ef fector region mutants or the GDP-bound form of p21(ras) is in the micr omolar range, which means that 100-fold lower affinity is not sufficie nt for signal transduction. The rate of the GTPase of p21(ras) is not modified by RED. Since P-i release is found not to be rate limiting, t he Ras-Raf signal of the cell may be terminated by the intrinsic GTPas e of p2l(ras).