Quantitative Analysis of the Complex between p21 and the Ras-binding Domain of the Human Raf-1 Protein Kinase

The Ras-binding domain (RBD) of human Raf-1 was purified from Escherichia coli , and its interaction with Ras was investigated. Its dissociation constant with p21 •guanyl-5′-yl imidodiphosphate was found to be 18 nM, with a slight preference for H-ras over K- and N-ras. Oncogenic forms bind with slightly lower affinity. The affinity of RBD for effector region mutants or the GDP-bound form of p21 is in the micromolar range, which means that 100-fold lower affinity is not sufficient for signal transduction. The rate of the GTPase of p21 is not modified by RBD. Since P i release is found not to be rate limiting, the Ras-Raf signal of the cell may be terminated by the intrinsic GTPase of p21 .