DIFFERENCE IN THE MECHANISM OF INTERACTION OF RAF-1 AND B-RAF WITH H-RAS

Ras is known to possess multiple cellular targets including Raf-l. Her e, we measured both direct binding of various H-Ras mutants to two rep resentative mammalian Ras targets, Raf-l and B-Raf, and the activity o f the mutants to stimulate Raf-l and B-Raf, and analysed the differenc e in their Ras-interaction mechanisms. B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-l by examining binding of the H-Ras mutants to Raf-l and B-Raf in the yeast two-hybrid and in v itro binding assays. Mutants, Y32F, A59E, and V45E bound to Raf-1 in S f9 cells coexpressing them, but failed to activate Raf-l. On the other hand, Y32F activated B-Raf in a cell-free system which consisted of r at brain cytosol and recombinant MEK. These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-l and B-Raf. (C) 1996 Academic Press, Inc.