MUTATIONS THAT ABOLISH THE ABILITY OF HA-RAS TO ASSOCIATE WITH RAF-1

Recent studies have revealed that Ras can associate physically with Ra f. In the present study, we tested 34 mutants of Ha-Ras carrying subst itution(s) in the region of residues 23- 71 for their ability to assoc iate with Raf-1. Mouse Ba/F3 cell lysates were incubated w ith each mu tant Ras protein, in either the guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)- or the guanosine 5'-[beta-thio]diphosphate (GDP beta S) -bound form, and the anti-Ras antibody Y13-238. The immunoprecipitates were analysed for the presence of Raf-1 by Western blotting with an a nti-Raf-1 antibody. Six mutants of Ras, E31K, P34G, T35S, D38N, D57A a nd A59T, failed to bind Raf-1. Mutations N26G, V29A, S39A, Y40W, R41A, V44A, V45E, L56A and T58A partially reduced the ability to bind Raf-1 . All the other mutants could associate with Raf-1 with nearly the sam e efficiency as that of wild-type Ras. Thus, the Raf-1-binding ability of Ras appears to be affected by mutations in the N-terminal region, and in particular, by those in and neighboring the effector region (re sidues 32-40) and in the region (residues 56-59) banking the N-termina l of Switch II. The abilities to bind Raf-1 and to induce neurite outg rowth of pheochromocytoma (PC) 12 cells correlate to each other for 22 Ras mutants. However, mutation A59T, which does not reduce the neurit e-inducing or transforming activities, abolishes the ability to bind R af-1. In contrast, mutations Y32F, K42A and L53A, which impair the neu rite-inducing activity of Ras, have no effect on the Ras.Raf-1 associa tion. Partially reduced Raf-1-binding ability was observed for mutants V29A, S39A, Y40W, R31A, V44A, L56A and T58A, which exhibit full neuri te-inducing activity, and also for mutant V45E, which has no activity of neurite induction.