DIFFERENTIAL STRUCTURAL REQUIREMENTS FOR INTERACTION OF RAS PROTEIN WITH ITS DISTINCT DOWNSTREAM EFFECTORS

Ras proteins have multiple effecters of distinct structures that do no t share significant structural homology at their Ras interaction sites . To prove possible differences in their recognition mechanisms of Ras , we screened 44 human Ha-Ras proteins carrying mutations in the effec tor region and its flanking sequences for interaction with human Raf-1 , Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenyly l cyclase. The Ras binding specificities were largely shared between R af-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their af finities for Byr2 selectively reduced. The only exception was Ras(D38N ), which lost the ability to bind Raf-1 while retaining the activity t o bind Byr2 and complement the Byr2(-) phenotype of S. pombe. On the o ther hand, adenylyl cyclase had quite distinct requirements for Ras re sidues; mutations P34G and T58A selectively abolished the ability to b ind and activate it without considerably affecting the interaction wit h Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In additi on, V45E mutation was found to impair the ability of Ras to activate b oth Raf-1 and adenylyl cyclase without significantly affecting the bin ding affinities for them. These results demonstrate that significant d ifferences exist in the recognition mechanisms by which the three effe ctor molecules associate with Ras and suggest that a region of Ras req uired for activation of the effecters in general may exist separately from that for binding the effectors.