MAMMALIAN RAS INTERACTS DIRECTLY WITH THE SERINE THREONINE KINASE RAF

We have identified proteins that interact with H-Ras using a two hybri d system screen of a mouse cDNA library. Approximately 50% of the clon es identified encoded portions of the c-Raf and A-Raf serine/threonine kinases. Overlaps among these clones define a conserved 81 residue re gion of the N-terminus of Raf as the Ras interaction region. We show t hat Raf interacts with wild-type and activated Ras, but not with an ef fector domain mutant of Ras or with a dominant-interfering Ras mutant. Using purified bacterially expressed fusion proteins, we show, furthe rmore, that Ras and the N-terminal region of Raf associate directly in vitro and that this interaction is dependent on GTP bound to Ras.