RAF-1 PHYSICALLY INTERACTS WITH RB AND REGULATES ITS FUNCTION - A LINK BETWEEN MITOGENIC SIGNALING AND CELL-CYCLE REGULATION

Cells initiate proliferation in response to growth factor stimulation, but the biochemical mechanisms linking signals received at the cell s urface receptors to the cell cycle regulatory molecules are not yet cl ear. In this study, we show that the signaling molecule Raf-1 can phys ically interact with Rb and p130 proteins in vitro and in vivo and tha t this interaction can be detected in mammalian cells without overexpr essing any component. The binding of Raf-1 to Rb occurs subsequent to mitogen stimulation, and this interaction can be detected only in prol iferating cells. Raf-1 can inactivate Rb function and can reverse Rb-m ediated repression of E2F1 transcription and cell proliferation effici ently. The region of Raf-1 involved in Rb binding spanned residues 1 t o 28 at the N terminus, and functional inactivation of Rb required a d irect interaction. Serum stimulation of quiescent human fibroblast HSF S cells led to a partial translocation of Raf-1 into the nucleus, wher e it colocalized with Rb. Further, Raf-1 was able to phosphorylate Rb in vitro quite efficiently. We believe that the physical interaction o f Raf-1 with Rb is a vital step in the growth factor-mediated inductio n of cell proliferation and that Raf-1 acts as a direct link between c ell surface signaling cascades and the cell cycle machinery.