ISOLATION OF A NEW-PROTEIN FACTOR REQUIRED FOR ACTIVATION OF RAF-1 BYHA-RAS - PARTIAL-PURIFICATION FROM RAT-BRAIN CYTOSOLS

Ras-mediated signaling pathways play a critical role in cellular proli feration and differentiation. Although it has been demonstrated that R as interacts with Raf-1 to stimulate the serine/threonine kinase activ ity of Raf-1, the precise mechanism by which Ras activates Raf-1 remai ns obscure. To address this question, we developed a cell-free system in which the activated form of H-Ras can induce Raf-1 activation. Usin g this system, we found the presence of a new protein factor, in cytos olic fractions of both human embryonic kidney 293 cells and rat brain tissues, that is required for Ras-dependent activation of Raf-1. The f actor was purified from rat brain cytosols through successive column c hromatographies on DEAE Sephacel, SP Sepharose and Sephacryl S-300, Th e approximate molecular weight of the activator was estimated as 40000 0 by gel filtration. Its activity was sensitive to heat and trypsin tr eatments. The purified activator did not contain Src, 14-3-3, protein kinase C, JAK2 or Ksr-1, as judged by immunoblotting, Further characte rization of the activator is underway.