SPECIFIC ASSOCIATION OF ACTIVATED MAP KINASE KINASE KINASE (RAF) WITHTHE PLASMA-MEMBRANES OF RAS-TRANSFORMED RETINAL CELLS

Plasma membrane-enriched fractions were prepared from human embryonic retinal cells transformed with either adenovirus El A and oncogenic ra s DNA, or E1A and E1B DNA. Ras comprised 5-10 % of the membrane protei n from the E1A/ras transformed cells, whereas the membranes from EIA/E 1B transformed cells did not overexpress Ras. The membranes from E1a/r as cells contained MAP kinase kinase kinase (MAPKKK) activity, even af ter washing in 0.5 M NaCl, whereas the membranes from E1A/EIB cells di d not. Neither membrane fraction contained MAP kinase kinase or MAP ki nase activity after washing with 0.5M NaCl. Immunoblotting experiments revealed about 10-fold more c-Raf in the membranes from EIA/ras cells than from E1A/E1B cells, and 50-60% of the MAPKKK activity in Triton X100-solubilised membranes from E1A/ras cells was immunoprecipitated w ith anti-Raf antibodies. A striking enrichment of c-Raf in the plasma membranes of E1A/ras cells was also demonstrated by immunocytochemistr y, where it was co-localized with Ras. The MAPKKK activity in E1A/ras membranes was unaffected by incubation with protein phosphatases or by inclusion of protein phosphatase inhibitors during isolation, nor was it activated by GTP-Ras or inhibited by GDP-Ras. The results support the view that Ras and c-Raf interact with one another, but that neithe r c-Raf phosphorylation nor its interaction with GTP-Ras are alone suf ficient for activation. The identification of MAPKKK activity in the m embranes of ras-transformed cells may prove useful in elucidating the mechanism by which Raf is activated by Ras.