Specific association of activated MAP kinase kinase kinase (Raf) with the plasma membranes of ras-transformed retinal cells

S. Traverse, P. Cohen, H. Paterson, C. Marshall, U. Rapp, R. J.A. Grand

    Research output: Contribution to journalArticlepeer-review

    101 Citations (Scopus)


    Plasma membrane-enriched fractions were prepared from human embryonic retinal cells transformed with either adenovirus E1A and oncogenic ras DNA, or E1A and E1B DNA. Ras comprised 5-10% of the membrane protein from the E1A/ras transformed cells, whereas the membranes from E1A/E1B transformed cells did not overexpress Ras. The membranes from E1A/ras cells contained MAP kinase kinase kinase (MAPKKK) activity, even after washing in 0.5 M NaCl, whereas the membranes from E1A/E1B cells did not. Neither membrane fraction contained MAP kinase kinase or MAP kinase activity after washing with 0.5 M NaCl. Immunoblotting experiments revealed about 10-fold more c-Raf in the membranes from E1A/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 with anti-Raf antibodies. A striking enrichment of c-Raf in the plasma membranes of E1A/ras cells was also demonstrated by immunocytochemistry, 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 neither c-Raf phosphorylation nor its interaction with GTP-Ras are alone sufficient for activation. The identification of MAPKKK activity in the membranes of ras-transformed cells may prove useful in elucidating the mechanism by which Raf is activated by Ras.

    Original languageEnglish
    Pages (from-to)3175-3181
    Number of pages7
    Issue number11
    Publication statusPublished - 1993

    ASJC Scopus subject areas

    • Molecular Biology
    • Genetics
    • Cancer Research


    Dive into the research topics of 'Specific association of activated MAP kinase kinase kinase (Raf) with the plasma membranes of ras-transformed retinal cells'. Together they form a unique fingerprint.

    Cite this