LOCALIZATION OF EPIDERMAL GROWTH FACTOR-STIMULATED RAS RAF-1 INTERACTION TO CAVEOLAE MEMBRANE/

An essential step in the epidermal growth factor (EGF)-dependent activ ation of MAP kinase is the recruitment of Raf-1 to the plasma membrane . Here we present evidence that caveolae are the membrane site where R af-1 is recruited. Caveolae fractions prepared from normal Rat-1 cells grown in the absence of serum were highly enriched in both EGF recept ors and Ras. Thirty seconds after EGF was added to these cells Raf-1 b egan to appear in caveolae but not in non caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant . The Raf-1 in this fraction was able to phosphorylate MAP kinase kina se, whereas cytoplasmic Raf-1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf-1 to caveolae. Overex pression of Ha-Ras(V12) caused the recruitment of Raf-1 to caveolae in dependently of EGF stimulation, and this was blocked by the farnesyltr ansferase inhibitor BZA-5B. Finally, prenylation appeared to be requir ed for localization of Ras to caveolae.