Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane.
An essential step in the epidermal growth factor (EGF)-dependent activation of MAP kinase is the recruitment of Raf-1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf-1 is recruited. Caveolae fractions prepared from normal Rat-1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf-1 began 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 kinase, whereas cytoplasmic Raf-1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf-1 to caveolae. Overexpression of Ha-RasV12 caused the recruitment of Raf-1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA-5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.
Mineo
C
C
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas 75235, USA.
James
G L
GL
Smart
E J
EJ
Anderson
R G
RG
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D018631:ras Proteins / Q000032:analysis*
D011518:Proto-Oncogene Proteins; D004815:Epidermal Growth Factor; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins