Cholesterol depletion from the plasma membrane triggers ligand-independent activation of the epidermal growth factor receptor.
We recently demonstrated that depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) caused activation of MAPK (Chen, X., and Resh, M. D. (2001) J. Biol. Chem. 276, 34617-34623). MAPK activation was phosphatidylinositol 3-kinase (PI3K)-dependent and involved increased tyrosine phosphorylation of the p85 subunit of PI3K. We next determined whether MbetaCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MbetaCD or filipin caused an increase in Tyr(P) levels of a 180-kDa protein that was identified as the epidermal growth factor receptor (EGFR). Cross-linking experiments showed that MbetaCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MbetaCD-induced tyrosine phosphorylation of EGFR, indicating that MbetaCD activation of EGFR is ligand-independent. Moreover, MbetaCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MbetaCD-induced phosphorylation of EGFR, SHC, phospholipase C-gamma, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MbetaCD causes ligand-independent activation of EGFR, resulting in MAPK activation by PI3K and Ras-dependent mechanisms. Moreover, these studies reveal a novel mode of action of MbetaCD, in addition to its ability to disrupt membrane rafts.
Chen
Xu
X
Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Resh
Marilyn D
MD
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003505:Cyclodextrins / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011799:Quinazolines; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D047392:beta-Cyclodextrins
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D003432:Cross-Linking Reagents; D016895:Culture Media, Serum-Free; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; C490897:Gab1 protein, mouse; D007527:Isoenzymes; D008024:Ligands; D010750:Phosphoproteins; D011506:Proteins; D011799:Quinazolines; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D020032:Tyrphostins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C101044:RTKI cpd; D014443:Tyrosine; D002784:Cholesterol; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma