| Abstract: |
We recently demonstrated that depletion of plasma membrane cholesterol with methyl-β-cyclodextrin (MβCD) 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 MβCD treatment induced tyrosine phosphorylation of other cellular proteins. Here we report that cholesterol depletion of serum-starved COS-1 cells with MβCD 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 MβCD induced dimerization of EGFR, indicative of receptor activation. Reagents that block release of membrane-bound EGFR ligands did not affect MβCD-induced tyrosine phosphorylation of EGFR, indicating that MβCD activation of EGFR is ligand-independent. Moreover, MβCD treatment resulted in increased tyrosine phosphorylation of EGFR downstream targets and Ras activation. Incubation of cells with the specific EGFR inhibitor AG4178 blocked MβCD-induced phosphorylation of EGFR, SHC, phospholipase C-γ, and Gab-1 as well as MAPK activation. We conclude that cholesterol depletion from the plasma membrane by MβCD 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 MβCD, in addition to its ability to disrupt membrane rafts. |
| Keywords: |
signal transduction; epidermal growth factor; mitogen activated protein kinase; protein phosphorylation; human cell; nonhuman; mass spectrometry; proteins; animal cell; animals; mice; models, biological; epidermal growth factor receptor; cell protein; protein binding; receptor, epidermal growth factor; enzyme activation; dose-response relationship, drug; tyrosine; cos cells; phosphorylation; phosphatidylinositol 3 kinase; animalia; blotting, western; enzyme inhibitors; cell membrane; 1-phosphatidylinositol 3-kinase; ligand; cell strain cos1; immunoblotting; adaptor proteins, signal transducing; phosphoproteins; ligands; dimerization; cholesterol; receptor affinity; mitogen-activated protein kinases; enzymes; isoenzymes; 3t3 cells; electrophoresis, polyacrylamide gel; membrane lipid; phospholipase c gamma; adaptor proteins, vesicular transport; cells; 4 (3 chloroanilino) 6,7 dimethoxyquinazoline; tyrphostins; methyl beta cyclodextrin; cross linking; beta-cyclodextrins; cross-linking reagents; membrane microdomains; biological membranes; culture media, serum-free; cyclodextrins; phospholipase c; precipitin tests; filipin; human; priority journal; article
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