Regulating c-Ras function: cholesterol depletion affects caveolin association, GTP loading, and signaling

Cholesterol-rich and caveolin-containing microdomains of the plasma membran e, termed "caveolae," have been implicated in signal transduction [1-4]. Ho wever, the role of caveolae in regulating the Ras-MAP kinase cascade is inc ompletely understood. The mammalian Ras isoforms (H, N, and K) use differen t membrane anchors to attach to the plasma membrane and thereby may localiz e to functionally distinct microdomains, which might explain isoform-specif ic signaling [5-9]. Here, we show that, in Cos epithelial cells, endogenous K-Ras colocalizes largely with caveolin, whereas N-Ras localizes to both c aveolar and noncaveolar subdomains; H-Ras localization was below detection limits. We find that epidermal growth factor (EGF) activates N-Ras but fail s to activate K-Ras in these cells. Extraction of cholesterol with methyl-b eta -cyclodextrin disrupts complex formation between caveolin and K- and N- Ras and, strikingy, enables EGF to activate both K-Ras and N-Ras. While cho lesterol depletion enhance's GTP-loading on total c-Ras, activation of the downstream MEK-MAP kinase cascade by EGF and lysophosphatidic acid but not that by phorbol ester is inhibited. Thus, plasma membrane cholesterol is es sential for negative regulation of c-Ras isoforms (complexed to caveolin), as well as for mitogenic signaling downstream of receptor-activated c-Ras.