The organization of the plasma membrane into distinct microdomains may be one mechanism controlling cellular signal transduction. Kranenburg et al. investigated how the association of Ras proteins with caveolin in the detergent-insoluble, cholesterol-rich membrane domain called caveolae influences activation of these guanosine triphosphatases (GTPases) by various stimuli in Cos7 cells. Cos cells express K-Ras, N-Ras, and very small amounts of H-Ras. K-Ras is farnesylated, as all Ras proteins are, and also has a polybasic region believed to interact with acidic phospholipids. N-Ras and H-Ras are farnesylated and palmitoylated. K-Ras appeared to be predominantly localized to caveolin-containing membrane domains, whereas N-Ras was located in membrane structures with and without caveolin. Epidermal growth factor (EGF) activated N-Ras, but failed to activate K-Ras unless cholesterol was depleted from the cells, disrupting the caveolae and the biochemical interaction between caveolin and the Ras proteins. Cholesterol depletion also potentiated activation of the Ras proteins (detected by a pan-Ras antibody) by the G protein-coupled receptor agonist lysophosphatidic acid and by the protein kinase C-activator phorbol ester. To the authors' surprise, cholesterol depletion influenced activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), downstream of Ras differently, depending on the initial stimulus. Cholesterol depletion inhibited receptor-initiated ERK activation, but had no effect of ERK activation induced by protein kinase C. The results suggest that cholesterol has two distinct roles in Ras signaling: (i) an inhibitory role through its formation of plasma membrane microdomains, which promotes the association of Ras with caveolin, and (ii) a positive role downstream of Ras that is needed for MAPK activation.
O. Kranenburg, I. Verlaan, W. H. Moolenaar, Regulating c-Ras function: Cholesterol depletion affects caveolin association, GTP loading, and signaling. Curr. Biol. 11, 1880-1884 (2001). [Online Journal]