Activation of mitogen-activated protein kinase by membrane-targeted Raf chimeras is independent of raft localization

Binding of proteins to the plasma membrane can be achieved with various mem brane targeting motifs, including combinations of fatty acids, isoprenoids, and basic domains. In this study, we investigate whether attachment of dif ferent membrane targeting motifs influences the signaling capacity of membr ane-bound signal transduction proteins by directing the proteins to differe nt membrane microdomains. We used c-Raf-1 as a model for a signaling protei n that is activated when membrane-bound. Three different membrane targeting motifs from K-Ras, Fyn, and Src proteins were fused to the N or C terminus of Raf-1. The ability of the modified Rafs to initiate MAPK signaling was then investigated. All three modified Raf-1 constructs activated MAPK to ne arly equivalent levels. The extent of localization of the Raf-l constructs to membrane microdomains known as rafts did not correlate with the level of MAPK activation. Moreover, treatment of cells with the raft disrupting dru g methyl-beta -cyclodextrin (M beta CD) caused activation of MAPK to levels equivalent to those achieved with membrane-targeted Raf constructs. The us e of pharmacological agents as well as dominant negative mutants revealed t hat MAPK activation by M beta CD proceeds via a phosphoinositide 3-kinase-d ependent mechanism that is Ras/Raf-independent. We conclude that cholestero l depletion from the plasma membrane by M beta CD constitutes an alternativ e pathway for activating MAPK.