Neurofibromin (Nfl) is a similar to 280 kDa protein having tumor suppressor function, presumably by virtue of its GTPase activating domain, but little is known regarding molecular aspects of its effector pathways. Caveolin-1 (Cav-1) regulates diverse signaling molecules and has itself been implicated as a tumor suppressor. Here we demonstrate that Nfl binds to Cav-1's scaffolding domain and co-immunoprecipitates with Cav-1. Analysis of Nfl's primary structure reveals four potential caveolin binding domains, and interestingly, in individuals with neurofibromatosis 1, missense mutations occur with high frequency in 3 of the 4 putative domains. We show that Nfl modulates ras, Akt, and focal adhesion kinase pathways, thereby affecting cytoskeletal organization; moreover, Nfl's effects on signaling are altered when lipid rafts and caveolae are disrupted by cholesterol depletion. These novel findings provide insight into possible signaling mechanisms of Nfl and suggest that together Nfl and Cav-1 may coordinately regulate cell growth and differentiation. (c) 2005 Elsevier Inc. All rights reserved.