Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and e ffector of Ras. Previously Ive have shown that RasGAP contains a C2 domain which mediates both Ca2+ dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP: these are the Ca2+-dependent lipid binding p rotein annexin VI (p70) and two previously unidentified proteins, p55 and p 120, Here we provide evidence that p55 is the Src family kinase Fyn and p12 0 is the focal adhesion kinase family member Pyk2. In addition, in vitro bi nding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that F yn, Pyk2, annexin VI and RasGAP can form a protein complex: in mammalian ce lls. (C) 2000 Federation of European Biochemical Societies.