Identification of a protein associated with p21ras by chemical crosslinking

Abstract

The products of the ras oncogenes (p21ras) are ubiquitous membrane-associated proteins that bind guanine nucleotides and possess an intrinsic GTPase activity. Because of their functional homologies with regulatory guanine nucleotide-binding proteins, they are thought to be involved in the control of cellular proliferation as transducers of incoming growth signals. In an effort to identify proteins interacting with p21ras, we have used in vivo crosslinking techniques on Rat-1 fibroblasts and derived cell lines overexpressing p21ras and immunoprecipitation with polyclonal anti-p21ras antibodies. Under those conditions, using the homobifunctional crosslinker dithiobis(succinimidyl propionate), a protein of Mr 60,000 (p60) is found to be associated with p21ras, and this association is enhanced by the treatment of quiescent cells with serum. Upon sedimentation of detergent extracts from crosslinked cells on sucrose gradients, a p21-p60 complex could be demonstrated with a Mr of 200,000-300,000, p60 does not appear to be related to pp60src nor to the cytosolic GTPase activating protein that interacts with p21ras to enhance its GTPase activity. The amount of p60 seems to be limiting relative to p21ras in fibroblasts, since similar levels of p60 are immunoprecipitated from Rat-1 cells and transfectants overexpressing Ha-, Ki-, and N-ras p21s; the same protein is also found to associate with p21ras in numerous mammalian cell lines. The relevance of this component to the role of ras proteins in signal transduction is discussed.