Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4007-4011, June 1989 Biochemistry Identification of a protein associated with p2lras by chemical crosslinking (guanine nucleotide-binding protein/oncogenes/signal transduction) JEAN DE GUNZBURG*t, REBECCA RIEHL, AND ROBERT A. WEINBERG Whitehead Institute for Biomedical Research, Cambridge, MA 02139, and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142 Contributed by Robert A. Weinberg, March 2, 1989 ABSTRACT The products ofthe ras oncogenes (p2l) are might act to transduce incoming growth signals to second ubiquitous membrane-associated proteins that bind guanine messenger-producing enzymes within the cell (10-14). To nucleotides and possess an intrinsic GTPase activity. Because date, GAP constitutes the only candidate for an effector of of their functional homologies with regulatory guanine nucle- p21l5 function, but its further roles in signaling remain otide-binding proteins, they are thought to be involved in the unknown. control of cellular proliferation as transducers of incoming Much effort has been devoted to identify yet other proteins growth signals. In an effort to identify proteins interacting with that might interact with p21's. In the case of other oncopro- p2lr', we have used in vivo crosslinking techniques on Rat-1 teins, functional associations have often been uncovered fibroblasts and derived cell lines overexpressing p2l and because interacting proteins may form stable complexes and immunoprecipitation with polyclonal anti-p21ra antibodies. coimmunoprecipitate (15-18); in the case of p2ls, such Under those conditions, using the homobifunctional crosslinker experiments have failed to identify any associated proteins. dithiobis(succinimidyl propionate), a protein of Mr 60,000 We therefore sought to chemically crosslink p21ISS to other (p60) is found to be associated with p2l¶, and this association functionally associated proteins in intact cells, thinking that is enhanced by the treatment of quiescent cells with serum. an in vivo crosslinking technique would make it possible to Upon sedimentation ofdetergent extracts from crosslinked cells preserve interactions that could otherwise be weak and on sucrose gradients, a p21-p60 complex could be demon- short-lived. A similar approach used in earlier studies has strated with a Mr of 200,000-300,000. p60 does not appear to allowed detection ofprotein-protein interactions inside living be related to pp60irC nor to the cytosolic GTPase activating cells (19-21). By using in vivo crosslinking techniques and protein that interacts with p2l to enhance its GTPase activ- immunoprecipitation with polyclonal anti-p21ras antibodies, ity. The amount of p60 seems to be limiting relative to p2l1 we report that a protein of Mr 60,000 (p60) is associated with in fibroblasts, since similar levels of p60 are immunoprecipi- p21's and that this association is enhanced by the treatment tated from Rat-i cells and transfectants overexpressing Ha-, of cells with serum. Ki-, and N-ras p21s; the same protein is also found to associate with p2iraS in numerous mammalian cell lines. The relevance MATERIALS of this component to the role of ras proteins in signal trans- AND METHODS duction is discussed. Cell Lines and Culture Conditions. Rat-1 fibroblasts and derived cell lines were grown in Dulbecco's modified Eagle's The mammalian Ha-, Ki-, and N-ras protooncogenes encode medium (DMEM) supplemented with 10% calf serum. All cell highly homologous Mr 21,000 membrane-associated proteins lines were derived from the parental line Rat-1 by transfection (p21's) that bind GDP and GTP and possess a low intrinsic and previously characterized (22). Rat-1.His2 carries the histi- GTPase activity. Point mutations in regions important for dinol selection marker only, whereas Rat-l.Hras3 and Rat- GTP binding and hydrolysis are responsible for activating 1.Hrasl3 overexpress p21c-Ha-ras 36- and 97-fold, respectively, these proteins to their oncogenic forms; examples include the relative to Rat-1 cells. Rat-1 EJ cells are transformed by the EJ ras proteins encoded by the acutely transforming Harvey and oncogene (23). Kirsten murine sarcoma viruses as well as those found in Antisera. Recombinant p21cHa-aS was produced in Esche- many human tumors (see ref. 1 for a review). p2lmS proteins richia coli and purified according to Gross et al. (24). Female are expressed in most if not all tissues and are thought to be New Zealand rabbits were immunized subcutaneously with 250 involved in the control of cellular proliferation, though their ,g of protein in Freund's complete adjuvant and given booster mode of action remains presently unclear. Recently, a cyto- injections with the same antigen in incomplete adjuvant. Mono- solic protein of Mr 116,000-125,000 has been identified that clonal antibody 147 elicited against a peptide encompassing is capable of stimulating >100-fold the GTPase activity of residues 157-180 of p21c.ras(4A) was obtained from the Na- normal p21ras but not that ofits oncogenic variants (2-5). The tional Cancer Institute Repository (Microbiological Associ- sequence of this protein, termed GAP, has been determined ates). The anti-peptide antibody RK2 reactive to murine epi- by analysis of cDNA clones (6, 7). As an apparent result of dermal growth factor receptor was a generous gift from Julian GAP activity, normal p2las molecules within the cell are Downward (Whitehead Institute). found to be almost exclusively associated with GDP, whereas Labeling and Chemical Crosslinking Conditions. Subcon- their oncogenic variants are largely associated with GTP. fluent cultures were labeled with 0.1 mCi of [35S]methionine This p21s-GTP complex is thought to constitute an active per ml (1200 Ci/mmol; 1 Ci = 37 GBq; Tran35S-label; ICN) signal-emitting form of the protein. Because of functional in methionine-free DMEM containing 5% calf serum for 3-18 homologies with regulatory guanine nucleotide-binding G proteins (8, 9), it has been proposed that p21's molecules Abbreviations: DSP, dithiobis(succinimidyl propionate); G protein, GTP-binding protein; GAP, GTPase activating protein. *To whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge tPresent address: Unite 248, Institut National de la Santd et de la payment. This article must therefore be hereby marked "advertisement" Recherche Medical, Faculte de Mddecine Lariboisiere Saint-Louis, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10 av. de Verdun, 75010 Paris, France. Downloaded by guest on October 1, 2021 4007 4008 Biochemistry: de Gunzburg et al. Proc. Natl. Acad. Sci. USA 86 (1989) hr. The dishes were washed free of medium with phosphate- cipitated with a polyclonal serum reactive with all species of buffered saline (PBS) and incubated with gentle shaking for p21ras (data not shown) rather than monoclonal antibodies to 30 min on ice with the thiol-cleavable homobifunctional avoid dependence on recognition of an epitope that may be crosslinker dithiobis(succinimidyl propionate) (DSP) at 1 mM masked in a complex. Analysis ofthe products (Fig. 1) shows diluted in PBS from 0.1 M solution in dimethyl sulfoxide that immune serum indeed recognizes p21's (lanes 1 and 2) (crosslinked cells) or solvent alone (uncrosslinked cells). and that crosslinking of the cells with DSP (lane 3) results in After further washing with PBS, the cells were lysed on ice the immunoprecipitation of an additional protein of p60. in 50 mM Hepes buffer (pH 7.4) containing 0.1 M NaCl, 1 mM Incubation of the immune complex with buffer alone prior to EGTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% denaturation and electrophoresis did not release any material NaDodSO4, and protease inhibitors (10 mM benzamidine, 1 from the immune complex (lanes 4 and 5); in contrast, when mM phenylmethylsulfonyl fluoride, and 10 gg each of apro- reducing agents were included in the elution buffer, the bulk tinin, leupeptin, and soybean trypsin inhibitor per ml). Debris of p60 could be separated from p211s (lanes 6 and 7), were eliminated by a 10-min centrifugation at 14,000 X g, and demonstrating that they were bound via the thiol-cleavable extracts were precleared by a 30-min incubation at 40C with crosslinker. A faint band migrating slightly faster p60 was normal rabbit serum and protein A-Sepharose. They were often observed in the absence of crosslinker (lane 2); two either used immediately for immunoprecipitation or stored at other proteins of Mrs 41,000 and 80,000 were immunopre- -800C. cipitated with preimmune or immune serum and partially Immunoprecipitation and Analysis of the Products. Lysates released from the immune complex by reducing agents, representing 1-5 x 107 cpm were incubated at 4°C with 1 Al irrespective of whether the cells had been crosslinked or not. of immune or preimmune rabbit serum for 3 hr; immune In contrast to p60 and p21, the immunoprecipitation of these complexes were collected on 10 ,l of protein A-Sepharose three proteins was not competitively inhibited by the addition beads, washed twice with 1 ml of RIPA buffer (10 mM Tris, of purified recombinant p2l,-Ha-ras (lanes 8 and 9) and was pH 7.5/5 mM EDTA/1% Triton X-100/1% sodium deoxy- therefore considered to be nonspecific. cholate/0.1% NaDodSO4) containing 0.5 M NaCl, and Detection of p60 was dependent on the presence of the washed twice with RIPA buffer alone. They were treated in crosslinker (Fig. 2): it increased with the concentration of the following ways prior to electrophoresis on 5-15% gradi- DSP (maximal at 1 mM, lanes 1-6) and incubation time with ent polyacrylamide gels in the presence of NaDodSO4 (25) the cells (up to 1 hr, lanes 7-9). Blocking the reactive groups and fluorography. For analysis of the total immunoprecipi- with excess ammonium ions prior to treatment of the cells tate, the beads were boiled in NaDodSO4 sample buffer abolished the appearance of p60 (lane 10).