Oncogene (2002) 21, 2425 ± 2432 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc SmgGDS displays dierential binding and exchange activity towards dierent Ras isoforms Haris G Vikis1,4, Scott Stewart1,3,4 and Kun-Liang Guan*,1,2 1Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, MI 48109-0606, USA; 2Institute of Gerontology, University of Michigan, Ann Arbor, Michigan, MI 48109-0606, USA Ras family GTPases play central roles in a wide variety (GEFs) that function to promote the dissociation of of biological responses, including cell proliferation, GDP and binding of GTP to the GTPase (Cher®ls and dierentiation, and oncogenic transformation. We Chardin, 1999). searched for novel guanine nucleotide exchange factors Activating mutations in Ras have been described in a of HRas and isolated small G-protein dissociation variety of human cancers (Barbacid, 1987; Lowy and stimulator (smgGDS), a guanine nucleotide exchange Willumsen, 1993). These mutations, which include the factor known to act on numerous Ras and Rho family G12V and Q61L mutations, result in a constitutively GTPases. SmgGDS speci®cally interacts with both GTP-bound and therefore active form of Ras (Barba- dominant negative and nucleotide free forms of H and cid, 1987; Lowy and Willumsen, 1993). These active NRas, but not with the corresponding oncogenic forms. mutants are used by investigators to mimic activation An eector domain mutant of HRas, HRasN17G37, of endogenous Ras. Similarly, dominant interfering selectively lost the ability to bind smgGDS. However, mutants of Ras, such as RasN17 and RasA15, are smgGDS does not catalyze guanine nucleotide exchange extensively used to determine whether a cellular process on either H or NRas in vitro. In contrast, substrates of depends on Ras (Chen et al., 1994; Feig and Cooper, smgGDS, such as KRas, Rac1, and RhoA, bind to 1988; Mulcahy et al., 1985; Stacey et al., 1991). Upon smgGDS in both active and inactive forms which ectopic expression, these molecules can interfere with requires the presence of poly-basic residues in the C- Ras activation by sequestering RasGEFs, thereby termini of the GTPases. Our data suggest that the C- preventing them from activating endogenous Ras (Feig terminal poly-basic region of small GTPases is important and Cooper, 1988; Stacey et al., 1991). for both binding and nucleotide exchange by smgGDS. We attempted to identify novel RasGEFs by Furthermore, these data underscore the idea that searching for proteins that speci®cally interact with mammalian Ras isoforms are not functionally equivalent. dominant negative Ras and puri®ed small G-protein Oncogene (2002) 21, 2425 ± 2432. DOI: 10.1038/sj/ GDP dissociation stimulator (smgGDS), an atypical onc/1205306 GEF with multiple Armadillo (ARM) repeats (Yama- moto et al., 1990). SmgGDS binds speci®cally to the Keywords: Ras; Rac; Rho; dominant negative mutants; dominant negative mutant (N17), but not to the smgGDS; guanine nucleotide exchange factor constitutively active mutant (V12) of both H and NRas. In contrast to dominant negative Ras, smgGDS associates with the active and inactive forms of its Ras family GTPases are involved in a variety of known substrates, Rac1, Rap1a, RhoA and KRas and physiological processes including cell growth, dier- this appears to be mediated by the poly-basic C- entiation, and malignant transformation (Barbacid, terminal region of the GTPase, a motif not present in 1987; Katz and McCormick, 1997; Lowy and Will- both H and NRas. The identi®cation of these unique umsen, 1993). Active Ras signals these events through interactions between smgGDS and small GTPases may GTP-dependent interactions with downstream eector suggest distinct modes by which smgGDS may regulate molecules, such as the protein kinase Raf and the lipid small GTPases. Furthermore, our observations indicate kinase PI-3 kinase (Katz and McCormick, 1997). The that many cellular responses observed with dominant activity of Ras and related GTPases are positively negative Ras mutants, RasN17 and RasA15, could be regulated by guanine nucleotide exchange factors due to sequestration of smgGDS. Results and Discussion *Correspondence: K-L Guan; E-mail: [email protected] 3Current address: Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305-5323, USA Identification of smgGDS as an HRasN17 binding protein 4These authors contributed equally to this study Received 4 October 2001; revised 2 January 2002; accepted 8 Dominant negative Ras mutants are common lab January 2002 reagents that are used to determine if a cellular process SmgGDS and small GTPases HG Vikis et al 2426 is dependent on the activation of Ras. RasN17 and we isolated smgGDS as protein capable of binding to RasA15 are two such mutants that, when over- dominant negative HRas, we reasoned that HRas expressed, bind and sequester endogenous Ras ex- would serve as a substrate for smgGDS, in contrast to change factors. Consequently, we used the binding previous reports (Hiraoka et al., 1992). We directly properties of these mutants to isolate novel exchange tested smgGDS activity towards several GTPases using factors of HRas. We searched for novel Ras exchange puri®ed, recombinant proteins. Consistent with earlier factors by using recombinant GST-HRasN17 as an ®ndings we observed that RhoA (as well as Rac1 and anity matrix. To this end, 35S-labeled COS1 whole Rac2, data not shown) served as substrates for cell extracts were incubated with recombinant GST- smgGDS, while H and NRas did not (Mizuno et al., HRasN17 followed by extensive washing and elution 1991) (Figure 1d). This result is puzzling since the with reduced glutathione. After SDS ± PAGE and dominant negative forms of H, K, and NRas all autoradiography, we observed that a protein of associate with smgGDS to similar extents (see below) 62 kDa co-puri®ed with GST-HRasN17, but not with and would therefore be predicted to act as substrates GST-HRasV12 or GST (Figure 1a). A 62 kDa protein for smgGDS. was reproducibly detected in GST-HRasN17 pull- One explanation for this observation is that downs using a variety of cell lysates, including COS1, dominant negative H and NRas binding to smgGDS HeLa, MCF-7 and 293T (data not shown) as well as is a gain-of-function associated with the mutant mouse brain. proteins only. We ruled out this possibility by using In order to determine the identity of this protein, wild-type H and NRas stripped of nucleotide in extracts from 30 mouse brains were applied to a GST- binding assays with smgGDS. Neither GDP- nor HRasN17 anity column. Following washing and GTPgS-loaded HRas interacted with recombinant elution we observed a 62 kDa band that puri®ed with His-smgGDS (Figure 2a, left panel). HRas binding to GST-HRasN17 that was readily detected by Coomassie smgGDS was observed only in the presence of EDTA. blue staining (Figure 1b). This band was excised and Similar results were observed with NRas (Figure 2a, subjected to protease digestion and Edman degradation right panel). Since EDTA prevents nucleotide binding which yielded the following two peptide sequences: to small GTPases by chelation of magnesium, it is KTGSDLMYL and KSVAQQAALTE, both of which likely that smgGDS preferentially binds to the were identical matches to amino acid sequences within nucleotide free form of H and NRas. It appears from the murine small GTPase guanine nucleotide dissocia- our results that the conformation adopted by the N17 tion stimulator (smgGDS) (Yamamoto et al., 1990). conformation is the same as the nucleotide free Interestingly, smgGDS has previously been shown to conformation. This suggests that although complexed function as a GEF on a subset of small GTPases, yet it to GDP in vivo RasN17 most likely resides in a bears no sequence homology to any known RasGEFs nucleotide free-like conformation (Stewart and Guan, (Hiraoka et al., 1992; Mizuno et al., 1991). We did not 2000). Alternatively, a fraction of RasN17 may exist in identify any known RasGEFs (e.g. SOS) in our anity a nucleotide free form in vivo. Nevertheless, the puri®cation which may be attributed to the relative observation that RasN17 adopts a nucleotide free abundance of smgGDS compared to SOS, or to the conformation agrees with the model whereby GEFs binding avidity under these conditions. promote exchange by binding and stabilizing the To con®rm the interaction between smgGDS and nucleotide free conformation of GTPases (Cher®ls HRasN17, we probed GST-HRasN17 pulldowns of and Chardin, 1999; Feig, 1999). 293 cell lysates with anti-GDS antibody. Consistent When expressed together in 293T cells, HRasN17 co- with our above results smgGDS was found to associate immunoprecipitated with HA-smgGDS and the inter- exclusively with HRasN17 (Figure 1c, upper panel). As action of HA-smgGDS with HRasN17G37 (see below) a control, HRasV12 bound to Raf under identical was signi®cantly diminished (Figure 2b). We also conditions (Figure 1c, lower panel). Furthermore, observed that HRasA15, another dominant negative HRasN17G37, an eector domain mutant (described mutant of Ras, associates more strongly with HA- below), no longer interacted with smgGDS. These smgGDS than does HRasN17 although it is expressed results demonstrate that smgGDS selectively interacts at signi®cantly lower levels than other Ras mutants with HRasN17 in vitro. (Figure 2c). HRasA15 displays a dramatically lower anity towards guanine nucleotide and it has been postulated that a signi®cant fraction of RasA15 exists SmgGDS interacts with the nucleotide free form of HRas in a nucleotide free form in vivo (Lai et al., 1993). and NRas Therefore, the results are consistent with the model SmgGDS bears no sequence homology to any known that smgGDS interacts with the nucleotide free form of GEFs and was originally isolated as a protein able to Ras. stimulate GDP dissociation on the small GTPase Rap1 SmgGDS most likely uses a mechanism quite distinct (Yamamoto et al., 1990).