MOLECULAR AND CELLULAR BIOLOGY, Feb. 1994, p. 1104-1112 Vol. 14, No. 2 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology Identification of Residues of the H-Ras Critical for Functional Interaction with Guanine Nucleotide Exchange Factors RAYMOND D. MOSTELLER, JAEWON HAN, AND DANIEL BROEK* Department ofBiochemistry and Molecular Biology, Kenneth Norris Jr. Cancer Hospital and Research Center, University of Southern California School ofMedicine, Los Angeles, California 90033 Received 10 September 1993/Returned for modification 21 October 1993/Accepted 29 October 1993

Ras are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene ofSaccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism ofCDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-alalS which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasva1l2 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sosl and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Ras protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

The eukaryotic RAS proteins are involved in a variety of distinct RAS-specific GEF which are capable of sensing pathways stimulating diverse changes in distinct extracellular stimuli. cell biology (2, 23). In most vertebrate cells, Ras proteins in A model for the mechanism by which CDC25GEF activates response to extracellular signals promote cell growth and RAS proteins has been proposed, using the genetic analysis division; in the pheochromocytoma PC12 cell line, Ras of a RAS2Ala-22 mutant and based on the mechanism of proteins induce terminal differentiation and neurite-like out- activation of transducin (20, 22, 32, 33, 43, 44). This model growth; in the yeast Schizosaccharomyces pombe, RAS is predicts that CDC25GEF binds to the RAS-GDP molecule involved in cell mating; in the yeast Saccharomyces cerevi- and that GDP dissociates to yield a nucleotide-free RAS- siae RAS proteins regulate cell growth and division; and in CDC25GEF reaction intermediate. Then, GTP binds to give Drosophila melanogaster RAS proteins are involved in eye rise to a CDC25GEF-RAS-GTP complex, which dissociates development (for reviews, see references 2 and 23). In each to yield CDC25GEF and the active RAS-GTP molecule. of these systems a recurring theme is apparent: RAS pro- Supporting this model is the evidence that CDC25GEF cata- teins bound to GTP induce phenotypic changes, whereas lyzes guanine nucleotide exchange of RAS-GDP to RAS- RAS proteins bound to GDP are inactive. The intracellular GTP much more effectively than it catalyzes the reverse levels of the RAS-GDP and RAS-GTP complexes are thus reaction, RAS-GTP to RAS-GDP (20, 22). This observation carefully regulated. Guanine nucleotide exchange factors is consistent with the observation that CDC25GEF has a (GEFs) of the CDC25GEF gene family convert inactive RAS higher affinity for the RAS-GDP complex relative to the proteins to their active, GTP-bound state (18, 20, 22, 39). RAS-GTP complex (29). Also, it has been demonstrated in a GEFs capable of activation of RAS proteins have been coprecipitation assay that CDC25GEF binds tightly to RAS identified in yeasts, D. melanogaster, mice, rats, and hu- proteins in their nucleotide-free state but not to RAS pro- mans (4, 5, 7, 11, 17, 25, 34, 39, 46). In mammals, at least one teins bound to GDP or GTP (20). GEF, cdc25, is tissue specific, showing expression only in We are interested in the mechanism by which Ras proteins cells of the nervous system (8, 25, 39, 46). It is likely that the are activated by GEFs. First, we sought to identify amino regulation of RAS proteins in various cell types involves acid positions in the H-Ras protein which are essential for CDC25GEF interaction. Second, we wished to determine the effect, if any, of the interaction of CDC25GEF with H-Ras on the relative affinities for GDP and GTP. To address the first * Corresponding author. Mailing address: NOR524, 1441 Eastlake question, we made use of a mutant human Ras protein, Ave., Los Angeles, CA 90033-0800. Phone: (213) 224-6562. Fax: H-RasAl-15, which dominantly interferes with RAS function (213) 224-6417. in yeasts. Expression of this protein in yeasts results in the 1104 VOL. 14, 1994 RAS ACTIVATION BY CDC25GEF 1105 cellular pool of CDC25GEF being sequestered into a stable, combining different fragments, we were able to separate inactive complex with the H-Ras a-15 protein (29, 33). double mutations and to construct derivatives containing a Consequently, endogenous RAS proteins cannot be con- glycine or valine codon at position 12 and a glycine or verted by CDC25GE to their active GTP-bound state and alanine codon at position 15. The template DNA in the first the yeast cells undergo growth arrest. To obtain mutants round of amplification was the original mutant, a wild-type of H-RasAa-l5 defective in functional interaction with H-ras-containing plasmid, or a plasmid containing the H-ras- CDC25GEF, we mutagenized a yeast plasmid harboring the Val-12 cDNA. For subcloning into pAD4, the oligonucleo- H-rasMlal15 cDNA under the control of the GALIO promoter tides used for PCR amplification included SalI and SacI and screened for second-site H-ras mutants which did not restriction sites flanking the N-terminal and C-terminal ends interfere with growth of wild-type yeast cells. We reasoned of the coding sequence, respectively. that the mutations in these intragenic revertants might fall into Interaction of monoclonal antibodies Y13-259 and Y13-238 several classes: (i) mutations which introduce premature stop with H-Ras. Nickel-agarose beads were incubated with a codons; (ii) mutations which result in the production of phosphate-buffered saline (PBS)-1% Triton X-100-solubi- unstable or otherwise nonfunctional proteins; (iii) mutations lized E. coli extract of His-ta-ed H-RasY57 expression which destroy the sites on H-Ras critical for functional system. The His-tagged H-Ras expression system was interaction with CDC25GEF; and (iv) mutations that reverse kindly provided by Vincent Jung and Michael Wigler. The the biological defect due to the Ala-15 mutation. From se- Y57 mutation in H-Ras does not inhibit its interaction with quence analysis, molecular genetic analysis, and biochemical GEFs (45a). The nickel-a arose beads were incubated with analysis of the mutants, we conclude that residues which lie an E. coli (His-H-Ras 57) extract for 1 h and then exten- on the external surface of H-Ras, encompassing residues 62 to sively washed for 1 h at 4°C. The beads were washed five 69, are required for activation by CDC25GEF. times with PBS-1% Triton X-100 (buffer A). His-tagged To address the second question, we made use of the H-Ras-Ni-agarose beads containing 4 pmol of H-Ras were observation that CDC25GEF binds tightly to RAS proteins in incubated for 1 h at 4°C in 1 ml of buffer A with 0, 50, 100, their nucleotide-free state, such that the two molecules can or 200 pmol of purified glutathione S- (GST)- be coprecipitated (20). We produced a complex of CDC25GEF (20) or GST-Sosl (22). The beads were then CDC25GEF and nucleotide-free RAS and determined that pelleted and washed five times with buffer A. The pellets this tight interaction could be readily disrupted by addition were resuspended in 1 ml of buffer A, 1 ,ul of either of 25 nM GTP, whereas GDP concentrations in excess of 250 monoclonal antibody Y13-259 or Y13-258 was added to the nM were required to effect a similar disruption of the reaction (14), and the mixture was incubated for 1 h at 4°C. CDC25GEF-RAS complex. From the results presented here The Ni-agarose beads were pelleted and washed five times we propose that residues 62, 63, 67, and 69 of H-Ras are with buffer A and once with PBS. The beads were repelleted critical for CDC25GEF-mediated conformational changes in and treated with 10 ,ul of sodium dodecyl sulfate-polyacryl- H-Ras that result in a decreased affinity for GDP and an amide gel electrophoresis (SDS-PAGE) sample buffer at increased affinity for GTP. 95°C for 10 min. The samples were analyzed by Western immunoblot analysis (22) with a rabbit anti-rat immunoglob- MATERIALS AND METHODS ulin G (IgG) antibody as the first antibody and goat anti- rabbit IgG conjugated to alkaline phosphatase as the second Mutagenesis of the H-ras gene. A 10-Rg portion of the yeast antibody. shuttle vector pSP30, containing the human H-rasAIa15 Vectors and yeast strains. The wild-type yeast strain SP1 cDNA, was mutagenized with 0.67 M hydroxylamine for 1 h (Mata his3 leu2 trpl ura3 ade8 canl) has been previously at 75°C (41) as modified by Hsiung and Nitiss (16a). The described (33). The yeast strains STS8 (Mata his3 leu2 trpl mutagenized DNA was used to transform Escherichia coli ura3 ade8 canl rasl:: URA3, ras2ts) and LV25-5 (Mata his3 HB101, and a pool of about 20,000 transformants was used leu2 trpl ura3 ade8 canl cdc25-Sts) were kindly provided by to make the final preparation of plasmid DNA. Roy-Marie Ballester and Michael Wigler. The human GAP Selection of H-ras mutants. Yeast strain SP1 was trans- cDNA, H-GAP, was integrated into strains STS8 and formed with the hydroxylamine-mutagenized pool of plas- LV25-5 by transformation with XhoI-digested plasmid mid pSP30 DNA and plated on SC-'u medium. Approxi- p[PGK] H-GAP DNA (1). Ade+ transformants were selected mately 2,000 transformants were obtained at 30°C and then and screened by PCR for integration of the human GAP screened by replica plating on SGal-Lu medium (38). Dif- cDNA into the yeast genome at the ade8 locus. Plasmid ferences in growth were observed after a second round of pSP30 is the YEp51 vector containing the human H-rasAal15 replica plating. Colonies which grew at 36°C on SGal-Iu cDNAwhose expression was driven by the GAL1O promoter medium were purified by streaking, retested for growth on (kindly provided by Scott Powers). SGal-'u medium, and used to make plasmid DNA prepa- Construction of two-hybrid system vectors and assay for rations. The rescued plasmids were reintroduced into strain 13-galactosidase. PCR-amplified H-ras cDNA sequences SP1. None of the reintroduced plasmids interfered with (codons 1 to 166) were subcloned into the GAL4 activation growth in the presence of galactose. domain vector pGADGH and the GAL4 DNA-binding do- Subcloning of H-ras cDNA into a yeast expression vector. main vector pGBT9 by using oligonucleotides encoding The H-ras cDNA was subcloned into the yeast expression EcoRI or SailI endonuclease restriction sites at the 5' or 3' vector pAD4 (47) by using PCR-amplified copies of the end of the coding sequence, respectively. In some cases the wild-type or mutant H-ras cDNAs flanked by restriction intact region of H-ras was amplified, and in other cases endonuclease sites. In some cases the intact coding se- overlapping fragments were amplified and then combined in quence was amplified, and in other cases overlapping frag- a second round of PCR amplification. The fragments gener- ments of the coding region were amplified and then com- ated corresponded to approximately codons 1 to 54 and bined by a second round of PCR amplification (46). The codons 49 to 166. The template DNA used was the original fragments used corresponded approximately to H-ras mutant derivatives of pSP30, a wild-type H-ras-containing codons 1 through 54, 49 through 105, and 97 through 189. By plasmid or the parental H-rasAal15 plasmid pSP30. 1106 MOSTELLER ET AL. MOL. CELL. BIOL.

The PCR-amplified yeast CDC25 cDNA sequence (codons with hydroxylamine. The pool of mutagenized plasmid DNA 1100 to 1589) was subcloned into the pGEM-T vector was transformed into wild-type yeast strain SP1 (which (Promega Corp.) by using oligonucleotides encoding Sall contains a defective leu2 gene). The approximately 2,000 and SacI endonuclease restriction sites at the 5' or 3' end of Leu+ colonies appearing after incubation at 30°C for 2 days the coding sequence, respectively. Subsequently, a SalI were replicated onto SGal-'u plates and incubated at 36°C restriction fragment of the resulting plasmid was further for 2 days to identify revertants. The plasmids were rescued subcloned into pGADGH. Transformants of yeast strain from revertants, and the entire H-ras coding sequences were PCY2 (9) containing the two-hybrid system vectors were subjected to DNA sequence analysis (see Materials and tested for ,B-galactosidase activity by patching SC-Leu-Trp Methods). Five of the isolated revertants contained prema- agar medium plates containing 50 mM KPO4 (pH 7.0), 2% ture stop codons. Previous reports have demonstrated that sucrose, and 100 ,g of 5-bromo-4-chloro-3-indolyl-13-D-ga- the C terminus of H-Ras is critical for localization to the lactopyranoside (X-Gal) per ml (10). Plasmids pGADGH and plasma membrane and that dominant interfering mutations in pGBT9 were obtained from Linda van Elst and Michael RAS must localize to the plasma membrane to effectively Wigler. block RAS function in vivo (23). Thus, the revertants RAS-CDC25GEF binding assay. GST-CDC25GEF (an ap- containing premature stop codons were not further ana- proximately 70-kDa fusion protein made up of GST and the lyzed. Sequence analysis of seven revertants revealed that catalytic domain of the S. cerevisiae CDC25GEF) and the S. point mutations corresponding to seven distinct amino acid cerevisiae RAS2 and human H-Ras proteins from E. coli codon changes had occurred in the coding region of the expression systems were prepared as previously described H-ras cDNA (see Table 1). (6, 7, 20, 42). RAS2 and GST-CDC25GE fusion protein were Determination of whether H-Ras proteins with the amino coprecipitated by using glutathione-agarose beads as previ- acid substitutions of the identified intragenic suppressors of ously described (20). The glutathione-agarose suspensions H-rasua.l5 were capable of activating the RAS pathway in S. were washed four times in a PBS solution containing 1% cerevisiae via a CDC25GEF dependent and/or independent Triton X-100. Dilutions of GDP and GTP were prepared mechanism. We predicted that missense revertants in H-Ras from concentrated stocks (Boehringer Mannheim). Western proteins might fall into distinct classes: (i) nonfunctional blot analysis of the RAS2 protein was carried out with a proteins; (ii) proteins which are disrupted in a functional rabbit polyclonal antibody directed against RAS2 proteins interaction with CDC25GEF; and (iii) proteins with mutations prepared from an E. coli expression system and by the use of which reverted the biochemical defect of the Ala-15 muta- the ECL Visualization Kit as described by the manufacturer tion. To begin to distinguish these classes of mutants, we (Amersham). Nucleotide-free H-Ras was prepared by treat- carried out the following molecular genetic assays. First, we ment with EDTA as previously described (20). examined whether expression of mutant H-Ras proteins Guanine nucleotide-binding assay. The complex of nucle- containing the missense substitution we identified but lack- otide-free RAS2 and the GST-CDC25 fusion protein bound ing the Ala-15 substitution could suppress the loss of RAS to glutathione-agarose was prepared as described above. function. We used a yeast strain carrying the human GT- The final pellet (glutathione-agarose-GST-CDC25-Ras) was Pase-activating protein (H-GAP) (26) cDNA to compensate resuspended in buffer G (50 mM Tris-HCl [pH 7.5], 5 mM for the fact that endogenous yeast GAPs (encoded by IRAI MgCl2, 1 mM dithiothreitol, 20 mM KCI). [3H]GDP- and and IRA2) are inactive on the human protein, H-Ras. In the [3H]GTP-binding assays were carried out by using a nitro- presence of human GAP expression, CDC25GEF must be cellulose filter-binding assay as previously described (6) with functional for H-Ras to promote S. cerevisiae growth (1). We modifications described in the text. [3H]GDP and [3H]GTP, therefore used a strain of S. cerevisiae carrying the H-GAP with specific activities of 8.0 and 9.9 Ci/mmol, respectively, cDNA and deficient in RAS function to assay for the activity were obtained from New England Nuclear. of our mutant H-Ras proteins in vivo. Other materials and methods. Oligonucleotides were pur- The yeast expression plasmids encoding H-Ras proteins chased from Operon Technologies, Alameda, Calif. Yeast with one of the missense substitutions G60D, E62K, E63K, transformations were performed as previously described (3, M67I, D69N, E76K, or G77D (but not the alanine 15 36, 38). E. coli transformations were performed by electro- mutation) could not suppress the temperature-sensitive de- poration as described by the manufacturer (Bio-Rad, Rich- fect of the rasl ras2ts H-GAP strain (STS8-H-GAP) (Table mond, Calif.). Yeast strains were grown in YPD medium or 1). This is in contrast to wild-type H-Ras, which can sup- synthetic complete (SC) medium (38). SGal-'u medium press the defect of strain STS8-H-GAP (Table 1). Further, contains 2% galactose substituted for glucose (38). E. coli the original mutant plasmids we identified with substitution strains were grown in LB medium (24) containing 100 p,g of at position 60, 62, 63, 67, 69, 76, or 77 in addition to the ampicillin per ml. DNA sequencing was performed by the Ala-15 substitution failed to suppress the temperature-sensi- method of Sanger et al. (35) with the Sequenase version 2.0 tive defect of strain STS8-H-GAP (data not shown). Our kit from United States Biochemical Corp., Cleveland, Ohio. results indicate that these missense mutations produce either nonfunctional proteins or mutant proteins which cannot be RESULTS stimulated by CDC25GEF in vivo, but none of these muta- tions reversed the biochemical defect of the Ala-15 mutation. Identification of intragenic suppressors of the H-rasMa-l5 To distinguish between the possibility that the mutation dominant negative mutant. We reasoned that some intragenic resulted in nonfunctional proteins or proteins with a specific suppressors of the dominant negative H-rasAal15 mutant defect in CDC25GEF interactions, we carried out the follow- might be defective in functional interaction with S. cerevi- ing genetic assays. We constructed yeast expression plas- siae CDC25GEF. To obtain such intragenic suppressors we mids which encoded H-Ras with each of these missense used plasmid pSP30, which encodes the human H-rasAIa15 mutations in addition to the glycine 12-to-valine substitution. dominant negative mutant, its expression driven by the yeast The H-Rasval12 protein has a defective GTPase activity (1, GAL10 promoter (with the selectable marker being the 21) and is known to be maintained in significant levels of the LEU2 gene), and mutagenized this plasmid by treatment active GTP-bound form in vivo, even in the absence of a VOL. 14, 1994 RAS ACTIVATION BY CDC25GEF 1107

TABLE 1. Suppression of ras' defect by mutant TABLE 2. Interaction of mutant and wild-type H-Ras proteins and wild-type H-Ras Mutation in ,-galactosidase activity of PCY2-CDC25': Suppression of": H-Ras Gly-15 (wild type) Ala-15 (mutant) Mutation STS8-H-GAP STS8 (Gly-12) None + + + Val-12 Gly-12 G60D + E62K - - G60D - E63K - - E62K +++ -+++ M671 - - E63K +++ +++ D69N - - M67I +++ -+++ D69N +++ ++++ I ,-Galactosidase activity was measured by using a strain containing E76K - - - plasmid pGADGH-CDC25 (PCY2-CDC25) in addition to plasmids expressing G77D - - _ fusion of the Gal4 activator domain and various H-Ras proteins, by using the None +++ +++ +++ X-Gal plate assay. The fusion of wild-type and the indicated mutant H-Ras proteins was driven by pGBT9-based vectors. Symbols: +, strong 3-galacto- a The ability of the indicated H-Ras proteins to suppress the temperature- sidase activity; + +, stronger 3-galactosidase activity; - indicates little or no sensitive defect of a rasl rasts H-GAP strain (STS8-H-GAP) or a rasl, ras2' detectable ,B-galactosidase activity. Five independent transformants of the strain (STS8) is presented. Symbols: +++, growth at wild-type levels; +, indicated strains yielded similar results. extremely weak suppression; -, no detectable growth at the nonpermissive temperature. The columns below Gly-12 and Val-12 indicate that the H-Ras protein contains the wild-type glycine or the valine substitution at position 12. independent mechanism (see Discussion). Interestingly, sub- stitutions in H-Ras at position 62 or 63 (glutamic acid-to- guanine nucleotide exchange factor, because of the slow histidine substitutions) have been shown to result in H-Ras intrinsic guanine nucleotide exchange activity of the H-Ras proteins whose nucleotide exchange activity cannot be acti- proteins (1, 7, 33). If a plasmid encoding an H-Rasvall2 vated by the CDC25-related CDC25GEF protein in vitro (28). protein in addition to one of the above missense mutations Further, all of these residues (E62, E63, M67, and D69) are can suppress the temperature-sensitive defect of the rasl located on the external surface of the H-Ras protein (27, 30) ras2ts H-GAP strain (STS8-H-GAP), this would indicate that (see Discussion). the missense mutation does allow an active H-Ras-GTP Binding of CDC25 to wild-type and H-Ras mutants. We complex to form in vivo. If one of these plasmids cannot wished to determine whether the mutation at position 62, 63, suppress the loss of RAS function, it would suggest that the 67, or 69 of H-Ras results in a defect in interaction with missense mutation results in a nonfunctional protein which CDC25. The yeast two-hybrid system has been successfully cannot adopt the correct active GTP-bound state. used to examine protein-protein interactions (9, 10, 13). The plasmids encoding H-Rasva'l12 in addition to one of Munder and Furst provided the first direct evidence for the the missense mutations G60D, E76K, or G77D were unable interaction of RAS and CDC25 proteins by using the two- to suppress the temperature-sensitive defect of the rasl hybrid system (29). The system makes use of two plasmids; ras2ts H-GAP strain (Table 1). These results suggest that one plasmid contains the GAL4 activation domain but lacks these missense mutations (at position 60, 76, or 77) result in the GALA DNA-binding domain, the other plasmid contains proteins which cannot adopt the active GTP-bound state in the GAL4 DNA-binding domain but lacks the GALA activa- vivo under these experimental conditions. In contrast, the tion domain. By insertion of sequences into these vectors plasmids encoding H-Rasval-12 and also containing one of the encoding two proteins that normally interact directly, the missense mutations E62K, E63K, M671, or D69N encode two fusion proteins can bind, allowing expression of the proteins which can adopt an active GTP-bound state in vivo, GAL4-dependent ,B-galactosidase reporter gene. The two- as indicated by their ability to suppress the rasl ras2ts hybrid system vectors harboring wild-type H-Ras or mutant H-GAP strains (Table 1). H-Ras as well as the catalytic domain of CDC25 were As discussed above, the ability of the human H-Ras to prepared as described in Materials and Methods. We initially suppress the loss of Ras function in a yeast strain expressing introduced plasmid pGADGH-CDC25 into strain PCY2 the human H-GAP is dependent on endogenous CDC25 (which contains the GAL4-dependent-o-galactosidase re- activity. In the absence of H-GAP, CDC25 activity is not porter gene) to produce strain PCY2-CDC25. This strain was required for H-Ras suppression of the loss of Ras function in transformed with either pGBT9-H-ras, pGBT9-H-rasG60D, strain STS8 (1). We therefore used this observation as the pGBT19H-rasE62K, pGBT9-H-rasE3K, pGBT9-H-rasM671, or basis of a second genetic assay to assess the activity of the pGBT9-H-rasD69N. Independent transformants from each H-Ras mutants we had identified. We introduced each of our resulting strain were patched onto selective plates containing mutant plasmids (encoding wild-type glycine at position 12) X-Gal (a colorometric substrate for 3-galactosidase). After 3 into the Ras-deficient strain, STS8, to determine whether to 5 days of incubation at 28°C, 1-galactosidase activity was they could suppress the loss of Ras function. The wild-type evident by the blue color of patches containing wild-type H-Ras, as well as H-Ras mutants with substitution at posi- H-Ras and H-RasCIOD (Table 2). In contrast, the strains tion 62, 63, 67, or 69, could suppress the temperature- harboring H-Ras mutants with substitution at position 62, 63, sensitive defect of strain STS8 (Table 1). In contrast, H-Ras 67, or 69 remained white, indicating the lack of 3-galactosi- mutants with substitution at position 60, 76, or 77 could not dase activity (Table 2). These results indicate that, in con- suppress the defect in STS8 (Table 1). These results, taken trast to H-Ras and CDC25, which interact directly under together with the results of the experiments presented these conditions, our mutants in H-Ras with mutation at above, led us to suggest that the amino acids at positions 62, position 62, 63, 67, or 69 are unable to bind to CDC25. 63, 67, and 69 in H-Ras are critical for functional interaction Relative to wild-type RAS2, the dominant interfering with the CDC25GEF but are not essential for function if RAS RAS2Aa-22 exhibits a higher affinity for CDC25 as judged by can achieve an active GTP-bound state via a CDC25GEF_ results obtained with the yeast two-hybrid system (29). This 1108 MOSTELLER ET AL. MOL. CELL. BIOL. suggests that the use of a dominant interfering mutant might result in a more sensitive assay for examining interactions A 1 2 3 4 5 6 7 8 between RAS and CDC25GEF. To test this prediction, we constructed plasmid pGBT9-H-RasAlal5. This plasmid was transformed into strain PCY2-CDC25. We found that this plasmid, containing the Ala-15 mutation, was more potent in activating the 3-galactosidase reporter gene than was the wild-type H-Ras-containing plasmid pGBT9-H-ras. Thus we wished to examine the effect of mutation in H-Ras at position 62, 63, 67, or 69 on interaction with CDC25 by using this more sensitive Ras-CDC25GEF interaction assay. We con- structed pGBT9-based plasmids containing the Ala-15 muta- tion in addition to H-Ras mutation at codon or 60, 62, 63, 67, 1 2 3 4 7 8 69. We transformed the pGBT9-based H-Ras plasmids into B 12345675 6 strain PCY2-CDC25. The plasmids pGBT9-HrasAIals in- ! rn duced significant levels of 3-galactosidase activity (see Ma- terials and Methods) (Table 2). Strains harboring pGBT9- HrasAal15 plasmids in addition to a mutation at positions 60, 62, 63, 67, or 69 yielded little or no detectable ,-galactosi- dase activity. We note that there is a difference in the ability of the G60D mutation to yield 3-galactosidase activity (indicating inter- FIG. 1. Binding of the mouse SoS1GEF or yeast CDC25GEF to action with CDC25G F) in the context of Gly-15 (wild-type) H-Ras blocks the binding of anti-Ras antibody Y13-259 but not or Ala-15 H-Ras. We have not examined possible reasons for Y13-238. A Western blot is shown which detects the rat monoclonal these differences. However, there is precedent for H-Ras antibody that can coprecipitate with H-Ras proteins that have or in vivo for which two have not been prebound with Ras-specific GEFs. A constant amount protein instability proteins contain of a His-tagged H-Ras protein (4 pmol) bound to nickel-agarose missense mutations, even when these missense mutations beads was incubated for 1 h with various amounts of the yeast alone do not result in protein instability in vivo. For exam- GST-CDC25GEF or mouse GST-Sosl (see Materials and Methods). ple, point mutants with the H-Ras mutation S17N or S89F The amounts of GST-SoslGEF used were 0, 50, 100, and 200 pmol are stable in vivo, but the double mutation, S17N S89F, (lanes 1, 2, 3, and 4, respectively). The amounts of yeast GST- results in an unstable protein (16). Nonetheless, these results CDC25 proteins used were 0, 50, 100, and 200 pmol (lanes 5, 6, 7, indicate that the substitutions we have identified at positions and 8, respectively). After incubation, material not complexed to the 62, 63, 67, and 69 result in H-Ras proteins with a dramati- agarose beads was removed by repeated washings. The samples cally reduced affinity for CDC25GEF. were then incubated for 2 h with anti-Ras monoclonal antibody Y13-238 but not Y13-259 can Y13-259 (A) or Y13-238 (B). After incubation, soluble material not Monoclonal antibody bind to complexed to the beads was removed by repeated washings. The a complex of H-Ras and the mouse sos) or yeast CDC25 gene resulting pelleted beads were prepared for SDS-PAGE and Western product. Monoclonal antibody Y13-259 is known to interact blot analysis by using a rabbit anti-rat IgG polyclonal antibody to with H-Ras at amino acids 63, 65, 66, 67, 70, and 73 (40). The determine fractions which retain or lose the ability to bind the epitope for the anti-Ras monoclonal antibody Y13-238 has monoclonal antibodies (see Materials and Methods). The arrows not been mapped, but it does not interact at the same site as indicate the position of migration of the heavy chain of rat IgG. We does antibody Y13-259 (14). The results presented above note that the goat anti-rat IgG antibody used here has a differential suggest that residues 62, 63, 67, and 69 of H-Ras are critical reactivity toward the light chains of Y13-238 and Y13-259, suggest- for a functional interaction with Ras-specific GEF related to ing that these monoclonal antibodies have different species of light the and with the chain (X versus K) and that the goat anti-rat IgG antibody preferen- yeast CDC25GEF overlap epitope for tially recognizes one of these species. Western blot analysis of each antibody Y13-259. Therefore, we wished to determine of the samples indicated no detectable loss of His-tagged H-RasY57 whether a complex of H-Ras protein and the catalytic protein (data not shown). domain of the human CDC25 or the mouse SoSlGEF retained the ability to bind monoclonal antibodies Y13-259 and Y13-238. For this purpose we bound a His-tagged H-Ras protein to nickel-agarose beads (see Materials and Methods). Y13-238 to bind to the H-Ras protein and subsequently Aliquots of the nickel-agarose His-tagged H-Ras beads (con- coprecipitate with the nickel-agarose complex (Fig. 1). Thus taining approximately 4 pmol of H-Ras proteins) were incu- we conclude that the binding of Ras-specific GEFs related to bated with 0, 50, 100, or 200 pmol of a purified GST-SoslGEF the yeast CDC25 gene product masks the epitope for anti- (mouse) or GST-CDC25GEF (yeast) fusion proteins. The body Y13-259 but not Y13-258. beads were washed to remove material not complexed to Disruption of a nucleotide-free RAS-CDC25GEF complex them (see Materials and Methods). Either monoclonal anti- with GDP and GTP. RAS proteins in their nucleotide-free body Y13-259 or Y13-238 was added to each of the reactions state bind tightly to the catalytic domain of the yeast CDC25 and allowed to incubate (see Materials and Methods). Again, gene product such that the GST-CDC25GEF and RAS pro- the beads were extensively washed to remove soluble mate- teins in a 1:1 molar ratio can be effectively coprecipitated by rial not complexed to them. The resulting pelleted beads using glutathione-agarose beads (20). Using this system, we were prepared for Western blot analysis by using rabbit examined the ability of increasing concentrations of GTP anti-rat IgG to detect antibody Y13-259 and Y13-258. Addi- and GDP to destabilize a complex of RAS2 and GST- tion of excess GST-SoslGE (mouse) or GST-CDC25GEF CDC25GEF proteins. A fusion protein containing the cata- (yeast) effectively blocks the binding of monoclonal antibody lytic domain of the yeast CDC25 gene product was purified Y13-259 (Fig. 1). The GST-SoslGF and GST-CDC25GEF by using a GST fusion protein expression system (20). This had little or no effect on the ability of monoclonal antibody GST-CDC25GEF fusion protein has been shown to possess in VOL. 14, 1994 RAS ACTIVATION BY CDC25GEF 1109

nM GDP nM GTP

0 IC0 CD CD o~ L) 0o 0) 0 So 0)6 L~C)o 00o .580- -u\rCM - C\ n - _ N CM LO - 60-~~ o 40 - M 4f- -- AS2 z 20

0 200 400 600 8000 1000 nM GTP Conc. (nM)

Ln FIG. 3. Nucleotide binding to the complex of nucleotide-free o0 Ln H-Ras and GST-CDC25 protein. Aliquots of a complex of nucleo- tide-free H-Ras and GST-CDC25GEF bound to glutathione-agarose were incubated at 22°C for 10 min with various concentrations of [3H]GTP (*) or [3H]GDP (5). The reactions were stopped by tS -RAS2 incubation at 0°C for 3 min, and the slurry was used in a nitrocel- lulose filter-binding assay to determine the amount of GTP or GDP bound to the complex. The values plotted represent the average of FIG. 2. Disruption of the complex of nucleotide-free RAS2 pro- duplicate data points. Similar results were obtained in two indepen- teins and the GST-CDC25OEF fusion protein by increasing concen- dent experiments. Nucleotide binding is expressed as a percentage trations of GDP and GTP. The complex of nucleotide-free RAS2 and of maximal binding activity. GST-CDC25GEF was bound to glutathione-agarose. Aliquots of this stable complex were exposed to 0, 1, 10, 25, 50, 100, 250, 500, or 1,000 nM GDP or GTP as indicated. After incubation at 22°C for 10 min, supernatants (containing RAS2 protein no longer bound to the 250 nM GDP in causing dissociation of RAS2 from GST- GST-CDC25GEF fusion protein) and pellets (containing RAS2 pro- CDC25GEF (Fig. 2). In a separate experiment we found final tein bound to the GST-CDC25GEF fusion protein) were collected. concentrations of 10 nM GTP to be considerably less effi- Western blot analysis of the supernatant fractions, with a rabbit cient than 25 nM GTP in disruption of the GST-CDC25- anti-RAS2 polyclonal antibody, is presented. Western blot analysis RAS2 complex (Fig. 2). of the pellet fractions indicated that no significant loss of RAS2 To ensure that sample was not lost during the experiment, proteins occurred during the experiments (data not shown). we also carried out Western blot analysis of the pellets to determine the amount of RAS2 protein remaining bound to the GST-CDC25GEF. At the highest concentrations of GDP vitro guanine nucleotide exchange activity specific for RAS or GTP more than 90% of RAS2 protein was released from proteins (yeast RAS2 protein and human H-Ras protein). CDC25GEF, and for all concentrations of GDP or GTP the An extract from E. coli expressing the GST-CDC25GEF sum of the levels of RAS2 protein seen in supernatants and fusion protein was incubated with glutathione-agarose beads pellets was constant, as expected (data not shown). Thus we at 4°C for 30 min. The beads were pelleted and washed. The conclude that GTP at low concentrations is more effective resulting ellet contained approximately 70 pmol of GST- than GDP at low concentrations at causing dissociation of CDC25" fusion protein as judged by SDS-PAGE analysis RAS2 from GST-CDC25GEF. of an aliquot of the pellet. A suspension of the glutathione- GDP and GTP binding to a nucleotide-free RAS-CDC25GEF agarose-GST-CDC25GEF beads was incubated with 500 complex. Because we found differences in the ability of GTP pmol of nucleotide-free RAS2 proteins for 30 min at 4°C and and GDP to disrupt a RAS-GST-CDC25GEF complex, we then pelleted and washed. The final pellet was resuspended investigated whether this complex has a higher affinity for in 250 ,ul of PBS containing 5 mM MgCl2. Then 20 ,ul of the GTP than it does for GDP. For this purpose we prepared suspension was aliquoted into 11 tubes, and 20 ,ul of a approximately 50 pmol of a complex of GST-CDC25GEF PBS-MgCl2 solution containing differing concentrations of fusion protein and human H-Ras protein. This complex was GDP or GTP was added to the various tubes. The final bound to glutathione-agarose beads in a suspension of 1.2 ml concentrations of the GDP and GTP used were 0, 1, 25, 100, of buffer G (see Materials and Methods). Then 50 ,ul of this 250, 500, and 1,000 nM. The reactions were incubated at suspension was aliquoted into 24 different tubes at 4°C. A 22°C for 10 min, vortexed, and then centrifuged to pellet the solution containing [3H]GDP or [3H]GTP at various concen- agarose beads. The resulting supernatants and pellets were trations was added to the agarose-GST-CDC25-Ras-con- separated and analyzed by Western blotting to determine the taining suspensions (50 ,ul of solution per suspension). Final relative amounts of RAS2 proteins in the supernatants (not concentrations of 0, 25, 50, 100, 175, 250, 375, 500, 600, 700, complexed with GST-CDC25GEF) and the pellets (com- 800, and 1,000 nM GDP and GTP were used. Reactions were plexed with GST-CDC25GEF). incubated for 10 min at 22°C. We had 1previously determined As shown in Fig. 2, final concentrations of 0, 1, 25, and 100 that 10 min was sufficient to saturate [ H]GDP binding to the nM GDP did not cause significant RAS2 protein to dissociate GST-CDC25GEF-ras complex when 1,000 nM GDP was used from GST-CDC25GEF and enter the supernatant fraction. (data not shown). The reactions were stopped by incubation Final concentrations of 250, 500, and 1,000 nM GDP did at 0°C for 3 min. The amount of [3H]GDP or [3H]GTP bound cause increasing amounts of RAS2 to dissociate from the to H-Ras in each of the reactions was determined by using a GST-CDC25GEF. Low concentrations of GTP were able to nitrocellulose filter-binding assay (see Materials and Meth- effectively cause RAS2 to dissociate from GST-CDC25GEF; ods). As shown in Fig. 3, under these conditions 50% concentrations as low as 25 nM caused dissociation of a saturation of binding was achieved at a GTP concentration of significant amount of RAS2 protein from GST-CDC25GEF approximately 30 nM. The saturation curve for [3H]GDP (Fig. 2). A GTP concentration of 25 nM was as effective as shows 50% saturation occurring at approximately 200 nM 1110 MOSTELLER ET AL. MOL. CELL. BIOL.

GDP. Similar saturation curves were obtained when using reduced affinity, as indicated by our yeast two-hybrid anal- the yeast RAS2 protein under similar assay conditions (data ysis, the E62H and E63H mutations exhibited an increased not shown). These results clearly indicate that the complex affinity for the CDC25-related SCD25 gene product (28). The of CDC25GEF and Ras has a significantly higher affinity for differences in affinities for GEFs to the distinct Ras mutant GTP than it does for GDP. with substitutions at positions 62 and 63 may result from the differences in the amino acid substitution. Clearly there is a DISCUSSION precedent for mutations in H-Ras at a single position to yield profoundly different phenotypes depending on the substi- In yeast strains expressing a human GAP cDNA, wild- tuted amino acid (12). type H-Ras can suppress the rasl ras2ts mutation but not the In a second published report regarding Ras mutants with cdc25ts mutation, whereas the H-Rasval-l2 mutant, with a altered interaction with GEFs, RAS2 proteins with muta- defective GTPase activity, can suppress the loss of both tions at positions corresponding to H-Ras residues 73 and 74 cdc25 and ras function even when the human H-GAP is were found to be insensitive to CDC25GEF in vitro and could expressed (1). These observations demonstrate that suppres- not suppress the loss of Ras function in yeasts but could sion of the loss of ras function by H-Ras requires CDC25GEF adopt an active conformation as judged by their ability to activity (present in the rasl ras2ts strain) to convert the stimulate the yeast adenylate cyclase in vitro (45). Because H-Ras protein to its active, GTP-bound state. Further, these of the proximity of our mutants with positions 73 and 74, it results indicate that H-Ras can suppress the loss of Ras seems likely that the domain of H-Ras encompassing resi- function if the H-Ras protein can achieve and activate the dues 62 to 74 is critical for CDC25GEF-mediated activation of GTP-bound state via a CDC25-independent mechanism. H-Ras. That is, in the absence of H-GAP, H-Ras becomes activated Each of the amino acid residues that we propose interacts as a result of the lack of adequate H-Ras-specific GTPase with CDC25GEF is located in a-helix 2 of the H-Ras protein activity and the slow intrinsic guanine nucleotide exchange (27, 30). Furthermore, the side groups of amino acid residues rate of the H-Ras protein. Alternatively, the H-Rasvall2 E62, E63, M67, and D69 are external residues, exposed on mutant has a defective GTPase activity and becomes acti- the surface of the protein (14, 27, 30). Residues correspond- vated, again as a result of the slow intrinsic guanine nucle- ing to positions 73 and 74 of H-Ras, proposed sites of otide exchange rate of H-Ras. interaction with the CDC25GEF-related SCD25GEF (45), also We used these suppression assays to test H-Ras mutants lie on the external surface of a-helix 2. Thus it is likely that described here for their ability to be activated by CDC25GEF. the residues on the surface of a-helix 2 of H-Ras, namely Among the mutant proteins which can adopt an active E62, E63, M67, D69, R73, and T74, are required for func- conformation in vivo, only those containing substitution tional interaction with GEFs related to CDC25GEF. E62K, E63K, M67I, or D69N were unable to suppress the Recently Segal et al. have suggested that residues 97 to rasl ras2ts mutation in the human GAP strain (Table 1). We 103 of H-Ras are critical for CDC25GEF mediated guanine conclude that these substitutions prevent activation of nucleotide exchange (37). These studies made use of H-Ras H-Ras by CDC25GEF and thus that positions 62, 63, 67, and deletion mutants which were biologically active in activating 69 are critical for the CDC25GEF_mediated activation of Ras. the downstream target of RAS in S. cerevisiae, adenylate Further, we demonstrate, using the yeast two-hybrid sys- cyclase, but could not serve as substrates for CDC25GEF. tem, that the mutations described here at positions 62, 63, Further, Segal et al. demonstrated that H-Ras mutants with 67, and 69 result in H-Ras proteins which are defective in deletion of residues 64 to 70 and 75 to 76 could not activate interaction with the yeast CDC25GEF under condition where adenylate cyclase, indicating an inability of the mutants to H-Ras groteins without these mutations can interact with adopt an active GTP-bound state (37). Our result, obtained CDC25 EF (Table 2). by using point mutations which probably have less deleteri- Several recent reports have suggested that there are ous effects on overall protein structure than do deletion distinct regions of Ras critical for interaction with nucleotide mutation, clearly indicate that certain amino acid substitu- exchange factors. These reports have yielded conflicting tions in this region, namely, E62K, E63K, M67I, and D69N, results, implicating residues 62 to 74 or 97 to 108 in GEF- can adopt an active GTP-bound state in vivo. The region mediated activation of Ras (16, 28, 37, 45). Our studies near position 97 of H-Ras may be critical for CDC25GEF_ demonstrate the involvement of residues 62 to 69 of H-Ras in mediated guanine nucleotide exchange. However, because CDC25GEF interactions and extend the finding of others. of the differences in these results obtained by using deletion Mistou et al. have shown that substitution at position 62 or analysis in the 64-to-70 region (37) and our point mutations in 63 of H-Ras results in proteins whose guanine nucleotide the 62-to-69 region of H-Ras, it seems possible that deletion exchange rate cannot be dramatically activated by the analysis of H-Ras is not the most sensitive means of assess- CDC25-related SCD25GEF in vitro (28). Amino acid residues ing regions of H-Ras critical for functional interaction with in the RAS2 protein corresponding to positions 73 and 74 of CDC25GEF. H-Ras have been implicated in interaction with CDC25GEF How might residues 62, 63, 67, and 69 affect interaction (45). The first of these studies relied solely on in vitro with CDC25GEF? First, CDC25GEF could interact directly biochemistry and did not address whether substitutions in with these residues on H-Ras, and thus certain changes in the region of positions 62 and 63 affected the ability of the the nature of the side groups at these positions could mutant protein to adopt a biologically active conformation in dramatically impair direct protein-protein interactions. Sec- vivo or in vitro. The results presented here indicate that ond, substitution at these residues could alter the structure indeed the mutations described here at positions 62, 63, 67, of a distal CDC25GEF- on the H-Ras protein and and 69 result in proteins which can adopt an active confor- thus affect protein-protein interactions. Although we cannot mation in vivo. We observed one dramatic difference be- absolutely rule out either of the possibilities, we currently tween the E62K and E63K mutations we identified and the favor the first model. E62H and E63H mutations reported by Mistou et al. (28): Several observations are consistent with the model that whereas the E62K and E63K mutations have a profoundly residues 62, 63, 67, and 69 interact directly with CDC25. VOL. 14, 1994 RAS ACTIVATION BY CDC25GEF 1111 First, the region of 62 to 74 of the Ras family, although not bound GDP and favor the binding of GTP. Our proposed directly involved in nucleotide binding, is highly conserved model is supported by the observation that CDC25GE is a from yeasts to mammals. Second, GEFs of the CDC25 potent stimulator of guanine nucleotide exchange of H-ras- family can distinguish the Ras-GDP complex from the Ras- GDP to H-ras-GTP but not of the reverse reaction (20, 22). GTP complex (20, 22). Evidence for this comes from the Further, RAS in a nucleotide-free state binds tightly to the observations that CDC25GEF and SoSlGEF can stimulate yeast CDC25GEF catalytic domain (20). We have shown here guanine nucleotide exchange on Ras proteins bound to GDP that this tight association can be significantly disrupted with but do so only modestly when Ras-GTP is used as the 25 nM GTP but requires a 10-fold higher concentration of substrate (20, 22). Also, with the two-hybrid system no GDP to effect similar disruption of the complex. Also, we detectable interaction with CDC25 was observed for H- report here that H-Ras complexed with CDC25GEF has a Rasval12 (29). Thus, structural features of the ras-GDP significantly higher affinity for GTP than for GDP. Impor- complex distinct from those of the ras-GTP complex may be tantly, the intracellular concentration of GDP is approxi- responsible for the recognition of CDC25 for Ras proteins mately 100 nM, whereas that of GTP is about 1,000 nM (31). bound to GDP but not bound to GTP. Interestingly, the Consequently, assuming that GTP pools are not seques- region of H-Ras (residues 62 to 69) that we propose to be tered, the complex of CDC25GEF and RAS in vivo finds itself critical for CDC25GEF interactions has dramatically different in an environment in which the GTP concentration greatly structures when bound to GDP and when bound to GTP (14, exceeds that required for saturation of the nucleotide-free 19, 20, 30). Thus, these distinct structural differences may RAS-CDC25 reaction intermediate whereas that of GDP is underlie the preferential recognition of CDC25 for the GDP- well below saturating conditions. The results presented here bound form of Ras relative to the GTP-bound form (20, 22). suggest that the mechanism by which CDC25GEF catalyzed Third, previous work has indicated that protein-protein exchange is more involved than simply catalyzing the release interactions involving residues 62 to 73 of H-Ras can pro- of bound nucleotide and passively allowing rebinding of foundly affect the affinities for bound guanine nucleotides. GDP or GTP. Rather, after CDC25 EF stimulates release, it Antibody Y13-259 interacts with residues 63, 65, 66, 67, 70, actively participates in the preferential binding of GTP to and 73 of H-Ras (40). Binding of Y13-259 to H-Ras proteins Ras. preloaded with 3H-labeled guanine nucleotide dramatically decreases the dissociation rate of the bound guanine nucle- ACKNOWLEDGMENTS otide (15). Thus, these residues, which are not directly in interaction with the bound guanine nucleotide, We are grateful to Eric Tong for technical assistance and Chan- involved ning Der for useful discussion. We are grateful to Arieh Warshell, can dramatically affect affinities for GDP and GTP. Fourth, Robert Maxson, Minnie McMillan, and Anne Erwin for helpful the Ras-related protein, Rapla, shares considerable se- discussions and critical reading of the manuscript. We are grateful to quence homology with the Ras family of proteins; however, Arianne Helenkamp, Esther Olivo, and Sarah Olivo for preparation it is not a substrate for the CDC25-like GEF(s) (39). With of the manuscript. respect to H-Ras, K-Ras, and N-Ras, Rapla has amino acid This work was supported by NCI grant CA50261 (to D.B.), grant differences at positions 63, 70, and 74. Divergence of the Ras 2RT0347 from the University of California Tobacco-Related Disease and Rap proteins in this region may account for the lack of Research Program (to D.B.), and grant FY93 170 from the Robert E. stimulation of guanine nucleotide exchange by CDC25 on the and May R. Wright Foundation (to R.M. and D.B.). Rapla protein. Fifth, data presented here clearly demon- strate that the ability of the Ras-specific antibody Y13-259 REFERENCES (but not the anti-Ras antibody Y13-238) to bind to H-Ras protein is blocked by the catalytic domain of the mouse sosl 1. Ballester, R., T. Michaeli, K. Ferguson, H.-P. Xu, F. McCor- or yeast CDC25 gene products. The masking of the epitope mick, and M. Wigler. 1989. Genetic analysis of mammalian GAP expressed in yeast. Cell 59:681-686. for Y13-259 (residues 63 to 73) by yeast and mouse 2. Barbacid, M. 1987. ras genes. Annu. Rev. 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