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Identification of Residues of the H-Ras Protein Critical for Functional Interaction with Guanine Nucleotide Exchange Factors RAYMOND D

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Identification of Residues of the H-Ras Protein Critical for Functional Interaction with Guanine Nucleotide Exchange Factors RAYMOND D 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 Protein 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 proteins 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 signal transduction 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-transferase (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.
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