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Selective Inhibition of Ras Interaction with Its Particular Effector by Synthetic Peptides Corresponding to the Ras Effector Region

1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês

10.1074/jbc.273.17.10210

ISSN

1083-351X

Autores

Masako Ohnishi, Yuriko Yamawaki‐Kataoka, Ken‐ichi Kariya, Masako Tamada, Chang‐Deng Hu, Tohru Kataoka,

Tópico(s)

Microbial Natural Products and Biosynthesis

Resumo

Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17–44 competitively inhibited Ras association with all the three effectors at the K i values of 1∼10 μm, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function. Ras proteins possess multiple downstream effectors of distinct structures. We and others demonstrated that Ha-Ras carrying certain effector region mutations could interact differentially with its effectors, implying that significant differences exist in their Ras recognition mechanisms. Here, by employing the fluorescence polarization method, we measured the activity of effector region synthetic peptides bearing various amino acid substitutions to inhibit association of Ras with the effectors human Raf-1 and Schizosaccharomyces pombe Byr2. The effect of these peptides on association with another effector Saccharomyces cerevisiae adenylyl cyclase was also examined by measuring inhibition of the Ras-dependent adenylyl cyclase activity. The peptide corresponding to the residues 17–44 competitively inhibited Ras association with all the three effectors at the K i values of 1∼10 μm, and the inhibition was considerably attenuated by the D38A mutation. The peptide with the D38N mutation inhibited association of Ha-Ras with Byr2 but not with the others, whereas that with the P34G mutation inhibited association of Ha-Ras with Raf-1 and Byr2 but not with adenylyl cyclase. Thus, the specificity observed with the whole Ras protein was retained in the effector region peptide. These results suggest that the effector region residues constitute a major determinant for differential recognition of the effector molecules, raising a possibility for selective inhibition of a particular Ras function. The ras genes are widely conserved from yeasts to mammals. Their protein products belong to a family of small guanine nucleotide-binding proteins that operate in key processes of intracellular signal transduction systems regulating cell growth and differentiation. In higher eukaryotes including vertebrates, Ras proteins bind directly and activate Raf (Raf-1, A-Raf, and B-Raf) proteins, which results in activation of the mitogen-activated protein kinase phosphorylation cascade (for reviews, see Refs. 1Avruch J. Zhang X.-F. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (543) Google Scholar and 2Daum G. Eisenmann-Tappe I. Fries H.-W. Troppmair J. Rapp U.R. Trends Biochem. Sci. 1994; 19: 474-480Abstract Full Text PDF PubMed Scopus (490) Google Scholar). In addition, other mammalian proteins have been shown to associate directly with Ras in a GTP-dependent manner. These include Ral-guanine nucleotide dissociation stimulator (3Kikuchi A. Demo S.D. Ye Z.-H. Chen Y.-W. Williams L. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (245) Google Scholar, 4Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (255) Google Scholar, 5Spaargarten M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (250) Google Scholar), phosphatidylinositol 3-kinase (6Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1733) Google Scholar), the ζ isoform of protein kinase C (7Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar), AF-6 (8Kuriyama M. Harada N. Kuroda S. Yamamoto T. Nakafuku M. Iwamatsu A. Yamamoto D. Prasad R. Croce C. Canaani E. Kaibuchi K. J. Biol. Chem. 1996; 271: 607-610Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), and Rin1 (9Han L. Colicelli J. Mol. Cell. Biol. 1995; 15: 1518-1523Google Scholar). However, the significance of their interaction with Ras is presently unclear. In the fission yeast Schizosaccharomyces pombe, its single Ras homologue, Ras1, is involved in signal transduction from mating pheromone receptors, and protein kinase Byr2 is its direct downstream effector (10Wang Y. Xu H.-P. Riggs M. Wigler M. Mol. Cell. Biol. 1991; 11: 3554-3563Crossref PubMed Scopus (150) Google Scholar, 11Masuda T. Kariya K. Shinkai M. Okada T. Kataoka T. J. Biol. Chem. 1995; 270: 1979-1982Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). On the other hand, in the budding yeast Saccharomyces cerevisiae, adenylyl cyclase is an immediate downstream effector of a pair of Ras proteins, Ras1 and Ras2 (for a review, see Ref. 12Gibbs J.B. Marshall M. Microbiol. Rev. 1989; 53: 171-185Crossref PubMed Google Scholar). Mutational studies of the effector molecules identified discrete Ras binding regions. The Ras binding site of mammalian Raf-1 was mapped to an 81-amino acid segment in its N-terminal regulatory domain (13Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1666) Google Scholar, 14Chuang E. Barnard D. Hettich J. Zhang X.-F. Avruch J. Marshall M.S. Mol. Cell. Biol. 1994; 14: 5318-5325Crossref PubMed Scopus (158) Google Scholar, 15Pumiglia K. Chow Y.-H. Fabian J. Morrison D. Decker S. Jove R. Mol. Cell. Biol. 1995; 15: 398-406Crossref PubMed Scopus (42) Google Scholar). Recently another Ras binding site was identified in Raf-1, corresponding to the cysteine-rich region (16Brtva T.R. Drugan J.K. Ghosh S. Terrell R.G. Campbell-Burk S. Bell R.M. Der C.J. J. Biol. Chem. 1995; 270: 9809-9812Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 17Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In yeast adenylyl cyclase, the leucine-rich-repeat domain, which is composed of tandemly repeated 23-amino acid leucine-rich motifs (18Kataoka T. Broek D. Wigler M. Cell. 1985; 43: 493-505Abstract Full Text PDF PubMed Scopus (266) Google Scholar), was shown to bind directly to Ras (19Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Abstract Full Text PDF PubMed Google Scholar). Byr2 contains a Ras binding site within its N-terminal 206 amino acid residues (11Masuda T. Kariya K. Shinkai M. Okada T. Kataoka T. J. Biol. Chem. 1995; 270: 1979-1982Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Also, discrete Ras binding domains were identified and mapped in other Ras effectors (3Kikuchi A. Demo S.D. Ye Z.-H. Chen Y.-W. Williams L. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (245) Google Scholar, 4Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (255) Google Scholar, 5Spaargarten M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (250) Google Scholar, 6Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1733) Google Scholar, 7Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar, 8Kuriyama M. Harada N. Kuroda S. Yamamoto T. Nakafuku M. Iwamatsu A. Yamamoto D. Prasad R. Croce C. Canaani E. Kaibuchi K. J. Biol. Chem. 1996; 271: 607-610Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 9Han L. Colicelli J. Mol. Cell. Biol. 1995; 15: 1518-1523Google Scholar). Comparison of the primary structures of these Ras binding domains revealed very little conservation among them except that those of Ral-guanine nucleotide dissociation stimulator, AF-6, and Rin1 share a weakly homologous region called RA domain (20Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (179) Google Scholar). However, recent studies on the Ras binding regions of Raf-1 and Ral-guanine nucleotide dissociation stimulator revealed that they exhibit similar three-dimensional structures (21Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (563) Google Scholar, 22Huang L. Weng X. Hofer F. 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This region almost matches with “switch I,” one of the two regions that take significantly different conformations between the GTP- and GDP-bound forms of Ras (32Milburn M.V. Tong L. deVos A.M. Brunger A. Yamaizumi Z. Nishimura S. Kim S.-H. Science. 1990; 247: 939-945Crossref PubMed Scopus (855) Google Scholar, 33Pai E.F. Krengel U. Petsko G.A. Goody R.S. Kabsch W. Wittinghofer A. EMBO J. 1990; 9: 2351-2359Crossref PubMed Scopus (969) Google Scholar). The x-ray crystallographic study on the complex between a Ras homologue Rap1A and Raf-1 Ras binding region has provided evidence for their association at the atomic level (21Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (563) Google Scholar). In addition, the residues 26–31 and 42–53 flanking the effector region were shown to be critical for effector activation even though they do not change their conformations upon GDP/GTP exchange (30Shirouzu M. Koide H. Fujita-Yoshigaki J. Oshio H. Toyama Y. Yamasaki K. Fuhrman S.A. Villafranca E. Kajiro Y. Yokoyama S. Oncogene. 1994; 9: 2153-2157PubMed Google Scholar, 31Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (194) Google Scholar, 34Fujita-Yoshigaki J. Shirouzu M. Ito Y. Hattori S. Furuyama S. Nishimura S. Yokoyama S. J. Biol. Chem. 1995; 270: 4661-4667Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). These residues have been proposed to constitute the “activator region” (31Marshall M.S. Trends Biochem. Sci. 1993; 18: 250-254Abstract Full Text PDF PubMed Scopus (194) Google Scholar) or “constitutive effector region” (34Fujita-Yoshigaki J. Shirouzu M. Ito Y. Hattori S. Furuyama S. Nishimura S. Yokoyama S. J. Biol. Chem. 1995; 270: 4661-4667Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), and this region was recently implicated in interaction with the cysteine-rich region of Raf-1 (17Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Recently, we and others found that certain Ras mutants carrying substitutions in the effector region could discriminate the effector molecules (35White M.A. Nicolette C. Minden A. Polverino A. Van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (628) Google Scholar, 36Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama Y. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 37Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar, 38Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar, 39Shinkai M. Masuda T. Kariya K. Tamada M. Shirouzu M. Yokoyama S. Kataoka T. Biochem. Biophys. Res. Commun. 1996; 223: 729-734Crossref PubMed Scopus (9) Google Scholar). Specifically, more than 50 Ha-Ras mutants were examined by us for interaction with Raf-1, Byr2, and yeast adenylyl cyclase, the three effectors whose functions are firmly established by both genetic and biochemical evidences (36Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama Y. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We found that the D38N mutant lost the ability to associate with both Raf-1 and adenylyl cyclase while retaining the activity to associate with and activate Byr2. Also, the P34G mutation selectively abolished the ability of Ha-Ras to bind and activate adenylyl cyclase without affecting the interaction with Raf-1 and Byr2, suggesting that significant differences exist in the recognition mechanisms by which distinct effector molecules associate with Ras (36Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama Y. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). These observations opened up a possibility for selective inhibition of the function of a particular effector by exploiting the differences in the Ras recognition mechanisms. Here, we demonstrate that short synthetic peptides corresponding to the effector region can be employed for this purpose by showing that they retain the ability to differentially recognize the effectors. Synthetic peptides used in this experiment are shown in Table I. They were synthesized with a solid-phase method and purified by C18 reversed-phase high performance liquid chromatography (purchased from Peptide Institute Inc., Osaka, Japan). The peptides were dissolved in buffer A (20 mm Tris/HCl (pH 7.4), 50 mm NaCl, 5 mm MgCl2, 1 mm EDTA) or in dimethylformamide and centrifuged at 100,000 × g for 30 min to remove insoluble materials. Their concentrations were determined spectroscopically by measurement of absorbance at 293 nm at pH 11.6. An MBP 1The abbreviations used are: MBP, maltose-binding protein; GST, glutathione S-transferase; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GDPβS, guanosine 5′-O-(2-thiotriphosphate); mP, millipolarization; Mes, 2-(N-morpholino)ethanesulfonic acid; SII, peptide encompassing the switch II region of Ha-Ras; AD, peptide encompassing a part of the activator region of Ha-Ras. fusion protein of the Raf-1 N-terminal 206 residues, MBP-Raf-1-(1–206), and a GST fusion protein of the Byr2 N-terminal 236 residues, GST-Byr2-(1–236), were described before (11Masuda T. Kariya K. Shinkai M. Okada T. Kataoka T. J. Biol. Chem. 1995; 270: 1979-1982Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 17Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 36Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama Y. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Yeast strain FS3–1 (MATα his3 leu2 trp1 ura3 ade8 cyr1–2 ras2::URA3) carrying plasmids pAD4-GST-CYR1-(606–2026) and YEP-HIS3-ADC1-CAP, which overexpressed a GST fusion protein of adenylyl cyclase and the cyclase-associated protein CAP, respectively, under the control of the ADC1 promoter, was described before (40Shima F. Yamawaki-Kataoka Y. Yanagihara Y. Tamada M. Okada Y. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (48) Google Scholar). The posttranslationally fully modified and unmodified forms of human Ha-Ras were extracted from Sf9 cells infected with the baculovirus carrying Ha-ras and purified as described previously (41Okada T. Masuda T. Shinkai M. Kariya K. Kataoka T. J. Biol. Chem. 1996; 271: 4671-4678Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar).Table IAmino acid sequences of Ha-Ras peptides used in this studyNameResiduesSequenceWild type17–44SALTI QLIQN HFVDE YDPTI EDSYR KQVP34G17–44—– —– —– –G– —– —D38N17–44—– —– —– —– -N— —D38A17–44—– —– —– —– -A— —SII58–76DTAGQ EEYSA MRDQY MRTGEAD39–51SYRKQ VVIDG ETC Open table in a new tab The purified unmodified form of Ha-Ras was labeled with fluorescein (*F) at its N-terminal α-amino group by reaction with fluorescein succinimidylester (Pan Vera Co., Madison, Wisconsin) at pH 7.0. The fluorescein-labeled Ha-Ras (*F-Ha-Ras) was separated from the unincorporated dye by gel filtration chromatography on Sephadex G-25. One microliter of *F-Ha-Ras (about 105 fluorescence units or 8 fmol/reaction) loaded with GTPγS or GDPβS was mixed with 1 ml of buffer A containing various concentrations of the purified MBP-Raf-1-(1–206) or GST-Byr2-(1–236) in a siliconized glass tube. The reaction mixture was incubated for 15 min at 30 °C in the darkness followed by measurement of the fluorescence polarization value of each tube with BEACON (Pan Vera Co.) at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. For assay of inhibition by the synthetic peptides, similar measurements were performed with 100 nm MBP-Raf-1-(1–206) or GST-Byr2-(1–236) in the presence of varying concentrations of the synthetic peptides. A fluorescence polarization value is given by a difference between the vertical and horizontal fluorescence intensities divided by a sum of the vertical and horizontal fluorescence intensities and expressed as mP units (42Perrin F. J. Phys. Radium. 1926; 7: 390-401Crossref Google Scholar, 43Checovich W.J. Bolger R.E. Burke T. Nature. 1995; 375: 254-256Crossref PubMed Scopus (165) Google Scholar). It reflects the molecular size of a fluorescently labeled molecule (42Perrin F. J. Phys. Radium. 1926; 7: 390-401Crossref Google Scholar, 44Weber G. Adv. Protein Chem. 1953; 8: 415-459Crossref PubMed Scopus (441) Google Scholar, 45Maeda H. Anal. Biochem. 1979; 92: 222-227Crossref PubMed Scopus (57) Google Scholar). There occurs a large increase in the molecular size of *F-Ha-Ras upon complex formation with the effector proteins, causing an increase in the fluorescence polarization value. Thus, the change in fluorescence polarization of *F-Ha-Ras is proportional to the amount of the complex formed with the effector molecule. Adenylyl cyclase was solubilized from the crude membrane fraction of the yeast FS3–1 carrying pAD4-GST-CYR1-(606–2026) and YEP-HIS3-ADC1-CAP with buffer C (50 mm Mes (pH 6.2), 0.1 mm MgCl2, 0.1 mm EGTA, 1 mm β-mercaptoethanol) containing 1% LubrolPX, 0.6 m NaCl, and 1 mmphenylmethylsulfonyl fluoride. After centrifugation at 100,000 ×g for 1 h at 4 °C, the resulting supernatant was used for the measurement of adenylyl cyclase activity in the presence of varying concentrations of the GTPγS-bound form of posttranslationally fully modified Ha-Ras as described before (19Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Abstract Full Text PDF PubMed Google Scholar, 40Shima F. Yamawaki-Kataoka Y. Yanagihara Y. Tamada M. Okada Y. Kariya K. Kataoka T. Mol. Cell. Biol. 1997; 17: 1057-1064Crossref PubMed Scopus (48) Google Scholar). For the measurement of inhibition of adenylyl cyclase activities by the synthetic peptides, varying amounts of the peptides were added to the reaction mixtures before commencing the reaction. We employed the fluorescence polarization method to quantitatively analyze the association of Ha-Ras with its various effectors. The change in fluorescence polarization value of *F-Ha-Ras is proportional to the amount of a complex formed with its effector molecule. In this experiment, *F-Ha-Ras was loaded with GTPγS or GDPβS and examined for association with various concentrations of MBP-Raf-1-(1–206) (Fig. 1 A) or GST-Byr2-(1–236) (Fig. 1 B). *F-Ha-Ras alone exhibited an mP value of approximately 100, and the complex formation of *F-Ha-Ras with the effector polypeptides was detectable at the effector concentration as low as 10 nm. The binding was almost saturated at 100 nm for MBP-Raf-1-(1–206) or at 500 nm for GST-Byr2-(1–236) with the increase of the mP value by approximately 30 units. The extent of increase was consistent with that predicted from the increase in molecular size upon the complex formation with the effector polypeptides based on the observation that the reciprocals of the mP values correlate linearly with the reciprocal values of the molecular weights of the fluorescently labeled proteins (44Weber G. Adv. Protein Chem. 1953; 8: 415-459Crossref PubMed Scopus (441) Google Scholar, 45Maeda H. Anal. Biochem. 1979; 92: 222-227Crossref PubMed Scopus (57) Google Scholar). This suggested that the binding reactions occurred stoichiometrically. The maximal bindings were reached within 10 min of incubation, and the values remained stable for the following 30 min (data not shown). The binding reactions were dependent on the GTP-bound configuration of *F-Ha-Ras, and almost no binding was observed for MBP or GST only (Fig. 1, A and B). Since the *F-Ha-Ras concentration used was very low at 8 pm, essentially all of MBP-Raf-1-(1–206) or GST-Byr2-(1–236) added to the reactions could be regarded as in a free form. When reciprocals of the fluorescence polarization increase values were plotted against reciprocals of the effector concentrations, a straight line could be drawn with an intersect with the horizontal axis giving K d values of 35 nm for MBP-Raf-1-(1–206) and 90 nm for GST-Byr2-(1–236) (Fig. 1 C). These values are comparable although somewhat higher than those estimated from the inhibition profiles of Ras-dependent adenylyl cyclase activity by the same effector fragments (11Masuda T. Kariya K. Shinkai M. Okada T. Kataoka T. J. Biol. Chem. 1995; 270: 1979-1982Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Minato T. Wang J. Akasaka K. Okada T. Suzuki N. Kataoka T. J. Biol. Chem. 1994; 269: 20845-20851Abstract Full Text PDF PubMed Google Scholar). We next examined the ability of synthetic peptides encompassing the Ha-Ras effector region with various single amino acid substitutions (Table I) to interfere with the Ha-Ras-Raf-1 and Ha-Ras-Byr2 associations. The peptide corresponding to the residues 17–44 was chosen because this peptide had been used successfully in inhibition of the Ras binding to Raf-1 or to protein kinase Cζ (7Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar, 46Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar). In addition, similar experiments were carried out with synthetic peptides SII and AD, corresponding to the switch II region (residues 58–76) of Ha-Ras and to a part of the activator region (residues 39–51), respectively (Table I). Fig. 2 shows inhibition profiles of the association of *F-Ha-Ras with MBP-Raf-1-(1–206) (Fig. 2 A) or with GST-Byr2-(1–236) (Fig. 2 B) by increasing concentrations of the various peptides. Wild-type effector region peptide effectively inhibited the Ras association with both of the effectors with 70% inhibition for Raf-1 and 55% inhibition for Byr2 at the concentration of 10 μm, whereas that carrying the D38A mutation was much less effective. In addition, the specificities of inhibition by the various peptides were clearly different between the two effectors. Although P34G substitution did not notably affect the inhibitory activity of the peptide against both Raf-1 and Byr2, D38N substitution selectively impaired the inhibitory activity against complex formation of *F-Ha-Ras with Raf-1-(1–206) but not with Byr2-(1–236). Both SII and AD peptides had no inhibitory effect up to the concentration of 10 μm. Kinetic analyses of the inhibition patterns were carried out as follows to determine K d values of each peptide for the two effectors. The fluorescence polarization value (Y) is proportional to the amount of *F-Ha-Ras bound to the effector and hence formulated as Y = Y max ·X/(K Ras + X), where Y max, X, and K Ras represent the maximal polarization value, the effector concentration, and the K d value of *F-Ha-Ras for the effector, respectively. Assuming that the association with *F-Ha-Ras is competitively inhibited by a peptide whose concentration (P) is in a vast excess over X, the concentration of the effector sequestered by the peptide is X · P/(K p + P), where K p represents the K d value of the peptide for the effector. Thus, the free effector concentration available for association with *F-Ha-Ras is given by X · (1 −P/(K p + P)). The fluorescence polarization value (Y p) in the presence of the peptide (P) can be formulated as Y p = Y max · X· (1 − P/(K p +P))/(K Ras + X · (1 − P/(K p + P))). From the two equations is derived Y/Y p = (K Ras/K p · (K Ras + X)) · P + 1. When Y/Y p was plotted against P for inhibition by the various peptides of association with Raf-1 (Fig. 2 C) or with Byr2 (Fig. 2 D), a straight line can be drawn for either case that converged on the longitudinal axis at Y/Y p = 1. The K p value can be calculated as −K Ras ·P 0/(K Ras + X), where P 0 represents the P value at the intersect on the horizontal axis (Fig. 2, C and D). Thus, the K p values for MBP-Raf-1-(1–206) of wild-type, P34G, D38N, and D38A peptides were calculated as 2.1, 4.4, 28, and 28 μm, respectively, given that K Ras is 35 nm and X is 100 nm. Similarly, the K p values of the same set of peptides for GST-Byr2-(1–236) were calculated as 3.8, 2.0, 4.7, and 39 μm, respectively. The effects of the various synthetic peptides on association between Ha-Ras and yeast adenylyl cyclase were examined by measuring inhibition of Ha-Ras-dependent adenylyl cyclase activity (Fig. 3). Wild-type peptide inhibited adenylyl cyclase activity obtained by the 8 nm post-translationally modified form of Ha-Ras by a half at approximately 21 μm(Fig. 3 A). A notable difference from Raf-1 and Byr2 was observed in the effect of P34G substitution, which was found totally ineffective in inhibition of adenylyl cyclase activity. Assuming that the inhibitory associations of the peptides are competitive with that of Ha-Ras, the inhibition constants (K i) for the peptides can be derived from a conventional equation V= V max ·X/(Km · (1 +I/K i) + X), where V, V max, X,Km, and I represent the adenylyl cyclase activity, the maximal adenylyl cyclase activity, the concentration of Ha-Ras, the K d value for Ha-Ras, and the inhibitor peptide concentration, respectively. Rearranging the equation according to Lineweaver and Burk gives 1/V = 1/V max + K m · (1 +I/K i)/V max ·X. Adenylyl cyclase activities dependent on various concentrations of Ha-Ras were subjected to inhibition by varying concentrations of the peptides, and a reciprocal of the activity was plotted against a reciprocal of the Ha-Ras concentration. This gave a series of straight lines that converged on the longitudinal axis for each concentration of the peptide, wild type (Fig. 3 B), D38N (Fig. 3 C), and D38A (Fig. 3 D), as predicted from the above equation. The intersect of each line on the horizontal axis corresponds to −1/K m · (1 + I/K i). Thus, K i for each peptide could be determined by plotting reciprocals of the intersect values against I (Fig. 3 E). The K i values could be estimated from intersects of the straight lines on the horizontal axis and was determined to be 10, 56, and 68 μm for wild-type, D38N, and D38A peptides, respectively. Synthetic peptides encompassing the Ras effector region, in particular the one corresponding to the residues 17–44, were used successfully to competitively inhibit association of Ras with its effector proteins including Raf-1 (46Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar) and the ζ isoform of protein kinase C (7Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar). Contact epitope scanning using a series of smaller peptides suggested that many residues across the residues 17–51 may be involved in direct interaction with Raf-1 N-terminal fragment (47Barnard D. Diaz B. Hettich L. Chuang E. Zhang X.-F. Avruch J. Marshall M. Oncogene. 1995; 10: 1283-1290PubMed Google Scholar). 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In contrast, P34G substitution almost abolished the interaction with adenylyl cyclase but did not appreciably affect that with Raf-1 or Byr2. These specificities, summarized in Table II, are in good coincidence with those observed with the whole Ha-Ras protein (36Akasaka K. Tamada M. Wang F. Kariya K. Shima F. Kikuchi A. Yamamoto M. Shirouzu M. Yokoyama Y. Kataoka T. J. Biol. Chem. 1996; 271: 5353-5360Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), indicating that residues 17–44 are sufficient to assume a specific conformation that interacts selectively with a particular set of the effectors depending on a single amino acid substitution. The results strongly suggest that the effector region residues by themselves constitute a major determinant for differential recognition of the effector molecules, although we cannot exclude the possibility that residues outside of the effector region may contribute to this recognition process. Since other Ras effector molecules such as Ral-guanine nucleotide dissociation stimulator and phosphatidylinositol 3-kinase were also known to possess distinct requirements for the effector region residues from Raf-1, Byr2, or adenylyl cyclase and from one another (37Khosravi-Far R. White M.A. Westwick J.K. Solski P.A. Chrzanowska-Wodnicka M. Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar, 38Rodriguez-Viciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (962) Google Scholar), it may be possible to find a synthetic peptide carrying certain amino acid substitutions that specifically recognize any member of the multiple Ras effector molecules.Table IISelective interaction of Ha-Ras mutant proteins and effector region synthetic peptides with Ras effectorsEffectorsWild typeP34GD38ND38AProtein interactionPeptide K iProtein interactionPeptide K iProtein interactionPeptide K iProtein interactionPeptide K iμmμmμmμmRaf-1+2.12-aThe K i value of this wild-type peptide with Raf-1 agrees well with the reported result of about 5 μm (Ref. 46).+4.4−28−2-bFrom Ref. 49.28Byr2+3.8+2.0+4.7ND2-cNot determined.39Adenylyl cyclase+10−>500−56−2-dFrom Ref. 24.682-a The K i value of this wild-type peptide with Raf-1 agrees well with the reported result of about 5 μm (Ref. 46Warne P.H. Viciana P.R. Downward J. Nature. 1993; 364: 352-355Crossref PubMed Scopus (584) Google Scholar).2-b From Ref. 49Winkler D.G. Johnson J.C. Cooper J.A. Vojtek A.B. J. Biol. Chem. 1997; 272: 24402-24409Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar.2-c Not determined.2-d From Ref. 24Sigal I.S. Gibbs J.B. D'Alonzo J.S. Scolnick E.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4725-4729Crossref PubMed Scopus (199) Google Scholar. Open table in a new tab Our results have also shown that it is possible to achieve selective inhibition of the association of Ras with a particular effector molecule without significantly affecting those with other effectors by employing the antagonist peptide with a relatively small molecular size around 3 kDa. This may open up a possibility to design a compound that mimics a specific conformation of the effector region peptide bearing the particular substitution, which is in theory deducible from the x-ray crystallographic or nuclear magnetic resonance spectroscopic analysis. From a viewpoint of effectiveness in cancer drug therapy, it is possible that the effector-specific inhibition, which could potentially be achieved by administration of such a compound, is superior to the total inhibition of Ras functions by, for example, a compound that directly inactivates Ras. In addition, the effector region peptides carrying a series of substitutions might be found useful as effector-specific inhibitors for analysis of individual cellular functions of the distinct effector molecules. We thank X.-H. Deng for skillful technical assistance and T. Okada, A. Seki, and A. Kawabe for help in preparation of this manuscript.

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