Abstract
Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adams A. E., Johnson D. I., Longnecker R. M., Sloat B. F., Pringle J. R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990 Jul;111(1):131–142. doi: 10.1083/jcb.111.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ahmed S., Lee J., Wen L. P., Zhao Z., Ho J., Best A., Kozma R., Lim L. Breakpoint cluster region gene product-related domain of n-chimaerin. Discrimination between Rac-binding and GTPase-activating residues by mutational analysis. J Biol Chem. 1994 Jul 1;269(26):17642–17648. [PubMed] [Google Scholar]
- Ammerer G. Sex, stress and integrity: the importance of MAP kinases in yeast. Curr Opin Genet Dev. 1994 Feb;4(1):90–95. doi: 10.1016/0959-437x(94)90096-5. [DOI] [PubMed] [Google Scholar]
- Avraham H., Weinberg R. A. Characterization and expression of the human rhoH12 gene product. Mol Cell Biol. 1989 May;9(5):2058–2066. doi: 10.1128/mcb.9.5.2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender A., Pringle J. R. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Mar;11(3):1295–1305. doi: 10.1128/mcb.11.3.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blumer K. J., Johnson G. L. Diversity in function and regulation of MAP kinase pathways. Trends Biochem Sci. 1994 Jun;19(6):236–240. doi: 10.1016/0968-0004(94)90147-3. [DOI] [PubMed] [Google Scholar]
- Chang E. C., Barr M., Wang Y., Jung V., Xu H. P., Wigler M. H. Cooperative interaction of S. pombe proteins required for mating and morphogenesis. Cell. 1994 Oct 7;79(1):131–141. doi: 10.1016/0092-8674(94)90406-5. [DOI] [PubMed] [Google Scholar]
- Chen W., Lim H. H., Lim L. The CDC42 homologue from Caenorhabditis elegans. Complementation of yeast mutation. J Biol Chem. 1993 Jun 25;268(18):13280–13285. [PubMed] [Google Scholar]
- Chenevert J., Corrado K., Bender A., Pringle J., Herskowitz I. A yeast gene (BEM1) necessary for cell polarization whose product contains two SH3 domains. Nature. 1992 Mar 5;356(6364):77–79. doi: 10.1038/356077a0. [DOI] [PubMed] [Google Scholar]
- Choi K. Y., Satterberg B., Lyons D. M., Elion E. A. Ste5 tethers multiple protein kinases in the MAP kinase cascade required for mating in S. cerevisiae. Cell. 1994 Aug 12;78(3):499–512. doi: 10.1016/0092-8674(94)90427-8. [DOI] [PubMed] [Google Scholar]
- Christianson T. W., Sikorski R. S., Dante M., Shero J. H., Hieter P. Multifunctional yeast high-copy-number shuttle vectors. Gene. 1992 Jan 2;110(1):119–122. doi: 10.1016/0378-1119(92)90454-w. [DOI] [PubMed] [Google Scholar]
- Crespo P., Xu N., Simonds W. F., Gutkind J. S. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature. 1994 Jun 2;369(6479):418–420. doi: 10.1038/369418a0. [DOI] [PubMed] [Google Scholar]
- Cross F. R., Tinkelenberg A. H. A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle. Cell. 1991 May 31;65(5):875–883. doi: 10.1016/0092-8674(91)90394-e. [DOI] [PubMed] [Google Scholar]
- Cross F., Hartwell L. H., Jackson C., Konopka J. B. Conjugation in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1988;4:429–457. doi: 10.1146/annurev.cb.04.110188.002241. [DOI] [PubMed] [Google Scholar]
- Dolan J. W., Fields S. Overproduction of the yeast STE12 protein leads to constitutive transcriptional induction. Genes Dev. 1990 Apr;4(4):492–502. doi: 10.1101/gad.4.4.492. [DOI] [PubMed] [Google Scholar]
- Errede B., Levin D. E. A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol. 1993 Apr;5(2):254–260. doi: 10.1016/0955-0674(93)90112-4. [DOI] [PubMed] [Google Scholar]
- Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
- Friesen H., Lunz R., Doyle S., Segall J. Mutation of the SPS1-encoded protein kinase of Saccharomyces cerevisiae leads to defects in transcription and morphology during spore formation. Genes Dev. 1994 Sep 15;8(18):2162–2175. doi: 10.1101/gad.8.18.2162. [DOI] [PubMed] [Google Scholar]
- Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
- Hannon G. J., Demetrick D., Beach D. Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 1993 Dec;7(12A):2378–2391. doi: 10.1101/gad.7.12a.2378. [DOI] [PubMed] [Google Scholar]
- Hart M. J., Eva A., Evans T., Aaronson S. A., Cerione R. A. Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product. Nature. 1991 Nov 28;354(6351):311–314. doi: 10.1038/354311a0. [DOI] [PubMed] [Google Scholar]
- Hart M. J., Eva A., Zangrilli D., Aaronson S. A., Evans T., Cerione R. A., Zheng Y. Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product. J Biol Chem. 1994 Jan 7;269(1):62–65. [PubMed] [Google Scholar]
- Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hasson M. S., Blinder D., Thorner J., Jenness D. D. Mutational activation of the STE5 gene product bypasses the requirement for G protein beta and gamma subunits in the yeast pheromone response pathway. Mol Cell Biol. 1994 Feb;14(2):1054–1065. doi: 10.1128/mcb.14.2.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirsch J. P., Cross F. R. Pheromone response in yeast. Bioessays. 1992 Jun;14(6):367–373. doi: 10.1002/bies.950140604. [DOI] [PubMed] [Google Scholar]
- Horii Y., Beeler J. F., Sakaguchi K., Tachibana M., Miki T. A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways. EMBO J. 1994 Oct 17;13(20):4776–4786. doi: 10.1002/j.1460-2075.1994.tb06803.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson D. I., Pringle J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol. 1990 Jul;111(1):143–152. doi: 10.1083/jcb.111.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kishi K., Sasaki T., Kuroda S., Itoh T., Takai Y. Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI). J Cell Biol. 1993 Mar;120(5):1187–1195. doi: 10.1083/jcb.120.5.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kumagai N., Morii N., Fujisawa K., Nemoto Y., Narumiya S. ADP-ribosylation of rho p21 inhibits lysophosphatidic acid-induced protein tyrosine phosphorylation and phosphatidylinositol 3-kinase activation in cultured Swiss 3T3 cells. J Biol Chem. 1993 Nov 25;268(33):24535–24538. [PubMed] [Google Scholar]
- Leberer E., Dignard D., Harcus D., Hougan L., Whiteway M., Thomas D. Y. Cloning of Saccharomyces cerevisiae STE5 as a suppressor of a Ste20 protein kinase mutant: structural and functional similarity of Ste5 to Far1. Mol Gen Genet. 1993 Nov;241(3-4):241–254. doi: 10.1007/BF00284675. [DOI] [PubMed] [Google Scholar]
- Leberer E., Dignard D., Harcus D., Thomas D. Y., Whiteway M. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components. EMBO J. 1992 Dec;11(13):4815–4824. doi: 10.1002/j.1460-2075.1992.tb05587.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leberer E., Dignard D., Hougan L., Thomas D. Y., Whiteway M. Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling. EMBO J. 1992 Dec;11(13):4805–4813. doi: 10.1002/j.1460-2075.1992.tb05586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
- Legrain P., Dokhelar M. C., Transy C. Detection of protein-protein interactions using different vectors in the two-hybrid system. Nucleic Acids Res. 1994 Aug 11;22(15):3241–3242. doi: 10.1093/nar/22.15.3241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu H., Styles C. A., Fink G. R. Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science. 1993 Dec 10;262(5140):1741–1744. doi: 10.1126/science.8259520. [DOI] [PubMed] [Google Scholar]
- Manser E., Leung T., Monfries C., Teo M., Hall C., Lim L. Diversity and versatility of GTPase activating proteins for the p21rho subfamily of ras G proteins detected by a novel overlay assay. J Biol Chem. 1992 Aug 15;267(23):16025–16028. [PubMed] [Google Scholar]
- Manser E., Leung T., Salihuddin H., Tan L., Lim L. A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42. Nature. 1993 May 27;363(6427):364–367. doi: 10.1038/363364a0. [DOI] [PubMed] [Google Scholar]
- Manser E., Leung T., Salihuddin H., Zhao Z. S., Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 1994 Jan 6;367(6458):40–46. doi: 10.1038/367040a0. [DOI] [PubMed] [Google Scholar]
- Marsh L., Neiman A. M., Herskowitz I. Signal transduction during pheromone response in yeast. Annu Rev Cell Biol. 1991;7:699–728. doi: 10.1146/annurev.cb.07.110191.003411. [DOI] [PubMed] [Google Scholar]
- Marshall C. J. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994 Feb;4(1):82–89. doi: 10.1016/0959-437x(94)90095-7. [DOI] [PubMed] [Google Scholar]
- Miki T., Smith C. L., Long J. E., Eva A., Fleming T. P. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature. 1993 Apr 1;362(6419):462–465. doi: 10.1038/362462a0. [DOI] [PubMed] [Google Scholar]
- Miller P. J., Johnson D. I. Cdc42p GTPase is involved in controlling polarized cell growth in Schizosaccharomyces pombe. Mol Cell Biol. 1994 Feb;14(2):1075–1083. doi: 10.1128/mcb.14.2.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miyamoto S., Ohya Y., Ohsumi Y., Anraku Y. Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae. Gene. 1987;54(1):125–132. doi: 10.1016/0378-1119(87)90354-4. [DOI] [PubMed] [Google Scholar]
- Miyamoto S., Ohya Y., Sano Y., Sakaguchi S., Iida H., Anraku Y. A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly. Biochem Biophys Res Commun. 1991 Dec 16;181(2):604–610. doi: 10.1016/0006-291x(91)91233-3. [DOI] [PubMed] [Google Scholar]
- Munemitsu S., Innis M. A., Clark R., McCormick F., Ullrich A., Polakis P. Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol Cell Biol. 1990 Nov;10(11):5977–5982. doi: 10.1128/mcb.10.11.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Musacchio A., Gibson T., Rice P., Thompson J., Saraste M. The PH domain: a common piece in the structural patchwork of signalling proteins. Trends Biochem Sci. 1993 Sep;18(9):343–348. doi: 10.1016/0968-0004(93)90071-t. [DOI] [PubMed] [Google Scholar]
- Müller L., Xu G., Wells R., Hollenberg C. P., Piepersberg W. LRG1 is expressed during sporulation in Saccharomyces cerevisiae and contains motifs similar to LIM and rho/racGAP domains. Nucleic Acids Res. 1994 Aug 11;22(15):3151–3154. doi: 10.1093/nar/22.15.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neiman A. M. Conservation and reiteration of a kinase cascade. Trends Genet. 1993 Nov;9(11):390–394. doi: 10.1016/0168-9525(93)90139-9. [DOI] [PubMed] [Google Scholar]
- Neiman A. M., Herskowitz I. Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3398–3402. doi: 10.1073/pnas.91.8.3398. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nielsen O., Davey J., Egel R. The ras1 function of Schizosaccharomyces pombe mediates pheromone-induced transcription. EMBO J. 1992 Apr;11(4):1391–1395. doi: 10.1002/j.1460-2075.1992.tb05184.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nielsen O. Signal transduction during mating and meiosis in S. pombe. Trends Cell Biol. 1993 Feb;3(2):60–65. doi: 10.1016/0962-8924(93)90162-t. [DOI] [PubMed] [Google Scholar]
- Rad M. R., Xu G., Hollenberg C. P. STE50, a novel gene required for activation of conjugation at an early step in mating in Saccharomyces cerevisiae. Mol Gen Genet. 1992 Dec;236(1):145–154. doi: 10.1007/BF00279653. [DOI] [PubMed] [Google Scholar]
- Ramer S. W., Davis R. W. A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):452–456. doi: 10.1073/pnas.90.2.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reid B. J., Hartwell L. H. Regulation of mating in the cell cycle of Saccharomyces cerevisiae. J Cell Biol. 1977 Nov;75(2 Pt 1):355–365. doi: 10.1083/jcb.75.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
- Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
- Shinjo K., Koland J. G., Hart M. J., Narasimhan V., Johnson D. I., Evans T., Cerione R. A. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9853–9857. doi: 10.1073/pnas.87.24.9853. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sloat B. F., Adams A., Pringle J. R. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1981 Jun;89(3):395–405. doi: 10.1083/jcb.89.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
- Surana U., Robitsch H., Price C., Schuster T., Fitch I., Futcher A. B., Nasmyth K. The role of CDC28 and cyclins during mitosis in the budding yeast S. cerevisiae. Cell. 1991 Apr 5;65(1):145–161. doi: 10.1016/0092-8674(91)90416-v. [DOI] [PubMed] [Google Scholar]
- Tkacz J. S., MacKay V. L. Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones. J Cell Biol. 1979 Feb;80(2):326–333. doi: 10.1083/jcb.80.2.326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Touhara K., Inglese J., Pitcher J. A., Shaw G., Lefkowitz R. J. Binding of G protein beta gamma-subunits to pleckstrin homology domains. J Biol Chem. 1994 Apr 8;269(14):10217–10220. [PubMed] [Google Scholar]
- Van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6213–6217. doi: 10.1073/pnas.90.13.6213. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
- Warne P. H., Viciana P. R., Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature. 1993 Jul 22;364(6435):352–355. doi: 10.1038/364352a0. [DOI] [PubMed] [Google Scholar]
- Whiteway M., Hougan L., Dignard D., Thomas D. Y., Bell L., Saari G. C., Grant F. J., O'Hara P., MacKay V. L. The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989 Feb 10;56(3):467–477. doi: 10.1016/0092-8674(89)90249-3. [DOI] [PubMed] [Google Scholar]
- Whiteway M., Hougan L., Thomas D. Y. Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae. Mol Cell Biol. 1990 Jan;10(1):217–222. doi: 10.1128/mcb.10.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xu H. P., White M., Marcus S., Wigler M. Concerted action of RAS and G proteins in the sexual response pathways of Schizosaccharomyces pombe. Mol Cell Biol. 1994 Jan;14(1):50–58. doi: 10.1128/mcb.14.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zheng Y., Cerione R., Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J Biol Chem. 1994 Jan 28;269(4):2369–2372. [PubMed] [Google Scholar]
- Ziman M., Preuss D., Mulholland J., O'Brien J. M., Botstein D., Johnson D. I. Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol Biol Cell. 1993 Dec;4(12):1307–1316. doi: 10.1091/mbc.4.12.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]