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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1994 Apr 12;91(8):3398–3402. doi: 10.1073/pnas.91.8.3398

Reconstitution of a yeast protein kinase cascade in vitro: activation of the yeast MEK homologue STE7 by STE11.

A M Neiman 1, I Herskowitz 1
PMCID: PMC43584  PMID: 8159759

Abstract

The mating-factor response pathway of Saccharomyces cerevisiae employs a set of protein kinase similar to kinases that function in signal transduction pathways of metazoans. We have purified the yeast protein kinases encoded by STE11, STE7, and FUS3 as fusions to glutathione S-transferase (GST) and reconstituted a kinase cascade in which STE11 phosphorylates and activates STE7, which in turn phosphorylates the mitogen-activated protein kinase FUS3. GST-STE11 is active even when purified from cells that have not been treated with alpha-factor. This observation raises the possibility that STE11 activity is governed by an inhibitor which is regulated by pheromone. We also identify a STE11-dependent phosphorylation site in STE7 which is required for activity of STE7. Conservation of this site in the mammalian STE7 homologue MEK and other STE7 relatives suggests that this may be a regulatory phosphorylation site in all MAP kinase kinases.

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Selected References

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  1. Blinder D., Bouvier S., Jenness D. D. Constitutive mutants in the yeast pheromone response: ordered function of the gene products. Cell. 1989 Feb 10;56(3):479–486. doi: 10.1016/0092-8674(89)90250-x. [DOI] [PubMed] [Google Scholar]
  2. Boulton T. G., Yancopoulos G. D., Gregory J. S., Slaughter C., Moomaw C., Hsu J., Cobb M. H. An insulin-stimulated protein kinase similar to yeast kinases involved in cell cycle control. Science. 1990 Jul 6;249(4964):64–67. doi: 10.1126/science.2164259. [DOI] [PubMed] [Google Scholar]
  3. Cairns B. R., Ramer S. W., Kornberg R. D. Order of action of components in the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase and the multiple phosphorylation of the STE7 kinase. Genes Dev. 1992 Jul;6(7):1305–1318. doi: 10.1101/gad.6.7.1305. [DOI] [PubMed] [Google Scholar]
  4. Crews C. M., Alessandrini A., Erikson R. L. Erks: their fifteen minutes has arrived. Cell Growth Differ. 1992 Feb;3(2):135–142. [PubMed] [Google Scholar]
  5. Crews C. M., Alessandrini A., Erikson R. L. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. doi: 10.1126/science.1411546. [DOI] [PubMed] [Google Scholar]
  6. Elion E. A., Satterberg B., Kranz J. E. FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1. Mol Biol Cell. 1993 May;4(5):495–510. doi: 10.1091/mbc.4.5.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Errede B., Gartner A., Zhou Z., Nasmyth K., Ammerer G. MAP kinase-related FUS3 from S. cerevisiae is activated by STE7 in vitro. Nature. 1993 Mar 18;362(6417):261–264. doi: 10.1038/362261a0. [DOI] [PubMed] [Google Scholar]
  8. Gartner A., Nasmyth K., Ammerer G. Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1. Genes Dev. 1992 Jul;6(7):1280–1292. doi: 10.1101/gad.6.7.1280. [DOI] [PubMed] [Google Scholar]
  9. Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
  10. Hicks J. B., Herskowitz I. Interconversion of Yeast Mating Types I. Direct Observations of the Action of the Homothallism (HO) Gene. Genetics. 1976 Jun;83(2):245–258. doi: 10.1093/genetics/83.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Irie K., Takase M., Lee K. S., Levin D. E., Araki H., Matsumoto K., Oshima Y. MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. Mol Cell Biol. 1993 May;13(5):3076–3083. doi: 10.1128/mcb.13.5.3076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  13. Kurjan J. Pheromone response in yeast. Annu Rev Biochem. 1992;61:1097–1129. doi: 10.1146/annurev.bi.61.070192.005313. [DOI] [PubMed] [Google Scholar]
  14. Lange-Carter C. A., Pleiman C. M., Gardner A. M., Blumer K. J., Johnson G. L. A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science. 1993 Apr 16;260(5106):315–319. doi: 10.1126/science.8385802. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Neiman A. M., Stevenson B. J., Xu H. P., Sprague G. F., Jr, Herskowitz I., Wigler M., Marcus S. Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms. Mol Biol Cell. 1993 Jan;4(1):107–120. doi: 10.1091/mbc.4.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Park H. O., Chant J., Herskowitz I. BUD2 encodes a GTPase-activating protein for Bud1/Rsr1 necessary for proper bud-site selection in yeast. Nature. 1993 Sep 16;365(6443):269–274. doi: 10.1038/365269a0. [DOI] [PubMed] [Google Scholar]
  20. Peter M., Gartner A., Horecka J., Ammerer G., Herskowitz I. FAR1 links the signal transduction pathway to the cell cycle machinery in yeast. Cell. 1993 May 21;73(4):747–760. doi: 10.1016/0092-8674(93)90254-n. [DOI] [PubMed] [Google Scholar]
  21. Peter M., Nakagawa J., Dorée M., Labbé J. C., Nigg E. A. Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase. Cell. 1990 Mar 9;60(5):791–801. doi: 10.1016/0092-8674(90)90093-t. [DOI] [PubMed] [Google Scholar]
  22. Rhodes N., Connell L., Errede B. STE11 is a protein kinase required for cell-type-specific transcription and signal transduction in yeast. Genes Dev. 1990 Nov;4(11):1862–1874. doi: 10.1101/gad.4.11.1862. [DOI] [PubMed] [Google Scholar]
  23. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  24. Stevenson B. J., Rhodes N., Errede B., Sprague G. F., Jr Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 1992 Jul;6(7):1293–1304. doi: 10.1101/gad.6.7.1293. [DOI] [PubMed] [Google Scholar]
  25. Teague M. A., Chaleff D. T., Errede B. Nucleotide sequence of the yeast regulatory gene STE7 predicts a protein homologous to protein kinases. Proc Natl Acad Sci U S A. 1986 Oct;83(19):7371–7375. doi: 10.1073/pnas.83.19.7371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Zhou Z., Gartner A., Cade R., Ammerer G., Errede B. Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases. Mol Cell Biol. 1993 Apr;13(4):2069–2080. doi: 10.1128/mcb.13.4.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]

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