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. 1997 Jul;17(7):3508–3519. doi: 10.1128/mcb.17.7.3508

Mkh1, a MEK kinase required for cell wall integrity and proper response to osmotic and temperature stress in Schizosaccharomyces pombe.

A S Sengar 1, N A Markley 1, N J Marini 1, D Young 1
PMCID: PMC232204  PMID: 9199286

Abstract

We have identified a Schizosaccharomyces pombe gene, mkh1, that encodes a MEK kinase (MEKK) homolog. The coding region of mkh1 is contained within a single exon encoding a 1,116-amino-acid protein. The putative catalytic domain of Mkh1 is 54% identical to the catalytic domain of S. cerevisiae Bck1, the most closely related protein. Deletion of mkh1 did not significantly affect cell growth or division under standard conditions. However, mkh1delta cell growth was inhibited by high KCl or NaCl concentrations. mkh1delta cells required a longer time to reenter the cell cycle after prolonged stationary-phase arrest. Also, mkh1delta cells exhibited a round cell shape, while overexpression of Mkh1 resulted in an elongated cell shape. mkh1delta cells exhibited a more dramatic phenotype when grown in nutrient-limiting conditions at high temperature or in hyperosmotic medium. In such conditions, completion of cytokinesis was inhibited, resulting in the growth of pseudohyphal filaments with multiple septa and nuclei. Also, mkh1delta cells were hypersensitive to beta-glucanase treatment. Together these results suggest that Mkh1 regulates cell morphology, cell wall integrity, salt resistance, cell cycle reentry from stationary-phase arrest, and filamentous growth in response to stress. These phenotypes are essentially identical to those exhibited by cells lacking Pmk1/Spm1, a recently identified mitogen-activated protein kinase. Our evidence suggests that Pmk1/Spm1 acts downstream from Mkh1 in a common pathway. Our results also suggest that Mkh1 and Pck2 act independently to maintain cell wall integrity, cell morphology, and salt resistance but act in opposition to regulate filamentous growth.

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Selected References

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  1. Basi G., Schmid E., Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene. 1993 Jan 15;123(1):131–136. doi: 10.1016/0378-1119(93)90552-e. [DOI] [PubMed] [Google Scholar]
  2. Brewster J. L., de Valoir T., Dwyer N. D., Winter E., Gustin M. C. An osmosensing signal transduction pathway in yeast. Science. 1993 Mar 19;259(5102):1760–1763. doi: 10.1126/science.7681220. [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. Chang F., Woollard A., Nurse P. Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring. J Cell Sci. 1996 Jan;109(Pt 1):131–142. doi: 10.1242/jcs.109.1.131. [DOI] [PubMed] [Google Scholar]
  5. Davenport K. R., Sohaskey M., Kamada Y., Levin D. E., Gustin M. C. A second osmosensing signal transduction pathway in yeast. Hypotonic shock activates the PKC1 protein kinase-regulated cell integrity pathway. J Biol Chem. 1995 Dec 15;270(50):30157–30161. doi: 10.1074/jbc.270.50.30157. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Fankhauser C., Simanis V. Cold fission: splitting the pombe cell at room temperature. Trends Cell Biol. 1994 Mar;4(3):96–101. doi: 10.1016/0962-8924(94)90182-1. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Garrett-Engele P., Moilanen B., Cyert M. S. Calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, is essential in yeast mutants with cell integrity defects and in mutants that lack a functional vacuolar H(+)-ATPase. Mol Cell Biol. 1995 Aug;15(8):4103–4114. doi: 10.1128/mcb.15.8.4103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  13. Herskowitz I. MAP kinase pathways in yeast: for mating and more. Cell. 1995 Jan 27;80(2):187–197. doi: 10.1016/0092-8674(95)90402-6. [DOI] [PubMed] [Google Scholar]
  14. Kamada Y., Jung U. S., Piotrowski J., Levin D. E. The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev. 1995 Jul 1;9(13):1559–1571. doi: 10.1101/gad.9.13.1559. [DOI] [PubMed] [Google Scholar]
  15. Kawamukai M., Gerst J., Field J., Riggs M., Rodgers L., Wigler M., Young D. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe. Mol Biol Cell. 1992 Feb;3(2):167–180. doi: 10.1091/mbc.3.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kobori H., Toda T., Yaguchi H., Toya M., Yanagida M., Osumi M. Fission yeast protein kinase C gene homologues are required for protoplast regeneration: a functional link between cell wall formation and cell shape control. J Cell Sci. 1994 May;107(Pt 5):1131–1136. doi: 10.1242/jcs.107.5.1131. [DOI] [PubMed] [Google Scholar]
  17. Krisak L., Strich R., Winters R. S., Hall J. P., Mallory M. J., Kreitzer D., Tuan R. S., Winter E. SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae. Genes Dev. 1994 Sep 15;8(18):2151–2161. doi: 10.1101/gad.8.18.2151. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Lee K. S., Levin D. E. Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. Mol Cell Biol. 1992 Jan;12(1):172–182. doi: 10.1128/mcb.12.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Levin D. E., Bishop J. M. A putative protein kinase gene (kin1+) is important for growth polarity in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8272–8276. doi: 10.1073/pnas.87.21.8272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Levin D. E., Bowers B., Chen C. Y., Kamada Y., Watanabe M. Dissecting the protein kinase C/MAP kinase signalling pathway of Saccharomyces cerevisiae. Cell Mol Biol Res. 1994;40(3):229–239. [PubMed] [Google Scholar]
  23. Levin D. E., Errede B. The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol. 1995 Apr;7(2):197–202. doi: 10.1016/0955-0674(95)80028-x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Matviw H., Yu G., Young D. Identification and genetic analysis of Schizosaccharomyces pombe cDNAs that suppress deletion of IRA1 in Saccharomyces cerevisiae. Gene. 1993 Jul 15;129(1):147–152. doi: 10.1016/0378-1119(93)90711-b. [DOI] [PubMed] [Google Scholar]
  26. Matviw H., Yu G., Young D. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins. Mol Cell Biol. 1992 Nov;12(11):5033–5040. doi: 10.1128/mcb.12.11.5033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  28. Mazzei G. J., Schmid E. M., Knowles J. K., Payton M. A., Maundrell K. G. A Ca(2+)-independent protein kinase C from fission yeast. J Biol Chem. 1993 Apr 5;268(10):7401–7406. [PubMed] [Google Scholar]
  29. Mertins P., Gallwitz D. Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element. EMBO J. 1987 Jun;6(6):1757–1763. doi: 10.1002/j.1460-2075.1987.tb02428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Millar J. B., Buck V., Wilkinson M. G. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast. Genes Dev. 1995 Sep 1;9(17):2117–2130. doi: 10.1101/gad.9.17.2117. [DOI] [PubMed] [Google Scholar]
  31. Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. [DOI] [PubMed] [Google Scholar]
  32. Nadin-Davis S. A., Nasim A. A gene which encodes a predicted protein kinase can restore some functions of the ras gene in fission yeast. EMBO J. 1988 Apr;7(4):985–993. doi: 10.1002/j.1460-2075.1988.tb02905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nadin-Davis S. A., Nasim A. Schizosaccharomyces pombe ras1 and byr1 are functionally related genes of the ste family that affect starvation-induced transcription of mating-type genes. Mol Cell Biol. 1990 Feb;10(2):549–560. doi: 10.1128/mcb.10.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Nurse P., Thuriaux P., Nasmyth K. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet. 1976 Jul 23;146(2):167–178. doi: 10.1007/BF00268085. [DOI] [PubMed] [Google Scholar]
  37. Paravicini G., Cooper M., Friedli L., Smith D. J., Carpentier J. L., Klig L. S., Payton M. A. The osmotic integrity of the yeast cell requires a functional PKC1 gene product. Mol Cell Biol. 1992 Nov;12(11):4896–4905. doi: 10.1128/mcb.12.11.4896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Roberts R. L., Fink G. R. Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 1994 Dec 15;8(24):2974–2985. doi: 10.1101/gad.8.24.2974. [DOI] [PubMed] [Google Scholar]
  40. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  41. Russell P., Nurse P. cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell. 1986 Apr 11;45(1):145–153. doi: 10.1016/0092-8674(86)90546-5. [DOI] [PubMed] [Google Scholar]
  42. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Seger R., Krebs E. G. The MAPK signaling cascade. FASEB J. 1995 Jun;9(9):726–735. [PubMed] [Google Scholar]
  44. Shiozaki K., Russell P. Cell-cycle control linked to extracellular environment by MAP kinase pathway in fission yeast. Nature. 1995 Dec 14;378(6558):739–743. doi: 10.1038/378739a0. [DOI] [PubMed] [Google Scholar]
  45. Shiozaki K., Russell P. Counteractive roles of protein phosphatase 2C (PP2C) and a MAP kinase kinase homolog in the osmoregulation of fission yeast. EMBO J. 1995 Feb 1;14(3):492–502. doi: 10.1002/j.1460-2075.1995.tb07025.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sidorova J., Breeden L. Analysis of the SWI4/SWI6 protein complex, which directs G1/S-specific transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1069–1077. doi: 10.1128/mcb.13.2.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Toda T., Dhut S., Superti-Furga G., Gotoh Y., Nishida E., Sugiura R., Kuno T. The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway. Mol Cell Biol. 1996 Dec;16(12):6752–6764. doi: 10.1128/mcb.16.12.6752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Toda T., Shimanuki M., Yanagida M. Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes Dev. 1991 Jan;5(1):60–73. doi: 10.1101/gad.5.1.60. [DOI] [PubMed] [Google Scholar]
  50. Toda T., Shimanuki M., Yanagida M. Two novel protein kinase C-related genes of fission yeast are essential for cell viability and implicated in cell shape control. EMBO J. 1993 May;12(5):1987–1995. doi: 10.1002/j.1460-2075.1993.tb05848.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wang Y., Xu H. P., Riggs M., Rodgers L., Wigler M. byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype. Mol Cell Biol. 1991 Jul;11(7):3554–3563. doi: 10.1128/mcb.11.7.3554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Warbrick E., Fantes P. A. The wis1 protein kinase is a dosage-dependent regulator of mitosis in Schizosaccharomyces pombe. EMBO J. 1991 Dec;10(13):4291–4299. doi: 10.1002/j.1460-2075.1991.tb05007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Xu H. P., Wang Y., Riggs M., Rodgers L., Wigler M. Biological activity of the mammalian RAP genes in yeast. Cell Regul. 1990 Sep;1(10):763–769. doi: 10.1091/mbc.1.10.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yoshida T., Toda T., Yanagida M. A calcineurin-like gene ppb1+ in fission yeast: mutant defects in cytokinesis, cell polarity, mating and spindle pole body positioning. J Cell Sci. 1994 Jul;107(Pt 7):1725–1735. doi: 10.1242/jcs.107.7.1725. [DOI] [PubMed] [Google Scholar]
  55. Yu G., Swiston J., Young D. Comparison of human CAP and CAP2, homologs of the yeast adenylyl cyclase-associated proteins. J Cell Sci. 1994 Jun;107(Pt 6):1671–1678. doi: 10.1242/jcs.107.6.1671. [DOI] [PubMed] [Google Scholar]
  56. Zaitsevskaya-Carter T., Cooper J. A. Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe. EMBO J. 1997 Mar 17;16(6):1318–1331. doi: 10.1093/emboj/16.6.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]

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