Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1993 May;12(5):1987–1995. doi: 10.1002/j.1460-2075.1993.tb05848.x

Two novel protein kinase C-related genes of fission yeast are essential for cell viability and implicated in cell shape control.

T Toda 1, M Shimanuki 1, M Yanagida 1
PMCID: PMC413420  PMID: 8491190

Abstract

Two novel protein kinase C (PKC)-like genes, pck1+ and pck2+ were isolated from fission yeast by PCR. Both contain common domains of PKC-related molecules, but lack a putative Ca(2+)-binding domain so that they may belong to the nPKC group. Gene disruption of pck1+ and pck2+ establishes that they share an overlapping essential function for cell viability. Cells of a single pck2 deletion display severe defects in cell shape; they are irregular and sometimes pear-like instead of cylindrical. In contrast, the induced overexpression of pck2+ is lethal, producing multiseptated and branched cells. These results suggest that fission yeast PKC-like genes are involved in the polarity of cell growth control. We show that pck2 is allelic to sts6, a locus we have previously identified by its supersensitivity to staurosporine, a potent protein kinase inhibitor [Toda et al. (1991) Genes Dev., 5, 60-73]. In addition, the lethal overexpression of pck2+ can be suppressed by staurosporine, indicating that fission yeast pck1 and pck2 are molecular targets of this inhibitor.

Full text

PDF
1988

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abe K., Yoshida M., Usui T., Horinouchi S., Beppu T. Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein kinases. Exp Cell Res. 1991 Jan;192(1):122–127. doi: 10.1016/0014-4827(91)90166-r. [DOI] [PubMed] [Google Scholar]
  2. Adachi Y., Yanagida M. Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crm1+ which encodes a 115-kD protein preferentially localized in the nucleus and its periphery. J Cell Biol. 1989 Apr;108(4):1195–1207. doi: 10.1083/jcb.108.4.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arndt K. T., Styles C. A., Fink G. R. A suppressor of a HIS4 transcriptional defect encodes a protein with homology to the catalytic subunit of protein phosphatases. Cell. 1989 Feb 24;56(4):527–537. doi: 10.1016/0092-8674(89)90576-x. [DOI] [PubMed] [Google Scholar]
  4. Bruno S., Ardelt B., Skierski J. S., Traganos F., Darzynkiewicz Z. Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. Cancer Res. 1992 Jan 15;52(2):470–473. [PubMed] [Google Scholar]
  5. Burns D. J., Bell R. M. Protein kinase C contains two phorbol ester binding domains. J Biol Chem. 1991 Sep 25;266(27):18330–18338. [PubMed] [Google Scholar]
  6. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chikashige Y., Kinoshita N., Nakaseko Y., Matsumoto T., Murakami S., Niwa O., Yanagida M. Composite motifs and repeat symmetry in S. pombe centromeres: direct analysis by integration of NotI restriction sites. Cell. 1989 Jun 2;57(5):739–751. doi: 10.1016/0092-8674(89)90789-7. [DOI] [PubMed] [Google Scholar]
  8. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  9. Cohen P. T., Brewis N. D., Hughes V., Mann D. J. Protein serine/threonine phosphatases; an expanding family. FEBS Lett. 1990 Aug 1;268(2):355–359. doi: 10.1016/0014-5793(90)81285-v. [DOI] [PubMed] [Google Scholar]
  10. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  11. Coussens L., Parker P. J., Rhee L., Yang-Feng T. L., Chen E., Waterfield M. D., Francke U., Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science. 1986 Aug 22;233(4766):859–866. doi: 10.1126/science.3755548. [DOI] [PubMed] [Google Scholar]
  12. Crissman H. A., Gadbois D. M., Tobey R. A., Bradbury E. M. Transformed mammalian cells are deficient in kinase-mediated control of progression through the G1 phase of the cell cycle. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7580–7584. doi: 10.1073/pnas.88.17.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drubin D. G. Development of cell polarity in budding yeast. Cell. 1991 Jun 28;65(7):1093–1096. doi: 10.1016/0092-8674(91)90001-f. [DOI] [PubMed] [Google Scholar]
  14. Fan J. B., Chikashige Y., Smith C. L., Niwa O., Yanagida M., Cantor C. R. Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome. Nucleic Acids Res. 1989 Apr 11;17(7):2801–2818. doi: 10.1093/nar/17.7.2801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  16. Fukui Y., Miyake S., Satoh M., Yamamoto M. Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the ras1 gene product. Mol Cell Biol. 1989 Dec;9(12):5617–5622. doi: 10.1128/mcb.9.12.5617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grimm C., Kohli J., Murray J., Maundrell K. Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet. 1988 Dec;215(1):81–86. doi: 10.1007/BF00331307. [DOI] [PubMed] [Google Scholar]
  18. Gschwendt M., Kittstein W., Marks F. Protein kinase C activation by phorbol esters: do cysteine-rich regions and pseudosubstrate motifs play a role? Trends Biochem Sci. 1991 May;16(5):167–169. doi: 10.1016/0968-0004(91)90064-3. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hirano T., Hiraoka Y., Yanagida M. A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J Cell Biol. 1988 Apr;106(4):1171–1183. doi: 10.1083/jcb.106.4.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. House C., Kemp B. E. Protein kinase C contains a pseudosubstrate prototope in its regulatory domain. Science. 1987 Dec 18;238(4834):1726–1728. doi: 10.1126/science.3686012. [DOI] [PubMed] [Google Scholar]
  22. Hubbard S. R., Bishop W. R., Kirschmeier P., George S. J., Cramer S. P., Hendrickson W. A. Identification and characterization of zinc binding sites in protein kinase C. Science. 1991 Dec 20;254(5039):1776–1779. doi: 10.1126/science.1763327. [DOI] [PubMed] [Google Scholar]
  23. Hughes D. A., Fukui Y., Yamamoto M. Homologous activators of ras in fission and budding yeast. Nature. 1990 Mar 22;344(6264):355–357. doi: 10.1038/344355a0. [DOI] [PubMed] [Google Scholar]
  24. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kikkawa U., Nishizuka Y. The role of protein kinase C in transmembrane signalling. Annu Rev Cell Biol. 1986;2:149–178. doi: 10.1146/annurev.cb.02.110186.001053. [DOI] [PubMed] [Google Scholar]
  26. Kinoshita N., Ohkura H., Yanagida M. Distinct, essential roles of type 1 and 2A protein phosphatases in the control of the fission yeast cell division cycle. Cell. 1990 Oct 19;63(2):405–415. doi: 10.1016/0092-8674(90)90173-c. [DOI] [PubMed] [Google Scholar]
  27. Knopf J. L., Lee M. H., Sultzman L. A., Kriz R. W., Loomis C. R., Hewick R. M., Bell R. M. Cloning and expression of multiple protein kinase C cDNAs. Cell. 1986 Aug 15;46(4):491–502. doi: 10.1016/0092-8674(86)90874-3. [DOI] [PubMed] [Google Scholar]
  28. Lee C. C., Wu X. W., Gibbs R. A., Cook R. G., Muzny D. M., Caskey C. T. Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science. 1988 Mar 11;239(4845):1288–1291. doi: 10.1126/science.3344434. [DOI] [PubMed] [Google Scholar]
  29. Lee M. G., Nurse P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature. 1987 May 7;327(6117):31–35. doi: 10.1038/327031a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Levin D. E., Fields F. O., Kunisawa R., Bishop J. M., Thorner J. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell. 1990 Jul 27;62(2):213–224. doi: 10.1016/0092-8674(90)90360-q. [DOI] [PubMed] [Google Scholar]
  32. Lund P. M., Hasegawa Y., Kitamura K., Shimoda C., Fukui Y., Yamamoto M. Mapping of the ras1 gene of Schizosaccharomyces pombe. Mol Gen Genet. 1987 Oct;209(3):627–629. doi: 10.1007/BF00331175. [DOI] [PubMed] [Google Scholar]
  33. Marks J., Hagan I. M., Hyams J. S. Growth polarity and cytokinesis in fission yeast: the role of the cytoskeleton. J Cell Sci Suppl. 1986;5:229–241. doi: 10.1242/jcs.1986.supplement_5.15. [DOI] [PubMed] [Google Scholar]
  34. Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990 Jul 5;265(19):10857–10864. [PubMed] [Google Scholar]
  35. Mitchison J. M., Nurse P. Growth in cell length in the fission yeast Schizosaccharomyces pombe. J Cell Sci. 1985 Apr;75:357–376. doi: 10.1242/jcs.75.1.357. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Nadin-Davis S. A., Nasim A., Beach D. Involvement of ras in sexual differentiation but not in growth control in fission yeast. EMBO J. 1986 Nov;5(11):2963–2971. doi: 10.1002/j.1460-2075.1986.tb04593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nakadate T., Jeng A. Y., Blumberg P. M. Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action. Biochem Pharmacol. 1988 Apr 15;37(8):1541–1545. doi: 10.1016/0006-2952(88)90016-0. [DOI] [PubMed] [Google Scholar]
  39. Nakano H., Kobayashi E., Takahashi I., Tamaoki T., Kuzuu Y., Iba H. Staurosporine inhibits tyrosine-specific protein kinase activity of Rous sarcoma virus transforming protein p60. J Antibiot (Tokyo) 1987 May;40(5):706–708. doi: 10.7164/antibiotics.40.706. [DOI] [PubMed] [Google Scholar]
  40. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  41. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  42. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  43. Ogita K., Miyamoto S., Koide H., Iwai T., Oka M., Ando K., Kishimoto A., Ikeda K., Fukami Y., Nishizuka Y. Protein kinase C in Saccharomyces cerevisiae: comparison with the mammalian enzyme. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5011–5015. doi: 10.1073/pnas.87.13.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ohkura H., Kinoshita N., Miyatani S., Toda T., Yanagida M. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell. 1989 Jun 16;57(6):997–1007. doi: 10.1016/0092-8674(89)90338-3. [DOI] [PubMed] [Google Scholar]
  45. Ohno S., Akita Y., Konno Y., Imajoh S., Suzuki K. A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family. Cell. 1988 Jun 3;53(5):731–741. doi: 10.1016/0092-8674(88)90091-8. [DOI] [PubMed] [Google Scholar]
  46. Ono Y., Fujii T., Igarashi K., Kuno T., Tanaka C., Kikkawa U., Nishizuka Y. Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4868–4871. doi: 10.1073/pnas.86.13.4868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. The structure, expression, and properties of additional members of the protein kinase C family. J Biol Chem. 1988 May 15;263(14):6927–6932. [PubMed] [Google Scholar]
  48. Osada S., Mizuno K., Saido T. C., Akita Y., Suzuki K., Kuroki T., Ohno S. A phorbol ester receptor/protein kinase, nPKC eta, a new member of the protein kinase C family predominantly expressed in lung and skin. J Biol Chem. 1990 Dec 25;265(36):22434–22440. [PubMed] [Google Scholar]
  49. Osada S., Mizuno K., Saido T. C., Suzuki K., Kuroki T., Ohno S. A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle. Mol Cell Biol. 1992 Sep;12(9):3930–3938. doi: 10.1128/mcb.12.9.3930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Ravid S., Spudich J. A. Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5877–5881. doi: 10.1073/pnas.89.13.5877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Schaeffer E., Smith D., Mardon G., Quinn W., Zuker C. Isolation and characterization of two new drosophila protein kinase C genes, including one specifically expressed in photoreceptor cells. Cell. 1989 May 5;57(3):403–412. doi: 10.1016/0092-8674(89)90915-x. [DOI] [PubMed] [Google Scholar]
  56. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  57. Shimanuki M., Goebl M., Yanagida M., Toda T. Fission yeast sts1+ gene encodes a protein similar to the chicken lamin B receptor and is implicated in pleiotropic drug-sensitivity, divalent cation-sensitivity, and osmoregulation. Mol Biol Cell. 1992 Mar;3(3):263–273. doi: 10.1091/mbc.3.3.263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Simon A. J., Milner Y., Saville S. P., Dvir A., Mochly-Rosen D., Orr E. The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae. Proc Biol Sci. 1991 Feb 22;243(1307):165–171. doi: 10.1098/rspb.1991.0027. [DOI] [PubMed] [Google Scholar]
  59. Smith D. P., Ranganathan R., Hardy R. W., Marx J., Tsuchida T., Zuker C. S. Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C. Science. 1991 Dec 6;254(5037):1478–1484. doi: 10.1126/science.1962207. [DOI] [PubMed] [Google Scholar]
  60. Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
  61. Toda T., Adachi Y., Hiraoka Y., Yanagida M. Identification of the pleiotropic cell division cycle gene NDA2 as one of two different alpha-tubulin genes in Schizosaccharomyces pombe. Cell. 1984 May;37(1):233–242. doi: 10.1016/0092-8674(84)90319-2. [DOI] [PubMed] [Google Scholar]
  62. 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]
  63. Umesono K., Toda T., Hayashi S., Yanagida M. Cell division cycle genes nda2 and nda3 of the fission yeast Schizosaccharomyces pombe control microtubular organization and sensitivity to anti-mitotic benzimidazole compounds. J Mol Biol. 1983 Aug 5;168(2):271–284. doi: 10.1016/s0022-2836(83)80018-7. [DOI] [PubMed] [Google Scholar]
  64. Yoshida S., Ikeda E., Uno I., Mitsuzawa H. Characterization of a staurosporine- and temperature-sensitive mutant, stt1, of Saccharomyces cerevisiae: STT1 is allelic to PKC1. Mol Gen Genet. 1992 Feb;231(3):337–344. doi: 10.1007/BF00292700. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES