Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Sep;13(9):5829–5842. doi: 10.1128/mcb.13.9.5829

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.

P Zheng 1, D S Fay 1, J Burton 1, H Xiao 1, J L Pinkham 1, D F Stern 1
PMCID: PMC360328  PMID: 8355715

Abstract

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Full text

PDF
5829

Images in this article

5834Image
on p.5834
5834Image
on p.5834
5835Image
on p.5835
5835Image
on p.5835
5836Image
on p.5836
5836Image
on p.5836
5837Image
on p.5837
5838Image
on p.5838
5839Image
on p.5839

Selected References

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

  1. Ammerer G., Hunter C. P., Rothman J. H., Saari G. C., Valls L. A., Stevens T. H. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol. 1986 Jul;6(7):2490–2499. doi: 10.1128/mcb.6.7.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrews B. J. Gene expression. Dialogue with the cell cycle. Nature. 1992 Jan 30;355(6359):393–394. doi: 10.1038/355393a0. [DOI] [PubMed] [Google Scholar]
  3. Andrews B. J., Herskowitz I. Regulation of cell cycle-dependent gene expression in yeast. J Biol Chem. 1990 Aug 25;265(24):14057–14060. [PubMed] [Google Scholar]
  4. Bell S. P., Stillman B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature. 1992 May 14;357(6374):128–134. doi: 10.1038/357128a0. [DOI] [PubMed] [Google Scholar]
  5. Ben-David Y., Letwin K., Tannock L., Bernstein A., Pawson T. A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators. EMBO J. 1991 Feb;10(2):317–325. doi: 10.1002/j.1460-2075.1991.tb07952.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brill S. J., Stillman B. Replication factor-A from Saccharomyces cerevisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 1991 Sep;5(9):1589–1600. doi: 10.1101/gad.5.9.1589. [DOI] [PubMed] [Google Scholar]
  7. Bueno A., Russell P. Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division. EMBO J. 1992 Jun;11(6):2167–2176. doi: 10.1002/j.1460-2075.1992.tb05276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Byers B., Goetsch L. Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb Symp Quant Biol. 1974;38:123–131. doi: 10.1101/sqb.1974.038.01.016. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  10. Dailey D., Schieven G. L., Lim M. Y., Marquardt H., Gilmore T., Thorner J., Martin G. S. Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity. Mol Cell Biol. 1990 Dec;10(12):6244–6256. doi: 10.1128/mcb.10.12.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Diffley J. F., Cocker J. H. Protein-DNA interactions at a yeast replication origin. Nature. 1992 May 14;357(6374):169–172. doi: 10.1038/357169a0. [DOI] [PubMed] [Google Scholar]
  12. Din S., Brill S. J., Fairman M. P., Stillman B. Cell-cycle-regulated phosphorylation of DNA replication factor A from human and yeast cells. Genes Dev. 1990 Jun;4(6):968–977. doi: 10.1101/gad.4.6.968. [DOI] [PubMed] [Google Scholar]
  13. Dirick L., Moll T., Auer H., Nasmyth K. A central role for SWI6 in modulating cell cycle Start-specific transcription in yeast. Nature. 1992 Jun 11;357(6378):508–513. doi: 10.1038/357508a0. [DOI] [PubMed] [Google Scholar]
  14. Dutta A., Stillman B. cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J. 1992 Jun;11(6):2189–2199. doi: 10.1002/j.1460-2075.1992.tb05278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fien K., Stillman B. Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex. Mol Cell Biol. 1992 Jan;12(1):155–163. doi: 10.1128/mcb.12.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fotedar R., Roberts J. M. Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. EMBO J. 1992 Jun;11(6):2177–2187. doi: 10.1002/j.1460-2075.1992.tb05277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Furter-Graves E. M., Hall B. D. DNA sequence elements required for transcription initiation of the Schizosaccharomyces pombe ADH gene in Saccharomyces cerevisiae. Mol Gen Genet. 1990 Sep;223(3):407–416. doi: 10.1007/BF00264447. [DOI] [PubMed] [Google Scholar]
  18. Gould K. L., Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. doi: 10.1038/342039a0. [DOI] [PubMed] [Google Scholar]
  19. Gómez N., Cohen P. Dissection of the protein kinase cascade by which nerve growth factor activates MAP kinases. Nature. 1991 Sep 12;353(6340):170–173. doi: 10.1038/353170a0. [DOI] [PubMed] [Google Scholar]
  20. Hahn S., Hoar E. T., Guarente L. Each of three "TATA elements" specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8562–8566. doi: 10.1073/pnas.82.24.8562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hanic-Joyce P. J. Mapping CDC mutations in the yeast S. cerevisiae by rad52-mediated chromosome loss. Genetics. 1985 Aug;110(4):591–607. doi: 10.1093/genetics/110.4.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Healy A. M., Helser T. L., Zitomer R. S. Sequences required for transcriptional initiation of the Saccharomyces cerevisiae CYC7 genes. Mol Cell Biol. 1987 Oct;7(10):3785–3791. doi: 10.1128/mcb.7.10.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Howell B. W., Afar D. E., Lew J., Douville E. M., Icely P. L., Gray D. A., Bell J. C. STY, a tyrosine-phosphorylating enzyme with sequence homology to serine/threonine kinases. Mol Cell Biol. 1991 Jan;11(1):568–572. doi: 10.1128/mcb.11.1.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jones J. S., Prakash L. Transcript levels of the Saccharomyces cerevisiae DNA repair gene RAD18 increase in UV irradiated cells and during meiosis but not during the mitotic cell cycle. Nucleic Acids Res. 1991 Feb 25;19(4):893–898. doi: 10.1093/nar/19.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
  29. Klekamp M. S., Weil P. A. Specific transcription of homologous class III genes in yeast-soluble cell-free extracts. J Biol Chem. 1982 Jul 25;257(14):8432–8441. [PubMed] [Google Scholar]
  30. 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]
  31. Letwin K., Mizzen L., Motro B., Ben-David Y., Bernstein A., Pawson T. A mammalian dual specificity protein kinase, Nek1, is related to the NIMA cell cycle regulator and highly expressed in meiotic germ cells. EMBO J. 1992 Oct;11(10):3521–3531. doi: 10.1002/j.1460-2075.1992.tb05435.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lindberg R. A., Quinn A. M., Hunter T. Dual-specificity protein kinases: will any hydroxyl do? Trends Biochem Sci. 1992 Mar;17(3):114–119. doi: 10.1016/0968-0004(92)90248-8. [DOI] [PubMed] [Google Scholar]
  33. Link A. J., Olson M. V. Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution. Genetics. 1991 Apr;127(4):681–698. doi: 10.1093/genetics/127.4.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lowndes N. F., Johnson A. L., Breeden L., Johnston L. H. SWI6 protein is required for transcription of the periodically expressed DNA synthesis genes in budding yeast. Nature. 1992 Jun 11;357(6378):505–508. doi: 10.1038/357505a0. [DOI] [PubMed] [Google Scholar]
  35. Lowndes N. F., McInerny C. J., Johnson A. L., Fantes P. A., Johnston L. H. Control of DNA synthesis genes in fission yeast by the cell-cycle gene cdc10+. Nature. 1992 Jan 30;355(6359):449–453. doi: 10.1038/355449a0. [DOI] [PubMed] [Google Scholar]
  36. Madura K., Prakash S. Nucleotide sequence, transcript mapping, and regulation of the RAD2 gene of Saccharomyces cerevisiae. J Bacteriol. 1986 Jun;166(3):914–923. doi: 10.1128/jb.166.3.914-923.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
  38. McIntosh E. M., Atkinson T., Storms R. K., Smith M. Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jan;11(1):329–337. doi: 10.1128/mcb.11.1.329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nakielny S., Cohen P., Wu J., Sturgill T. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase. EMBO J. 1992 Jun;11(6):2123–2129. doi: 10.1002/j.1460-2075.1992.tb05271.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Nasmyth K., Dirick L. The role of SWI4 and SWI6 in the activity of G1 cyclins in yeast. Cell. 1991 Sep 6;66(5):995–1013. doi: 10.1016/0092-8674(91)90444-4. [DOI] [PubMed] [Google Scholar]
  41. Neigeborn L., Mitchell A. P. The yeast MCK1 gene encodes a protein kinase homolog that activates early meiotic gene expression. Genes Dev. 1991 Apr;5(4):533–548. doi: 10.1101/gad.5.4.533. [DOI] [PubMed] [Google Scholar]
  42. Oechsner U., Magdolen V., Zoglowek C., Häcker U., Bandlow W. Yeast adenylate kinase is transcribed constitutively from a promoter in the short intergenic region to the histone H2A-1 gene. FEBS Lett. 1988 Dec 19;242(1):187–193. doi: 10.1016/0014-5793(88)81013-5. [DOI] [PubMed] [Google Scholar]
  43. Parker L. L., Atherton-Fessler S., Lee M. S., Ogg S., Falk J. L., Swenson K. I., Piwnica-Worms H. Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner. EMBO J. 1991 May;10(5):1255–1263. doi: 10.1002/j.1460-2075.1991.tb08067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Parker L. L., Piwnica-Worms H. Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science. 1992 Sep 25;257(5078):1955–1957. doi: 10.1126/science.1384126. [DOI] [PubMed] [Google Scholar]
  45. Shero J. H., Hieter P. A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1). Genes Dev. 1991 Apr;5(4):549–560. doi: 10.1101/gad.5.4.549. [DOI] [PubMed] [Google Scholar]
  46. Siede W., Friedberg E. C. Regulation of the yeast RAD2 gene: DNA damage-dependent induction correlates with protein binding to regulatory sequences and their deletion influences survival. Mol Gen Genet. 1992 Mar;232(2):247–256. doi: 10.1007/BF00280003. [DOI] [PubMed] [Google Scholar]
  47. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Stern D. F., Zheng P., Beidler D. R., Zerillo C. Spk1, a new kinase from Saccharomyces cerevisiae, phosphorylates proteins on serine, threonine, and tyrosine. Mol Cell Biol. 1991 Feb;11(2):987–1001. doi: 10.1128/mcb.11.2.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Woolford C. A., Daniels L. B., Park F. J., Jones E. W., Van Arsdell J. N., Innis M. A. The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases. Mol Cell Biol. 1986 Jul;6(7):2500–2510. doi: 10.1128/mcb.6.7.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES