Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 May;14(5):2958–2965. doi: 10.1128/mcb.14.5.2958

Characterization of tobacco protein kinase NPK5, a homolog of Saccharomyces cerevisiae SNF1 that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae.

T Muranaka 1, H Banno 1, Y Machida 1
PMCID: PMC358663  PMID: 8164654

Abstract

We have isolated a cDNA (cNPK5) that encodes a protein kinase of 511 amino acids from suspension cultures of tobacco cells. The predicted kinase domain of NPK5 is 65% identical in terms of amino acid sequence to that of the SNF1 serine/threonine protein kinase of Saccharomyces cerevisiae, which plays a central role in catabolite repression in yeast cells. SNF1 positively regulates transcription of various glucose-repressible genes of the yeast, such as the SUC2 gene for a secreted invertase, in response to glucose deprivation: snf1 mutants cannot utilize sucrose as a carbon source. Expression of cNPK5 in yeast cells allowed the snf1 mutant cells to utilize sucrose for growth and caused constitutive expression of the SUC2 gene in wild-type cells even in the presence of glucose, an indication that the NPK5 protein is present in a constitutively active form in S. cerevisiae. On the other hand, expression of cNPK5 failed to suppress the growth defect of the snf4 mutant cells in the presence of sucrose and to induce expression of the SUC2 gene. These results indicate that SNF4 is required for the induction of SUC2 expression by NPK5, as by SNF1, even if NPK5 is constitutively active in S. cerevisiae. The recombinant NPK5 protein is capable of autophosphorylation in vitro in a reaction that requires Mn2+ rather than Mg2+ ions but is inhibited by Ca2+ ions. Both dicotyledonous and monocotyledonous plants have several copies of the NPK5-related gene, which probably constitute a small gene family. NPK5-related genes were found to be expressed in the roots, leaves, and stems of tobacco plants. The high degree of structural conservation and the functional similarity of NPK5 to SNF1 lead us to speculate that NPK5 (or a related kinase) also plays a role in sugar metabolism in higher plants.

Full text

PDF
2958

Images in this article

2962Image
on p.2962
2962Image
on p.2962
2963Image
on p.2963

Selected References

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

  1. Alderson A., Sabelli P. A., Dickinson J. R., Cole D., Richardson M., Kreis M., Shewry P. R., Halford N. G. Complementation of snf1, a mutation affecting global regulation of carbon metabolism in yeast, by a plant protein kinase cDNA. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8602–8605. doi: 10.1073/pnas.88.19.8602. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banno H., Hirano K., Nakamura T., Irie K., Nomoto S., Matsumoto K., Machida Y. NPK1, a tobacco gene that encodes a protein with a domain homologous to yeast BCK1, STE11, and Byr2 protein kinases. Mol Cell Biol. 1993 Aug;13(8):4745–4752. doi: 10.1128/mcb.13.8.4745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  4. Carlson M., Osmond B. C., Botstein D. Mutants of yeast defective in sucrose utilization. Genetics. 1981 May;98(1):25–40. doi: 10.1093/genetics/98.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carlson M. Regulation of sugar utilization in Saccharomyces species. J Bacteriol. 1987 Nov;169(11):4873–4877. doi: 10.1128/jb.169.11.4873-4877.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Celenza J. L., Carlson M. A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science. 1986 Sep 12;233(4769):1175–1180. doi: 10.1126/science.3526554. [DOI] [PubMed] [Google Scholar]
  7. Celenza J. L., Carlson M. Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):49–53. doi: 10.1128/mcb.4.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Celenza J. L., Carlson M. Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein. Mol Cell Biol. 1989 Nov;9(11):5034–5044. doi: 10.1128/mcb.9.11.5034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Celenza J. L., Marshall-Carlson L., Carlson M. The yeast SNF3 gene encodes a glucose transporter homologous to the mammalian protein. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2130–2134. doi: 10.1073/pnas.85.7.2130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Denis C. L., Gallo C. Constitutive RNA synthesis for the yeast activator ADR1 and identification of the ADR1-5c mutation: implications in posttranslational control of ADR1. Mol Cell Biol. 1986 Nov;6(11):4026–4030. doi: 10.1128/mcb.6.11.4026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Estruch F., Treitel M. A., Yang X., Carlson M. N-terminal mutations modulate yeast SNF1 protein kinase function. Genetics. 1992 Nov;132(3):639–650. doi: 10.1093/genetics/132.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldstein A., Lampen J. O. Beta-D-fructofuranoside fructohydrolase from yeast. Methods Enzymol. 1975;42:504–511. doi: 10.1016/0076-6879(75)42159-0. [DOI] [PubMed] [Google Scholar]
  13. Halford N. G., Vicente-Carbajosa J., Sabelli P. A., Shewry P. R., Hannappel U., Kreis M. Molecular analyses of a barley multigene family homologous to the yeast protein kinase gene SNF1. Plant J. 1992 Sep;2(5):791–797. [PubMed] [Google Scholar]
  14. 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]
  15. Hirayama T., Oka A. Novel protein kinase of Arabidopsis thaliana (APK1) that phosphorylates tyrosine, serine and threonine. Plant Mol Biol. 1992 Nov;20(4):653–662. doi: 10.1007/BF00046450. [DOI] [PubMed] [Google Scholar]
  16. Hoshino S., Miyazawa H., Enomoto T., Hanaoka F., Kikuchi Y., Kikuchi A., Ui M. A human homologue of the yeast GST1 gene codes for a GTP-binding protein and is expressed in a proliferation-dependent manner in mammalian cells. EMBO J. 1989 Dec 1;8(12):3807–3814. doi: 10.1002/j.1460-2075.1989.tb08558.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Karrer E. E., Rodriguez R. L. Metabolic regulation of rice alpha-amylase and sucrose synthase genes in planta. Plant J. 1992 Jul;2(4):517–523. [PubMed] [Google Scholar]
  18. Laurière C., Laurière M., Sturm A., Faye L., Chrispeels M. J. Characterization of beta-fructosidase, an extracellular glycoprotein of carrot cells. Biochimie. 1988 Nov;70(11):1483–1491. doi: 10.1016/0300-9084(88)90285-4. [DOI] [PubMed] [Google Scholar]
  19. Le Guen L., Thomas M., Bianchi M., Halford N. G., Kreis M. Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase. Gene. 1992 Oct 21;120(2):249–254. doi: 10.1016/0378-1119(92)90100-4. [DOI] [PubMed] [Google Scholar]
  20. Miyajima I., Nakafuku M., Nakayama N., Brenner C., Miyajima A., Kaibuchi K., Arai K., Kaziro Y., Matsumoto K. GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell. 1987 Sep 25;50(7):1011–1019. doi: 10.1016/0092-8674(87)90167-x. [DOI] [PubMed] [Google Scholar]
  21. Neigeborn L., Carlson M. Genes affecting the regulation of SUC2 gene expression by glucose repression in Saccharomyces cerevisiae. Genetics. 1984 Dec;108(4):845–858. doi: 10.1093/genetics/108.4.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Onouchi H., Yokoi K., Machida C., Matsuzaki H., Oshima Y., Matsuoka K., Nakamura K., Machida Y. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res. 1991 Dec 11;19(23):6373–6378. doi: 10.1093/nar/19.23.6373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sarokin L., Carlson M. Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Oct;5(10):2521–2526. doi: 10.1128/mcb.5.10.2521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sherman F. Getting started with yeast. Methods Enzymol. 1991;194:3–21. doi: 10.1016/0076-6879(91)94004-v. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Sturm A., Chrispeels M. J. cDNA cloning of carrot extracellular beta-fructosidase and its expression in response to wounding and bacterial infection. Plant Cell. 1990 Nov;2(11):1107–1119. doi: 10.1105/tpc.2.11.1107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Thompson-Jaeger S., François J., Gaughran J. P., Tatchell K. Deletion of SNF1 affects the nutrient response of yeast and resembles mutations which activate the adenylate cyclase pathway. Genetics. 1991 Nov;129(3):697–706. doi: 10.1093/genetics/129.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Weil M., Rausch T. Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin. Plant Physiol. 1990 Dec;94(4):1575–1581. doi: 10.1104/pp.94.4.1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Winston F., Carlson M. Yeast SNF/SWI transcriptional activators and the SPT/SIN chromatin connection. Trends Genet. 1992 Nov;8(11):387–391. doi: 10.1016/0168-9525(92)90300-s. [DOI] [PubMed] [Google Scholar]
  30. Yang X., Hubbard E. J., Carlson M. A protein kinase substrate identified by the two-hybrid system. Science. 1992 Jul 31;257(5070):680–682. doi: 10.1126/science.1496382. [DOI] [PubMed] [Google Scholar]
  31. Yu S. M., Kuo Y. H., Sheu G., Sheu Y. J., Liu L. F. Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice. J Biol Chem. 1991 Nov 5;266(31):21131–21137. [PubMed] [Google Scholar]

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

RESOURCES