Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Mar;171(3):1417–1422. doi: 10.1128/jb.171.3.1417-1422.1989

Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin.

T Miyakawa 1, Y Oka 1, E Tsuchiya 1, S Fukui 1
PMCID: PMC209761  PMID: 2537817

Abstract

A Ca2+- and calmodulin (CaM)-dependent protein kinase of Saccharomyces cerevisiae was partially purified by CaM affinity chromatography of the soluble fraction, and the properties of the enzyme were investigated. The protein kinase activity of the affinity-purified preparation was stimulated at least eightfold by the simultaneous presence of Ca2+ and CaM. The enzyme stimulation was strongly inhibited by trifluoperazine (TFP), a CaM antagonist. When the kinase was incubated in the presence of ATP, Ca2+, and CaM before the assay, the enzyme showed activity even in the presence of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and TFP. The conversion to this Ca2+- and CaM-independent form occurred very rapidly under the incubation conditions required for protein phosphorylation by the kinase. At the highest level of conversion, Ca2+- and CaM-independent kinase activity, which was measured in the presence of EGTA and TFP, was nearly equal to the total kinase activity, which was measured in the presence of Ca2+ and CaM. A protein with a molecular weight of 58,000 was the major species that was phosphorylated in a Ca2+- and CaM-dependent manner by incubation of the CaM affinity-purified proteins with [gamma-32P]ATP. The protein kinase activity of the protein with the same molecular weight was demonstrated by in situ protein phosphorylation in sodium dodecyl sulfate-polyacrylamide gels by using casein as the substrate, after removal of the detergent from electrophoresed CaM-binding proteins. These data indicate that phosphorylation of the kinase is responsible for the conversion of enzyme activity. Enzyme regulation by this mode may play an important role in integrating cellular functions during the cell cycle. A possible role for the Ca2+-and CaM-dependent protein kinase in the signal transduction of the mating pheromone alpha factor is also discussed.

Full text

PDF
1422

Images in this article

1420Image
on p.1420

Selected References

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

  1. Abe K., Kusaka I., Fukui S. Morphological change in the early stages of the mating process of Rhodosporidium toruloides. J Bacteriol. 1975 May;122(2):710–718. doi: 10.1128/jb.122.2.710-718.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Davis T. N., Urdea M. S., Masiarz F. R., Thorner J. Isolation of the yeast calmodulin gene: calmodulin is an essential protein. Cell. 1986 Nov 7;47(3):423–431. doi: 10.1016/0092-8674(86)90599-4. [DOI] [PubMed] [Google Scholar]
  3. Geahlen R. L., Anostario M., Jr, Low P. S., Harrison M. L. Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem. 1986 Feb 15;153(1):151–158. doi: 10.1016/0003-2697(86)90074-6. [DOI] [PubMed] [Google Scholar]
  4. Gopalakrishna R., Anderson W. B. Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1982 Jan 29;104(2):830–836. doi: 10.1016/0006-291x(82)90712-4. [DOI] [PubMed] [Google Scholar]
  5. Hemmings B. A., Zubenko G. S., Hasilik A., Jones E. W. Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Jan;78(1):435–439. doi: 10.1073/pnas.78.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Lai Y., Nairn A. C., Greengard P. Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4253–4257. doi: 10.1073/pnas.83.12.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Londesborough J., Nuutinen M. Ca2+/calmodulin-dependent protein kinase in Saccharomyces cerevisiae. FEBS Lett. 1987 Jul 13;219(1):249–253. doi: 10.1016/0014-5793(87)81226-7. [DOI] [PubMed] [Google Scholar]
  10. Lou L. L., Lloyd S. J., Schulman H. Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase by autophosphorylation: ATP modulates production of an autonomous enzyme. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9497–9501. doi: 10.1073/pnas.83.24.9497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miller S. G., Kennedy M. B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell. 1986 Mar 28;44(6):861–870. doi: 10.1016/0092-8674(86)90008-5. [DOI] [PubMed] [Google Scholar]
  12. Miyakawa T., Kadota T., Okubo Y., Hatano T., Tsuchiya E., Fukui S. Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica. J Bacteriol. 1984 Jun;158(3):814–819. doi: 10.1128/jb.158.3.814-819.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Miyakawa T., Nishihara M., Tsuchiya E., Fukui S. Role of metabolism of the mating pheromone in sexual differentiation of the heterobasidiomycete Rhodosporidium toruloides. J Bacteriol. 1982 Sep;151(3):1184–1194. doi: 10.1128/jb.151.3.1184-1194.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Miyakawa T., Tachikawa T., Jeong Y. K., Tsuchiya E., Fukui S. Transient increase of Ca2+ uptake as a signal for mating pheromone-induced differentiation in the heterobasidiomycetous yeast Rhodosporidium toruloides. J Bacteriol. 1985 Jun;162(3):1304–1306. doi: 10.1128/jb.162.3.1304-1306.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nairn A. C., Hemmings H. C., Jr, Greengard P. Protein kinases in the brain. Annu Rev Biochem. 1985;54:931–976. doi: 10.1146/annurev.bi.54.070185.004435. [DOI] [PubMed] [Google Scholar]
  16. Ohya Y., Uno I., Ishikawa T., Anraku Y. Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae. Eur J Biochem. 1987 Oct 1;168(1):13–19. doi: 10.1111/j.1432-1033.1987.tb13380.x. [DOI] [PubMed] [Google Scholar]
  17. Schworer C. M., Colbran R. J., Soderling T. R. Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism. J Biol Chem. 1986 Jul 5;261(19):8581–8584. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES