Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1988 Nov;85(21):7962–7966. doi: 10.1073/pnas.85.21.7962

Phosphorylation of yeast phosphatidylserine synthase in vivo and in vitro by cyclic AMP-dependent protein kinase.

A J Kinney 1, G M Carman 1
PMCID: PMC282333  PMID: 2847149

Abstract

Evidence is presented that demonstrates that phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) from Saccharomyces cerevisiae is phosphorylated in vivo and in vitro by cAMP-dependent protein kinase. Phosphatidylserine synthase activity in cell extracts was reduced in the bcy1 mutant (which has high cAMP-dependent protein kinase activity) and elevated in the cyr1 mutant (which has low cAMP-dependent protein kinase activity) when compared with wild-type cells. The reduced phosphatidylserine synthase activity in the bcy1 mutant correlated with elevated levels of a phosphorylated form of the phosphatidylserine synthase Mr 23,000 subunit. The elevated phosphatidylserine synthase activity in the cyr1 mutant correlated with reduced levels of the phosphorylated form of the enzyme. There was negligible phosphorylation of the phosphatidylserine synthase Mr 23,000 subunit from stationary-phase cells. Pure phosphatidylserine synthase was phosphorylated by the cAMP-dependent protein kinase catalytic subunit, which resulted in a 60-70% reduction in phosphatidylserine synthase activity. The cAMP-dependent protein kinase catalytic subunit catalyzed the incorporation of 0.7 mol of phosphate per mol of phosphatidylserine synthase Mr 23,000 subunit. The specific cAMP-dependent protein kinase inhibitor prevented the phosphorylation of phosphatidylserine synthase and the inhibition of its activity by the catalytic subunit. Analysis of peptides derived from protease-treated labeled phosphatidylserine synthase showed only one labeled peptide. Phospho amino acid analysis of labeled phosphatidylserine synthase showed that the enzyme was phosphorylated at a serine residue.

Full text

PDF
7962

Images in this article

7963Image
on p.7963
7963Image
on p.7963
7965Image
on p.7965
7965Image
on p.7965
7965Image
on p.7965
7965Image
on p.7965

Selected References

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

  1. Atkinson K. D., Jensen B., Kolat A. I., Storm E. M., Henry S. A., Fogel S. Yeast mutants auxotrophic for choline or ethanolamine. J Bacteriol. 1980 Feb;141(2):558–564. doi: 10.1128/jb.141.2.558-564.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bae-Lee M. S., Carman G. M. Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase. J Biol Chem. 1984 Sep 10;259(17):10857–10862. [PubMed] [Google Scholar]
  3. Bailis A. M., Poole M. A., Carman G. M., Henry S. A. The membrane-associated enzyme phosphatidylserine synthase is regulated at the level of mRNA abundance. Mol Cell Biol. 1987 Jan;7(1):167–176. doi: 10.1128/mcb.7.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bell G. I., Valenzuela P., Rutter W. J. Phosphorylation of yeast DNA-dependent RNA polymerases in vivo and in vitro. Isolation of enzymes and identification of phosphorylated subunits. J Biol Chem. 1977 May 10;252(9):3082–3091. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Carman G. M., Matas J. Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae. Can J Microbiol. 1981 Nov;27(11):1140–1149. doi: 10.1139/m81-179. [DOI] [PubMed] [Google Scholar]
  8. Carson M. A., Atkinson K. D., Waechter C. J. Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium. J Biol Chem. 1982 Jul 25;257(14):8115–8121. [PubMed] [Google Scholar]
  9. Cheng H. C., Kemp B. E., Pearson R. B., Smith A. J., Misconi L., Van Patten S. M., Walsh D. A. A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. J Biol Chem. 1986 Jan 25;261(3):989–992. [PubMed] [Google Scholar]
  10. Chock P. B., Rhee S. G., Stadtman E. R. Interconvertible enzyme cascades in cellular regulation. Annu Rev Biochem. 1980;49:813–843. doi: 10.1146/annurev.bi.49.070180.004121. [DOI] [PubMed] [Google Scholar]
  11. Cobon G. S., Crowfoot P. D., Linnane A. W. Biogenesis of mitchondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions. Biochem J. 1974 Nov;144(2):265–275. doi: 10.1042/bj1440265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Corbin J. D., Reimann E. M. Assay of cyclic AMP-dependent protein kinases. Methods Enzymol. 1974;38:287–290. doi: 10.1016/0076-6879(74)38044-5. [DOI] [PubMed] [Google Scholar]
  13. Culbertson M. R., Henry S. A. Inositol-requiring mutants of Saccharomyces cerevisiae. Genetics. 1975 May;80(1):23–40. doi: 10.1093/genetics/80.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haid A., Suissa M. Immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:192–205. doi: 10.1016/s0076-6879(83)96017-2. [DOI] [PubMed] [Google Scholar]
  15. Hartzell H. C., Glass D. B. Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases. J Biol Chem. 1984 Dec 25;259(24):15587–15596. [PubMed] [Google Scholar]
  16. Hemmings B. A. Purification and properties of the phospho and dephospho forms of yeast NAD-dependent glutamate dehydrogenase. J Biol Chem. 1980 Aug 25;255(16):7925–7932. [PubMed] [Google Scholar]
  17. Henry S. A., Atkinson K. D., Kolat A. I., Culbertson M. R. Growth and metabolism of inositol-starved Saccharomyces cerevisiae. J Bacteriol. 1977 Apr;130(1):472–484. doi: 10.1128/jb.130.1.472-484.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Homann M. J., Bailis A. M., Henry S. A., Carman G. M. Coordinate regulation of phospholipid biosynthesis by serine in Saccharomyces cerevisiae. J Bacteriol. 1987 Jul;169(7):3276–3280. doi: 10.1128/jb.169.7.3276-3280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Homann M. J., Poole M. A., Gaynor P. M., Ho C. T., Carman G. M. Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae. J Bacteriol. 1987 Feb;169(2):533–539. doi: 10.1128/jb.169.2.533-539.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hromy J. M., Carman G. M. Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles. Modulation of activity by phospholipids. J Biol Chem. 1986 Nov 25;261(33):15572–15576. [PubMed] [Google Scholar]
  21. Johnson K. E., Cameron S., Toda T., Wigler M., Zoller M. J. Expression in Escherichia coli of BCY1, the regulatory subunit of cyclic AMP-dependent protein kinase from Saccharomyces cerevisiae. Purification and characterization. J Biol Chem. 1987 Jun 25;262(18):8636–8642. [PubMed] [Google Scholar]
  22. KANFER J., KENNEDY E. P. METABOLISM AND FUNCTION OF BACTERIAL LIPIDS. II. BIOSYNTHESIS OF PHOSPHOLIPIDS IN ESCHERICHIA COLI. J Biol Chem. 1964 Jun;239:1720–1726. [PubMed] [Google Scholar]
  23. Kiyono K., Miura K., Kushima Y., Hikiji T., Fukushima M., Shibuya I., Ohta A. Primary structure and product characterization of the Saccharomyces cerevisiae CHO1 gene that encodes phosphatidylserine synthase. J Biochem. 1987 Nov;102(5):1089–1100. doi: 10.1093/oxfordjournals.jbchem.a122147. [DOI] [PubMed] [Google Scholar]
  24. Klig L. S., Homann M. J., Carman G. M., Henry S. A. Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant. J Bacteriol. 1985 Jun;162(3):1135–1141. doi: 10.1128/jb.162.3.1135-1141.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  26. Kuchler K., Daum G., Paltauf F. Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae. J Bacteriol. 1986 Mar;165(3):901–910. doi: 10.1128/jb.165.3.901-910.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Letts V. A., Henry S. A. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae. J Bacteriol. 1985 Aug;163(2):560–567. doi: 10.1128/jb.163.2.560-567.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Letts V. A., Klig L. S., Bae-Lee M., Carman G. M., Henry S. A. Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7279–7283. doi: 10.1073/pnas.80.23.7279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matsumoto K., Uno I., Ishikawa T. Control of cell division in Saccharomyces cerevisiae mutants defective in adenylate cyclase and cAMP-dependent protein kinase. Exp Cell Res. 1983 Jun;146(1):151–161. doi: 10.1016/0014-4827(83)90333-6. [DOI] [PubMed] [Google Scholar]
  31. Matsumoto K., Uno I., Ishikawa T. Initiation of meiosis in yeast mutants defective in adenylate cyclase and cyclic AMP-dependent protein kinase. Cell. 1983 Feb;32(2):417–423. doi: 10.1016/0092-8674(83)90461-0. [DOI] [PubMed] [Google Scholar]
  32. Matsumoto K., Uno I., Oshima Y., Ishikawa T. Isolation and characterization of yeast mutants deficient in adenylate cyclase and cAMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2355–2359. doi: 10.1073/pnas.79.7.2355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nikawa J., Tsukagoshi Y., Kodaki T., Yamashita S. Nucleotide sequence and characterization of the yeast PSS gene encoding phosphatidylserine synthase. Eur J Biochem. 1987 Aug 17;167(1):7–12. doi: 10.1111/j.1432-1033.1987.tb13297.x. [DOI] [PubMed] [Google Scholar]
  34. Otaka E., Kumazaki T., Matsumoto K. In vivo phosphorylation of Saccharomyces cerevisiae ribosomal protein S10 by cyclic-AMP-dependent protein kinase. J Bacteriol. 1986 Aug;167(2):713–715. doi: 10.1128/jb.167.2.713-715.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pajares M. A., Villalba M., Mato J. M. Purification of phospholipid methyltransferase from rat liver microsomal fraction. Biochem J. 1986 Aug 1;237(3):699–705. doi: 10.1042/bj2370699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Poole M. A., Fischl A. S., Carman G. M. Enzymatic detection of phospholipid biosynthetic enzymes after electroblotting. J Bacteriol. 1985 Feb;161(2):772–774. doi: 10.1128/jb.161.2.772-774.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Poole M. A., Homann M. J., Bae-Lee M. S., Carman G. M. Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors. J Bacteriol. 1986 Nov;168(2):668–672. doi: 10.1128/jb.168.2.668-672.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Raetz C. R., Carman G. M., Dowhan W., Jiang R. T., Waszkuc W., Loffredo W., Tsai M. D. Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast. Biochemistry. 1987 Jun 30;26(13):4022–4027. doi: 10.1021/bi00387a042. [DOI] [PubMed] [Google Scholar]
  39. Rittenhouse J., Moberly L., Marcus F. Phosphorylation in vivo of yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase at the cyclic AMP-dependent site. J Biol Chem. 1987 Jul 25;262(21):10114–10119. [PubMed] [Google Scholar]
  40. Toda T., Cameron S., Sass P., Zoller M., Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987 Jul 17;50(2):277–287. doi: 10.1016/0092-8674(87)90223-6. [DOI] [PubMed] [Google Scholar]
  41. Zinker S., Warner J. R. The ribosomal proteins of Saccharomyces cerevisiae. Phosphorylated and exchangeable proteins. J Biol Chem. 1976 Mar 25;251(6):1799–1807. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES