Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1989 Aug 15;262(1):335–339. doi: 10.1042/bj2620335

Identification of high levels of type 1 and type 2A protein phosphatases in higher plants.

C MacKintosh 1, P Cohen 1
PMCID: PMC1133265  PMID: 2554892

Abstract

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.

Full text

PDF
335

Selected References

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

  1. Bennett J. Chloroplast phosphoproteins. Evidence for a thylakoid-bound phosphoprotein phosphatase. Eur J Biochem. 1980 Feb;104(1):85–89. doi: 10.1111/j.1432-1033.1980.tb04403.x. [DOI] [PubMed] [Google Scholar]
  2. Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blowers D. P., Trewavas A. J. Autophosphorylation of plasma membrane bound calcium calmodulin dependent protein kinase from pea seedlings and modification of catalytic activity by autophosphorylation. Biochem Biophys Res Commun. 1987 Mar 13;143(2):691–696. doi: 10.1016/0006-291x(87)91409-4. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  6. Cohen P., Foulkes J. G., Holmes C. F., Nimmo G. A., Tonks N. K. Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 1988;159:427–437. doi: 10.1016/0076-6879(88)59042-0. [DOI] [PubMed] [Google Scholar]
  7. Cohen P., Klumpp S., Schelling D. L. An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 1989 Jul 3;250(2):596–600. doi: 10.1016/0014-5793(89)80803-8. [DOI] [PubMed] [Google Scholar]
  8. Cohen P., Schelling D. L., Stark M. J. Remarkable similarities between yeast and mammalian protein phosphatases. FEBS Lett. 1989 Jul 3;250(2):601–606. doi: 10.1016/0014-5793(89)80804-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Echevarría C., Yidal J., Le Maréchal P., Brulfert J., Ranjeva R., Gadal P. The phosphorylation of Sorghum leaf phosphoenolpyruvate carboxylase is a Ca++-calmodulin dependent process. Biochem Biophys Res Commun. 1988 Sep 15;155(2):835–840. doi: 10.1016/s0006-291x(88)80571-0. [DOI] [PubMed] [Google Scholar]
  11. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  12. Ingebritsen T. S., Stewart A. A., Cohen P. The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur J Biochem. 1983 May 2;132(2):297–307. doi: 10.1111/j.1432-1033.1983.tb07362.x. [DOI] [PubMed] [Google Scholar]
  13. Kuret J., Bell H., Cohen P. Identification of high levels of protein phosphatase-1 in rat liver nuclei. FEBS Lett. 1986 Jul 28;203(2):197–202. doi: 10.1016/0014-5793(86)80741-4. [DOI] [PubMed] [Google Scholar]
  14. Lin P. P. Phosphoprotein Phosphatase of Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES . Plant Physiol. 1980 Sep;66(3):368–374. doi: 10.1104/pp.66.3.368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McGowan C. H., Cohen P. Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+-dependent enzyme. Methods Enzymol. 1988;159:416–426. doi: 10.1016/0076-6879(88)59041-9. [DOI] [PubMed] [Google Scholar]
  16. Polya G. M., Haritou M. Purification and characterization of two wheat-embryo protein phosphatases. Biochem J. 1988 Apr 15;251(2):357–363. doi: 10.1042/bj2510357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ranjeva R., Refeno G., Boudet A. M., Marmé D. Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5222–5224. doi: 10.1073/pnas.80.17.5222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Stewart A. A., Cohen P. Protein phosphatase-2B from rabbit skeletal muscle: a Ca2+-dependent, calmodulin-stimulated enzyme. Methods Enzymol. 1988;159:409–416. doi: 10.1016/0076-6879(88)59040-7. [DOI] [PubMed] [Google Scholar]
  19. Stewart A. A., Hemmings B. A., Cohen P., Goris J., Merlevede W. The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities. Eur J Biochem. 1981 Mar 16;115(1):197–205. doi: 10.1111/j.1432-1033.1981.tb06217.x. [DOI] [PubMed] [Google Scholar]
  20. Strålfors P., Hiraga A., Cohen P. The protein phosphatases involved in cellular regulation. Purification and characterisation of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle. Eur J Biochem. 1985 Jun 3;149(2):295–303. doi: 10.1111/j.1432-1033.1985.tb08926.x. [DOI] [PubMed] [Google Scholar]
  21. Suganuma M., Fujiki H., Suguri H., Yoshizawa S., Hirota M., Nakayasu M., Ojika M., Wakamatsu K., Yamada K., Sugimura T. Okadaic acid: an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1768–1771. doi: 10.1073/pnas.85.6.1768. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES