Skip to main content
SpringerLink
Log in

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

  • Published:
Acta Biologica Hungarica Aims and scope Submit manuscript

Abstract

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Alberts, A. S., Thorburn, A. M., Shenolikar, S., Mumby, M. C., Feramisco, J. R. (1993) Regulation of the cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc. Natl. Acad. Sci. USA 90, 388–392.

    Article  CAS  Google Scholar 

  2. Antoniw, J. F., Cohen, P. (1976) Separation of two Phosphorylase kinase phosphatases from rabbit skeletal muscle. Eur. J. Biochem. 68, 45–54.

    Article  CAS  Google Scholar 

  3. Barford, D. (1996) Molecular mechanisms of the protein serine/threonine phosphatases. TIBS 21, 407–412.

    CAS  PubMed  Google Scholar 

  4. Cohen, P. (1989) The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58, 453–508.

    Article  CAS  Google Scholar 

  5. Cohen, P., Foulkes, G. J., Holmes, C. F. B., Nimmo, G. A., Tonks, N. K. (1988) Protein phosphatase inhibitor-1 and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 159, 427–437.

    Article  CAS  Google Scholar 

  6. Cohen, P., Klump, S., Schelling, D. L. (1989) An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 250, 596–600.

    Article  CAS  Google Scholar 

  7. Davis, R. H., De Serres, F. J. (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol. 17A, 79–143.

    Article  Google Scholar 

  8. Doonan, J. H., Morris, R. N. (1989) The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase-1. Cell 57, 987–996.

    Article  CAS  Google Scholar 

  9. Durfee, T., Becherer, K., Chen, P-L., Yeh, S-H., Yang, Y., Kilburn, A. E., Lee, W-H., Elledge, S. J. (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 7, 555–569.

    Article  CAS  Google Scholar 

  10. Erdődi, F., Csortos, C, Sparks, L., Murányi, A., Gergely, P. (1992) Purification and characterization of three distinct types of protein phosphatase catalytic subunits in bovine platelets. Arch. Biochem. Biophys. 298, 682–687.

    Article  Google Scholar 

  11. Erdődi, F., Rokolya, A., Bárány, M., Bárány, K. (1989) Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle. Biochim. Biophys. Acta 1011, 67–74.

    Article  Google Scholar 

  12. Feng, Z. H., Wilson, S. E., Peng, Z. Y., Schlender, K. K., Reimann, E. M., Trumbly, R. J. (1991) The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. J. Biol. Chem. 266, 23796–23801.

    CAS  PubMed  Google Scholar 

  13. Fischer, E. H., Krebs, E. G. (1962) Muscle Phosphorylase b. Methods Enzymol. 5, 369–373.

    Article  CAS  Google Scholar 

  14. François, J. M., Thompson-Jaeger, S., Skroch, J., Zellenka, U., Spevak, W., Tatchell, K. (1992) GAC1 may encode a regulatory subunit for protein phospharase type 1 in Saccharomyces cerevisiae. EMBO J. 11, 87–96.

    Article  Google Scholar 

  15. Gergely, P., Erdődi, F., Bot, G. (1984) Heparin inhibits the activity of protein phosphatase-1. FEBS Lett. 169, 45–48.

    Article  CAS  Google Scholar 

  16. Higuchi, S., Tamura, J., Giri, P. R., Potti, J. W., Kinkaid, R. L. (1991) Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit. J. Biol. Chem. 266, 18104–18112.

    CAS  PubMed  Google Scholar 

  17. Hisamoto, N., Sugimoto, K., Matsumoto, K. (1994) The GLC7 type-1 protein phosphatase of Saccharomyces cerevisiae is required for cell cycle progression in G2/M. Mol. Cell. Biol. 14, 3158–3165.

    Article  CAS  Google Scholar 

  18. Krebs, E. G., Fischer, E. H. (1962) Phosphorylase b kinase from rabbit skeletal muscle. Methods Enzymol. 5, 373–376.

    Article  CAS  Google Scholar 

  19. Lammli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  Google Scholar 

  20. Lee, E. Y. C., Silberman, S. R., Ganapathi, M. K., Petrovic, S., Paris, H. (1980) The phosphoprotein phosphatases: Properties of the enzymes involved in the regulation of glycogen metabolism. Adv. Cycl. Nucl. Res. 13, 95–131.

    CAS  Google Scholar 

  21. McGowan, C. H., Cohen, P. (1987) Identification of two isoenzymes of protein phosphatase 2C in both rabbit skeletal muscle and liver. Eur. J. Biochem. 166, 713–722.

    Article  CAS  Google Scholar 

  22. Merril, C. R., Dunau, M. L., Goldman, D. (1981) A rapid sensitive silver stain for polypeptides in Polyacrylamide gels. Anal. Biochem. 110, 201–207.

    Article  CAS  Google Scholar 

  23. Prokisch, H., Yarden, O., Kincaid, R., Barthelmess, I-B. (1995) Calcineurin, a calmodulin-dependent protein phosphatase is essential for hyphal elongation and involved in the action of cyclosporin A and FK506 in Neurospora crassa. Fung. Genet. Newsl. 42A, 25.

    Google Scholar 

  24. Read, S. M., Northcote, D. H. (1981) Minimization of variation in the response to different proteins of the Coomassie Blue G dye-binding assay for protein. Anal. Biochem. 116, 53–64.

    Article  CAS  Google Scholar 

  25. Silberman, S. R., Speth, M., Nemani, R., Ganapathi, M. K., Dombrádi, V., Paris, H., Lee, E. Y. C. (1984) Isolation and characterization of rabbit skeletal muscle protein phosphatases CI and CII. J. Biol. Chem. 259, 2913–2922.

    CAS  PubMed  Google Scholar 

  26. Stone, E. M., Yamano, H., Kinoshita, N., Yanagida, M. (1993) Mitotic regulation of protein phosphatases by fission yeast sds22 protein. Curr. Biol. 3, 13–26.

    Article  CAS  Google Scholar 

  27. Szöőr, B., Fehér, Z., Bakó, É., Erdődi, F., Szabó, G., Gergely P., Dombrádi, V. (1995) Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa. Comp. Biochem. Physiol. 112B, 515–522.

    Article  Google Scholar 

  28. Szöőr, B., Fehér, Z., Szabó, G., Gergely, P., Dombrádi, V. (1994) Detection of Ser/Thr protein phosphatases in Neurospora crassa. Fung. Genet. Newsl. 41, 82–84.

    Google Scholar 

  29. Yanagida, M., Kinoshita, N., Stone, E. M., Yamano, H. (1992) Protein phosphatases and cell division cycle control. In: Regulation of the Eukaryotic Cell Cycle (Ciba Foundation Symposium 170). Wiley, Chichester, pp. 130–146.

    Google Scholar 

  30. Yatzkan, E., Yarden, O. (1995) Inactivation of a single type-2A phosphoprotein phosphatase is lethal in Neurospora crassa. Curr. Genet. 28, 458–466.

    Article  CAS  Google Scholar 

  31. Zapella, P. D. A., da-Silva, A. M., da-Costa-Maia, J. C., Terenzi, H. F. (1996) Serine/threonine protein phosphatases and a protein phosphatase inhibitor from Neurospora crassa. Braz. J. Med. Biol. Res. 29, 599–604.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Chemistry, University Medical School of Debrecen, Bern tér 18/B, H-4026, Debrecen, Hungary

    B. Szöőr,  V. Dombrádi & P. Gergely

  2. Department of Biology, University Medical School of Debrecen, Debrecen, Hungary

    Z. Fehér

Authors
  1. B. Szöőr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. V. Dombrádi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. P. Gergely
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Z. Fehér
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Dedicated to the memory of Professor Gábor Szabó.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Szöőr, B., Dombrádi, V., Gergely, P. et al. Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa. BIOLOGIA FUTURA 48, 289–302 (1997). https://doi.org/10.1007/BF03543201

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03543201

Keywords

Search

Navigation