BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

The nonconserved N-terminus of protein phosphatases 1 influences its active site

  • Xie, XiuJie (Tianjin Key Laboratory of Environmental Remediation and Pollution Control/College of Environmental Science and Engineering, Nankai University) ;
  • Huang, Wei (Department of Biochemistry and Molecular Biology, Beijing Normal University) ;
  • Xue, ChengZhe (Department of Biochemistry and Molecular Biology, Beijing Normal University) ;
  • Wei, Qun (Department of Biochemistry and Molecular Biology, Beijing Normal University)
  • Published : 2008.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Protein phosphatase 1 consists of a secondary structure arrangement, conserved in the serine/threonine protein phosphatase gene family, flanked by nonconserved N-terminal and C-terminal domains. The deletion mutant of PP1 with the 8 nonconserved N-terminal residues removed was designated PP1-(9-330). PP1-(9-330) had a higher activity and affinity than PP1 when assayed against four different substrates, and it also demonstrated a 6-fold higher sensitivity to the inhibitor okadaic acid. This suggested that the N-terminal domain suppresed the activity of PP1 and interfered with its inhibition by okadaic acid. The ANS fluorescence intensity of PP1-(9-330) was greater than that of PP1, which implies that the hydrophobic groove running from active site in the truncated PP1 was more hydrophobic than in PP1. Our findings provide evidence that the nonconserved N-terminus of PP1 functions as an important regulatory domain that influences the active site and its pertinent properties.

Keywords

References

  1. Cohen, P.T.W. (2002) Protein phosphatase 1 - targeted in many directions. J.Cell Sci.115, 241-256.
  2. Terrak, M., Kerff, F., Langsetmo, K., Tao, T. and Dominguez, R. (2004) Structural basis of protein phosphatase 1 regulation. Nature 429, 780-784. https://doi.org/10.1038/nature02582
  3. Watanabe, T., Huang, H. B., Horiuchi, A., Silva, E. F., Wilson, L. H., Allen, P. B., Shenolikar, S., Greengard, P. and Nairn, A. C. (2001) Protein phosphatase 1 regulation by inhibitors and targeting subunits. PNAS. 98, 3080- 3085. https://doi.org/10.1073/pnas.051003898
  4. Gibbons, J. A., Weiser, D. C. and Shenolikar, S. (2005) Importance of a surface hydrophobic pocket on protein phosphatase-1 catalytic subunit in recognizing cellular regulators. J. Biol. Chem. 280, 15903-15911. https://doi.org/10.1074/jbc.M500871200
  5. Colbran, R. J., Carmody, L. C., Bauman, P. A., Wadzinski, B. E. and Bass, M. A. (2003) Analysis of specific interactions of native protein phosphatase 1 isoforms with targeting subunits. Methods Enzymol. 366, 156-175. https://doi.org/10.1016/S0076-6879(03)66014-3
  6. Maynes, J. T., Bateman, K. S., Cherney, M. M., Das, A. K., Luu, H. A., Holmes, C. F. B. and James, M. N. G. (2001) Crystal Structure of the Tumor-promoter Okadaic Acid Bound to Protein Phosphatase-1. J. Biol. Chem. 276, 44078-44082. https://doi.org/10.1074/jbc.M107656200
  7. Goldberg, J., Huang, H., Kwon, Y., Greengard, P., Nairn, A. and Kuriyan, J. (1995) Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase- 1. Nature 376, 745-753. https://doi.org/10.1038/376745a0
  8. Egloff, M. P., Cohen, P. T. W., Reinemer, P. and Barford, D. (1995) Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J. Mol. Biol. 254, 942-959.
  9. Xing, Y., Xu, Y., Chen, Y., Jeffrey, P. D., Chao, Y., Lin, Z., Li, Z., Strack, S., Stock, J. B. and Shi Y. (2006) Structure of Protein Phosphatase 2A Core Enzyme Bound to Tumor-Inducing Toxins. Cell 127, 341-353. https://doi.org/10.1016/j.cell.2006.09.025
  10. Barton, G. J., Cohen, P. T. W. and Barford, D. (1994) Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Eur. J. Biochem. 220, 225-237. https://doi.org/10.1111/j.1432-1033.1994.tb18618.x
  11. Xie, X. J., Xue, C. Z., Huang, W., Yu, D.Y. and Wei, Q. (2006) The $\beta_{12}-\beta_{13}$ loop is a key regulatory element for the activity and properties of the catalytic domain of protein phosphatase 1 and 2B. Biol. Chem. 387, 1461-1467. https://doi.org/10.1515/BC.2006.183
  12. Xie, X. J., Xue, C. Z., Huang, W. and Wei, Q. (2006)The $\beta_{12}-\beta_{13}$ loop of Protein Phosphatase-1 is Involved in Activity Regulation. IUBMB. Life 58, 487-492. https://doi.org/10.1080/15216540600838224
  13. Liu, P., Huang, C., Wang, H.L., Zhou, K., Xiao, F.X. and Wei, Q. (2004) The importance of loop 7 for the activity of calcineurin. FEBS Lett. 577, 205-208
  14. Liu, P., Huang, C., Jia, Z. C., Yi, F., Yu, D. Y. and Wei, Q. (2005) Non-catalytic domains of subunit A negatively regulate the activity of calcineurin. Biochimie. 87, 215-221. https://doi.org/10.1016/j.biochi.2004.10.009
  15. Maynes, J. T., Perreault, K. R., Cherney, M. M., Luu, H. A., James, M. N. G. and Holmes, C. F. B. (2004) Crystal Structure and Mutagenesis of a Protein Phosphatase-1: Calcineurin Hybrid Elucidate the Role of the $\beta 12-\beta 13$ Loop in Inhibitor Binding. J. Biol. Chem. 279, 43198- 43206. https://doi.org/10.1074/jbc.M407184200
  16. Zhang, Z. J., Zhao, S. M., Bai, G. and Lee, E. Y. C. (1994) Characterization of deletion mutants of the catalytic subunit of protein phosphatase-1. J. Biol. Chem. 269, 13766- 13700.
  17. Azad, M. A. K., Sawa, Y., Ishikawa, T. and Shibata, H. (2006) Purification and Characterization of Protein Phosphatase 2A from Petals of the Tulip Tulipa gesnerina. BMB reports. 39, 671-676. https://doi.org/10.5483/BMBRep.2006.39.6.671
  18. Zhang, Z., Bai, G., Deans-Zirattu, S. Browener, M. F. and Lee, E. Y. C. (1992) Expression of the catalytic subunit of phosphorylase phosphatase (protein phosphatase-1) in Escherichia coli. J. Biol. Chem. 267, 1484-1490.
  19. Cohen, P., Alemany, S., Hemmings, B., AResink, T.J., Stralfors, P. and Tung, H. Y. L. (1988) Protein phosphatase- 1 and protein phosphatase-2A from rabbit muscle. Methods enzylol. 159, 390-409. https://doi.org/10.1016/0076-6879(88)59039-0
  20. Owen,D. J., Papageorgiou, A. C., Garman, E. F., Noble, M. E. and Johnson, L. N.(1995) Expression, purification and crystallisation of phosphorylase kinase catalytic domain. J. Mol. Biol. 99, 243-250.
  21. Blumenthal, D. K., Takio, K., Hansen, R. S. and Krebs, E G. (1986) Dephosphorylation of CAMP-dependent Protein Kinase Regulatory Subunit (Type11) by Calmodulin-dependent Protein Phosphatase. J. Biol. Chem. 261, 8140- 8145.
  22. Instruction manual for Protein Serine/Threonine Phosphatase (PSP) Assay System provided by NEB Inc.
  23. Wei, Q. and Lee, E. Y. C. (1997) Mutagenesis of the L7loop connecting $\beta$ strand 12 and 13 of calcineurin: evidence for a structure role in activity change. Biochemistry 36, 7418-7424. https://doi.org/10.1021/bi962703s
  24. McCready, T. L, Islam, B. F., Schmitz, F. J., Luu, H. A., Dawson, J. F. and Holmes, C. F. B. (2000) Inhibition of Protein Phosphatase-1 by Clavosines A and B. NOVEL MEMBERS OF THE CALYCULIN FAMILY OF TOXINS. J.Biol.Chem. 275, 4192- 4198. https://doi.org/10.1074/jbc.275.6.4192
  25. Zhang, Z., Bai, G., Shima, S., Zhao, S., Nagao, M. and Lee, E. Y. C. (1993) Expression and characterization of rat protein phosphatases-1 alpha, -1 gamma 1, -1 gamma 2, and -1 delta. Arch. Biochem. Biophys. 303, 402-406. https://doi.org/10.1006/abbi.1993.1301