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Analysis of Protein Interactions Between Protein Phosphatase 1 and Noncatalytic Subunits Using the Yeast Two-Hybrid Assay

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Book cover Protein Phosphatase Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 93))

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Abstract

A large variety of methods exist to identify protein-protein interactions. The two hybrid method is unique in that interactions are assayed in vivo. The method can be used “analytically” or “preparatively.” In the analytical application, one surveys interactions between two proteins known to interact by comparing interaction of mutant proteins. The preparative two hybrid method allows one to identify putative proteins that interact with a given protein (called the “bait”) by screening libraries. This chapter will focus only on the analytical application of the two hybrid assay to analyze interactions of mutant protein phosphatase 1 (PP1) molecules with some PP1 noncatalytic subunits. For examples of the latter screening method, readers are referred to original papers that employed the method to isolate PP1 interacting proteins (1–3).

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References

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

    Article  PubMed  CAS  Google Scholar 

  2. Tu, J., Song, W., and Carlson, M. (1996) Protein phosphatase type 1 interacts with proteins required for meiosis and other cellular processes in Saccharomyces cerevisiae. Mol. Cell Biol 16, 4199–4206.

    PubMed  CAS  Google Scholar 

  3. Frederick, D L and Tatchell, K (1996) The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth Mol Cell Biol 16, 2922–2931

    PubMed  CAS  Google Scholar 

  4. Fields, S and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.

    Article  PubMed  CAS  Google Scholar 

  5. Dang, C V, Barrett, J., Villa-Garcia, M, Resar, L. M, Kato, G J, and Fearon, E R (1991) Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization Mol Cell Biol 11, 954–962

    PubMed  CAS  Google Scholar 

  6. Johnston, M (1987) A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae Microbiol Rev 51, 458–476

    PubMed  CAS  Google Scholar 

  7. Cannon, J. F., Pringle, J. R., Fiechter, A, and Khalil, M (1994) Characterization of glycogen-deficient glc mutants of Saccharomyces cerevisiae Genetics 136, 485–503

    PubMed  CAS  Google Scholar 

  8. Hisamoto, N, Sugimoto, K, and 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

    PubMed  CAS  Google Scholar 

  9. Cannon, J. F., Clemens, K. E., Morcos, P. A., Nair, B M., Pearson, J L, and Khalil, M (1995) Type 1 protein phosphatase systems of yeast Adv Prot Phosphatases 9, 215–236

    Google Scholar 

  10. Feng, Z., Wilson, S. E., Peng, Z.-Y., Schelnder, K K, Reimann, E M, and Trumbly, R J (1991) The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. J. Biol. Chem 226, 23,796–23,801

    Google Scholar 

  11. Wek, R C, Cannon, J F, Dever, T E, and Hinnebusch, A. G (1992) Truncated protein phosphatase GLC7 restores translational activation of GCN4 expression in yeast mutants defective for the eIF-2 alpha kinase GCN2. Mol Cell Biol 12, 5700–5710

    PubMed  CAS  Google Scholar 

  12. Black, S, Andrews, P D., Sneddon, A. A, and Stark, M J (1995) A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1 Yeast 11, 747–759.

    Article  PubMed  CAS  Google Scholar 

  13. Tu, J. and Carlson, M. (1994) The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae Mol Cell Biol 14, 6789–6796

    PubMed  CAS  Google Scholar 

  14. Tu, J. and Carlson, M (1995) REG 1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae EMBO J 14, 5939–5946

    PubMed  CAS  Google Scholar 

  15. Francois, J M, Thompson-Jaeger, S., Skroch, J, Zellenka, V, Spevak, W, and Tatchell, K (1992) GAC1 may encode a regulatory subunit for protein phosphatase type 1 in Saccharomyces cerevisiae EMBO J 11, 87–96

    PubMed  CAS  Google Scholar 

  16. Tang, P. M., Bondor, J A, Swiderek, K M, and DePaoli-Roach, A A (1991) Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase. J Biol Chem 266, 15782–15789

    PubMed  CAS  Google Scholar 

  17. Foulkes, J G and Cohen, P. (1980) The regulation of glycogen metabolism Purification and properties of protein phosphatase mhibitor-2 from rabbit skeletal muscle. Eur J Biochem 105, 195–203

    Article  PubMed  CAS  Google Scholar 

  18. MacKelvie, S H, Andrews, P. D, and Stark, M J (1995) The Saccharomyces cerevisiae gene SDS22 encodes a potential regulator of the mitotic function of yeast type 1 protein phosphatase Mol Cell Biol 15, 3777–3785

    PubMed  CAS  Google Scholar 

  19. Hisamoto, N., Frederick, D. L, Sugimoto, K., Tatchell, K., and Matsumoto, K (1995) The EGP1 gene may be a positive regulator of protein phosphatase type 1 in the growth control of Saccharomyces cerevisiae Mol Cell Biol 15, 3767–3776

    PubMed  CAS  Google Scholar 

  20. Renouf, S, Beullens, M, Wera, S, Van, E A, Sikela, J, Stalmans, W, and Bollen, M (1995) Molecular cloning of a human polypeptide related to yeast sds22, a regulator of protein phosphatase-1 FEBS Lett 375, 75–78.

    Article  PubMed  CAS  Google Scholar 

  21. Ramaswamy, N T, Li, L, Khalil, M, and Cannon, J. F (1997) Regulation of yeast glycogen metabolism and separation by Glc7p protein phosphatase Genetics (submitted).

    Google Scholar 

  22. Dalley, B. K. and Cannon, J F (1996) Novel, activated RAS mutations alter protein-protein interactions. Oncogene 13, 1209–1220

    PubMed  CAS  Google Scholar 

  23. Goldberg, J., Huang, H B, Kwon, Y. G., Greengard, P, Nairn, A. C, and Kunyan, J (1995) Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-1 Nature 376, 745–753

    Article  PubMed  CAS  Google Scholar 

  24. 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

    Article  PubMed  CAS  Google Scholar 

  25. Yang, X., Hubbard, E. J. A, and Carlson, M (1992) A protein kinase substrate identified by the two-hybrid system Science 257, 680–682

    Article  PubMed  CAS  Google Scholar 

  26. Elble, R (1992) A simple and efficient procedure for transformation of yeasts Bio Techniques 13, 18–20

    CAS  Google Scholar 

  27. Breeden, L. and Nasmyth, K (1985) Regulation of the yeast HO gene Cold Spring Harb Symp Quant Biol 50, 643–650

    PubMed  CAS  Google Scholar 

  28. Yocum, R. R., Hanley, S., West, R, and Ptashne, M (1984) Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae Mol. Cell Biol. 4, 1985–1998

    PubMed  CAS  Google Scholar 

  29. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  30. Bartel, P. L and Fields, S. (1995) Analyzing protein-protein interactions using two-hybrid system Meth Enzymol 254, 241–263

    Article  PubMed  CAS  Google Scholar 

  31. Stuart, J S, Frederick, D L., Varner, C. M., and Tatchell, K. (1994) The mutant type 1 protein phosphatase encoded by glc7−1 from Saccharomyces cerevisiae fails to interact productively with the GAC1-encoded regulatory subunit Mol Cell Biol 14, 896–905

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO

    Nadja T. Ramaswamy, Brian K. Dalley & John F. Cannon

Authors
  1. Nadja T. Ramaswamy
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  2. Brian K. Dalley
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  3. John F. Cannon
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Editor information

Editors and Affiliations

  1. University of Rochester Cencer Center, Rochester, NY

    John W. Ludlow

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© 1998 Humana Press Inc.

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Ramaswamy, N.T., Dalley, B.K., Cannon, J.F. (1998). Analysis of Protein Interactions Between Protein Phosphatase 1 and Noncatalytic Subunits Using the Yeast Two-Hybrid Assay. In: Ludlow, J.W. (eds) Protein Phosphatase Protocols. Methods in Molecular Biology™, vol 93. Humana Press. https://doi.org/10.1385/0-89603-468-2:251

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  • DOI: https://doi.org/10.1385/0-89603-468-2:251

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-468-6

  • Online ISBN: 978-1-59259-267-8

  • eBook Packages: Springer Protocols

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