Skip to main content
SpringerLink
Log in

An In Vivo Assay to Quantify Stable Protein Phosphatase 2A (PP2A) Heterotrimeric Species

  • Protocol
Protein Phosphatase Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 365))

  • 1713 Accesses

Abstract

Protein phosphatase 2A (PP2A) regulates a broad spectrum of cellular processes. The enzyme is, in fact, largely a collection of varied heterotrimeric species composed of a catalytic (C) subunit and regulatory (B-type) subunit bound together by a structural (A) subunit. One important feature of the C subunit is that its carboxy-terminus can be modified by phosphorylation and methylation. The mechanisms that trigger such posttranslational modifications, as well as their consequences, are still under investigation. However, data collected thus far indicate that these modifications alter the binding to B subunits for an AC dimer, thereby affecting the makeup of the PP2A species in the cell. In this chapter, we describe an in vivo assay for assessing stable PP2A heterotrimer formation that is based on specific subcellular localizations of PP2A heterotrimers. This assay can be used to study the impact of a wide variety of alterations (such as mutations and covalent modifications) on PP2A heterotrimer formation. We specifically describe the use of this assay to quantify the effects of methylation on the stable formation of PP2ARts1p and PP2ACdc55p heterotrimers.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mumby, M. C. and Walter, G. (1993) Protein serine/threonine phosphatases: structure, regulation, and functions in cell growth. Physiol. Rev. 73, 673–699.

    PubMed  CAS  Google Scholar 

  2. Schonthal, A. H. (1998) Role of PP2A in intracellular signal transduction pathways. Front. Biosci. 3, D1262–D1273.

    PubMed  CAS  Google Scholar 

  3. Millward, T. A., Zolnierowicz, S., and Hemmings, B. A. (1999) Regulation of protein kinase cascades by protein phosphatase 2A. Trends Biochem. Sci. 24, 186–191.

    Article  PubMed  CAS  Google Scholar 

  4. Virshup, D. M. (2000) Protein phosphatase 2A: a panoply of enzymes. Curr. Opin. Cell. Biol. 12, 180–185.

    Article  PubMed  CAS  Google Scholar 

  5. Janssens, V. and Goris, J. (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417–439.

    Article  PubMed  CAS  Google Scholar 

  6. van Zyl, W., Huang, W., Sneddon, A. A., et al. (1992) Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 4946–4959.

    PubMed  Google Scholar 

  7. van Zyl, W. H., Wills, N., and Broach, J. R. (1989) A general screen for mutant of Saccharomyces cerevisiae deficient in tRNA biosynthesis. Genetics 123, 55–68.

    PubMed  Google Scholar 

  8. Ronne, H., Carlberg, M., Hu, G. Z., and Nehlin, J. O. (1991) Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol. Cell. Biol. 11, 4876–4884.

    PubMed  CAS  Google Scholar 

  9. Healy, A. M., Zolnierowicz, S., Stapleton, A. E., Goebl, M., DePaoli-Roach, A. A., and Pringle, J. R. (1991) CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase. Mol. Cell. Biol. 11, 5767–5780.

    PubMed  CAS  Google Scholar 

  10. Shu, Y. and Hallberg, R. L. (1995) SCS1, a multicopy suppressor of hsp60-ts mutant alleles, does not encode a mitochondrially targeted protein. Mol. Cell. Biol. 15, 5618–5626.

    PubMed  CAS  Google Scholar 

  11. Shu, Y., Yang, H., Hallberg, E., and Hallberg, R. (1997) Molecular genetic analysis of Rts1p, a B′ regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A. Mol. Cell. Biol. 17, 3242–3253.

    PubMed  CAS  Google Scholar 

  12. Chen, J., Martin, B. L., and Brautigan, D. L. (1992) Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257, 1261–1264.

    Article  PubMed  CAS  Google Scholar 

  13. Guo, H. and Damuni, Z. (1993) Autophosphorylation-activated protein kinase phosphorylates and inactivates protein phosphatase 2A. Proc. Natl. Acad. Sci. USA 90, 2500–2504.

    Article  PubMed  CAS  Google Scholar 

  14. Turowski, P., Fernandez, A., Favre, B., Lamb, N. J., and Hemmings, B. A. (1995) Differential methylation and altered conformation of cytoplasmic and nuclear forms of protein phosphatase 2A during cell cycle progression. .J Cell. Biol. 129, 397–410.

    Article  PubMed  CAS  Google Scholar 

  15. Kowluru, A., Seavey, S. E., Rabaglia, M. E., Nesher, R., and Metz, S. A. (1996) Carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting cells: evidence for functional consequences on enzyme activity and insulin secretion. Endocrinology 137, 2315–2323.

    Article  PubMed  CAS  Google Scholar 

  16. Bryant, J. C., Westphal, R. S., and Wadzinski, B. E. (1999) Methylated C-terminal leucine residue of PP2A catalytic subunit is important for binding of regulatory Balpha subunit. Biochem. J. 339(Pt. 2), 241–246.

    Article  PubMed  CAS  Google Scholar 

  17. Gentry, M. S., Li, Y., Wei, H., et al. (2005) A Novel Assay for Protein Phosphatase 2A (PP2A) Complexes In Vivo Reveals Differential Effects of Covalent Modifications on Different Saccharomyces cerevisiae PP2A Heterotrimers. Eukaryot. Cell. 4, 1029–1040.

    Article  PubMed  CAS  Google Scholar 

  18. Wei, H., Ashby, D. G., Moreno, C. S., et al. (2001) Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p. J. Biol. Chem. 276, 1570–1577.

    Article  PubMed  CAS  Google Scholar 

  19. Wu, J., Tolstykh, T., Lee, J., Boyd, K., Stock, J. B., and Broach, J. R. (2000) Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. EMBO J. 19, 5672–5681.

    Article  PubMed  CAS  Google Scholar 

  20. Tolstykh, T., Lee, J., Vafai, S., and Stock, J. B. (2000) Carboxyl methylation regulates phosphoprotein phosphatase 2A by controlling the association of regulatory B subunits. EMBO J. 19, 5682–5691.

    Article  PubMed  CAS  Google Scholar 

  21. Yu, X. X., Du, X., Moreno, C. S., et al. (2001) Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen. Mol. Biol. Cell. 12, 185–199.

    PubMed  CAS  Google Scholar 

  22. Gentry, M. S. and Hallberg, R. L. (2002) Localization of Saccharomyces cerevisiae protein phosphatase 2A subunits throughout mitotic cell cycle. Mol. Biol. Cell. 13, 3477–3492.

    Article  PubMed  CAS  Google Scholar 

  23. Ogris, E., Gibson, D. M., and Pallas, D. C. (1997) Protein phosphatase 2A subunit assembly: the catalytic subunit carboxy terminus is important for binding cellular B subunit but not polyomavirus middle tumor antigen. Oncogene 15, 911–917.

    Article  PubMed  CAS  Google Scholar 

  24. Dobbelaere, J., Gentry, M. S., Hallberg, R. L., and Barral, Y. (2003) Phosphorylation-dependent regulation of septin dynamics during the cell cycle. Dev. Cell 4, 345–357.

    Article  PubMed  CAS  Google Scholar 

  25. Rines, D. R., He, X., and Sorger, P. K. (2002) Quantitative microscopy of green fluorescent protein-labeled yeast. Methods Enzymol. 351, 16–34.

    Article  PubMed  CAS  Google Scholar 

  26. Stotz, A. and Linder, P. (1990) The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors. Gene 95, 91–98.

    Article  PubMed  CAS  Google Scholar 

  27. Sherman, F. (2002) Getting started with yeast. Methods Enzymol. 350, 3–41.

    Article  PubMed  CAS  Google Scholar 

  28. Tatchell, K. and Robinson, L. C. (2002) Use of green fluorescent protein in living yeast cells. Methods Enzymol. 351, 661–683.

    Article  PubMed  CAS  Google Scholar 

  29. Gietz, R. D. and Sugino, A. (1988) New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534.

    Article  PubMed  CAS  Google Scholar 

  30. Sikorski, R. S. and Hieter, P. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.

    PubMed  CAS  Google Scholar 

  31. Cai, T., Reilly, T. R., Cerio, M., and Schmitt, M. E. (1999) Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation. Mol. Cell. Biol. 19, 7857–7869.

    PubMed  CAS  Google Scholar 

  32. Fox, T. D., Folley, L. S., Mulero, J. J., et al. (1991) Analysis and manipulation of yeast mitochondrial genes. Methods Enzymol. 194, 149–165.

    Article  PubMed  CAS  Google Scholar 

  33. Davis, L. I. and Fink, G. R. (1990) The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell 61, 965–978.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology, University of California, San Diego, California, USA

    Matthew S. Gentry

  2. Department of Biology, Syracuse University, Syracuse, New York, USA

    Richard L. Hallberg

  3. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA

    David C. Pallas

  4. Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia, USA

    David C. Pallas

Authors
  1. Matthew S. Gentry
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Richard L. Hallberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David C. Pallas
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada

    Greg Moorhead

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc.

About this protocol

Cite this protocol

Gentry, M.S., Hallberg, R.L., Pallas, D.C. (2007). An In Vivo Assay to Quantify Stable Protein Phosphatase 2A (PP2A) Heterotrimeric Species. In: Moorhead, G. (eds) Protein Phosphatase Protocols. Methods in Molecular Biology, vol 365. Springer, Totowa, NJ. https://doi.org/10.1385/1-59745-267-X:71

Download citation

  • DOI: https://doi.org/10.1385/1-59745-267-X:71

  • Publisher Name: Springer, Totowa, NJ

  • Print ISBN: 978-1-58829-711-2

  • Online ISBN: 978-1-59745-267-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation