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PTEN pp 155–165Cite as

In Cell and In Vitro Assays to Measure PTEN Ubiquitination

Part of the Methods in Molecular Biology book series (MIMB,volume 1388)

Abstract

The lipid and protein tyrosine phosphatase, PTEN, is one of the most frequently mutated tumor suppressors in human cancers and is essential for regulating the oncogenic pro-survival PI3K/AKT signaling pathway. Because of its diverse physiological functions, PTEN has attracted great interest from researchers in multiple research fields. The functional diversity of PTEN demands a collection of delicate regulatory mechanisms, including transcriptional control and posttranslational mechanisms that include ubiquitination. Addition of ubiquitin to PTEN can have several effects on PTEN function, potentially regulating its stability, localization, and activity. In cell and in vitro ubiquitination assays are employed to study the ubiquitination-mediated regulation of PTEN. However, PTEN ubiquitination assays are challenging to perform and the data published from these assays has been of mixed quality. Here we describe protocols to detect PTEN ubiquitination in cultured cells expressing epitope tagged ubiquitin (in cell PTEN ubiquitination assay) and also using purified proteins (in vitro PTEN ubiquitination assay).

Key words

  • Phosphatase
  • PTEN
  • Ubiquitin
  • Phosphoinositide
  • Cancer
  • Tumor suppressor

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References

  1. Komander D, Rape M (2012) The ubiquitin code. Ann Rev Biochem 81:203–229

    CAS  CrossRef  PubMed  Google Scholar 

  2. Popovic D, Vucic D, Dikic I (2014) Ubiquitination in disease pathogenesis and treatment. Nat Med 20:1242–1253

    CAS  CrossRef  PubMed  Google Scholar 

  3. Dikic I, Robertson M (2012) Ubiquitin ligases and beyond. BMC Biol 10:22–24

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  4. Leslie NR, Batty IH, Maccario H et al (2008) Understanding PTEN regulation: PIP2, polarity and protein stability. Oncogene 27:5464–5476

    CAS  CrossRef  PubMed  Google Scholar 

  5. Alimonti A, Carracedo A, Clohessy JG et al (2010) Subtle variations in Pten dose determine cancer susceptibility. Nat Genet 42:454–458

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Trotman LC, Wang X, Alimonti A et al (2007) Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128:141–156

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Wang X, Trotman LC, Koppie T et al (2007) NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128:129–139

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. Song MS, Salmena L, Carracedo A et al (2008) The deubiquitinylation and localization of PTEN are regulated by a HAUSP-PML network. Nature 455:813–817

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Zhang J, Zhang P, Wei Y et al (2013) Deubiquitylation and stabilization of PTEN by USP13. Nat Cell Biol 15:1486–1494

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  10. Van Themsche C, Leblanc V, Parent S et al (2009) X-linked inhibitor of apoptosis protein (XIAP) regulates PTEN ubiquitination, content, and compartmentalization. J Biol Chem 284:20462–20466

    CrossRef  PubMed  PubMed Central  Google Scholar 

  11. Maddika S, Kavela S, Rani N et al (2011) WWP2 is an E3 ubiquitin ligase for PTEN. Nat Cell Biol 13:728–733

    CrossRef  PubMed  PubMed Central  Google Scholar 

  12. Maccario H, Perera NM, Gray A et al (2010) Ubiquitination of PTEN (phosphatase and tensin homolog) inhibits phosphatase activity and is enhanced by membrane targeting and hyperosmotic Stress. J Biol Chem 285:12620–12628

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  13. McConnachie G, Pass I, Walker SM et al (2003) Interfacial kinetic analysis of the tumour suppressor phosphatase, PTEN: evidence for activation by anionic phospholipids. Biochem J 371:947–955

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We would like to thank the members of the NRL for constructive discussions. This work was funded by the Medical Research Council (grant code G0801865).

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Correspondence to Nicholas R. Leslie .

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Gupta, A., Maccario, H., Kriplani, N., Leslie, N.R. (2016). In Cell and In Vitro Assays to Measure PTEN Ubiquitination. In: Salmena, L., Stambolic, V. (eds) PTEN. Methods in Molecular Biology, vol 1388. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3299-3_11

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  • DOI: https://doi.org/10.1007/978-1-4939-3299-3_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3297-9

  • Online ISBN: 978-1-4939-3299-3

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