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).
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References
Komander D, Rape M (2012) The ubiquitin code. Ann Rev Biochem 81:203–229
Popovic D, Vucic D, Dikic I (2014) Ubiquitination in disease pathogenesis and treatment. Nat Med 20:1242–1253
Dikic I, Robertson M (2012) Ubiquitin ligases and beyond. BMC Biol 10:22–24
Leslie NR, Batty IH, Maccario H et al (2008) Understanding PTEN regulation: PIP2, polarity and protein stability. Oncogene 27:5464–5476
Alimonti A, Carracedo A, Clohessy JG et al (2010) Subtle variations in Pten dose determine cancer susceptibility. Nat Genet 42:454–458
Trotman LC, Wang X, Alimonti A et al (2007) Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128:141–156
Wang X, Trotman LC, Koppie T et al (2007) NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell 128:129–139
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
Zhang J, Zhang P, Wei Y et al (2013) Deubiquitylation and stabilization of PTEN by USP13. Nat Cell Biol 15:1486–1494
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
Maddika S, Kavela S, Rani N et al (2011) WWP2 is an E3 ubiquitin ligase for PTEN. Nat Cell Biol 13:728–733
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
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
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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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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