In cell and in vitro assays to measure PTEN ubiquitination

Amit Gupta, Helene Maccario, Nisha Kriplani, Nicholas R. Leslie

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Citations (Scopus)

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 prourvival 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).

Original languageEnglish
Title of host publicationPTEN
Subtitle of host publicationMethods and Protocols
PublisherHumana Press
Pages155-165
Number of pages11
ISBN (Electronic)9781493932993
ISBN (Print)9781493932979
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume1388
ISSN (Print)1064-3745

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Gupta, A., Maccario, H., Kriplani, N., & Leslie, N. R. (2016). In cell and in vitro assays to measure PTEN ubiquitination. In PTEN: Methods and Protocols (pp. 155-165). (Methods in Molecular Biology; Vol. 1388). Humana Press. https://doi.org/10.1007/978-1-4939-3299-3_11