title: In Cell and In Vitro Assays to Measure PTEN Ubiquitination.
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).
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot Watt University, Edinburgh, EH144AS, UK.
authors.forename: Amit
authors.initials: A
authors.lastname: Gupta
authors.affiliation: Faculte de Pharmacie, Aix Marseille Universite, Marseille, UK.
authors.forename: Helene
authors.initials: H
authors.lastname: Maccario
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot Watt University, Edinburgh, EH144AS, UK.
authors.forename: Nisha
authors.initials: N
authors.lastname: Kriplani
authors.affiliation: Institute of Biological Chemistry, Biophysics and Bioengineering, Heriot Watt University, Edinburgh, EH144AS, UK. n.r.leslie@hw.ac.uk.
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Leslie
mesh_terms: D001671:Biochemistry / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods; D051059:PTEN Phosphohydrolase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D054875:Ubiquitination
chemical_list: D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Cancer; PTEN; Phosphatase; Phosphoinositide; Tumor suppressor; Ubiquitin