title: Determination of VPS34/PIK3C3 Activityin vitroUtilising32P-γATP.
abstract: VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhouet al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereauxet al., 2013). Assaying VPS34 activity directlyin vitrocan be a key stage in delineating the action of a particular stimulus.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Munson
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Ganley