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 (Zhou et 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 (Devereaux et al., 2013). Assaying VPS34 activity directly in vitro can be a key stage in delineating the action of a particular stimulus.
Keywords: Phosphatidylinositol-3-phosphate, VPS34, PIK3C3, Lipid kinase assay, TLC
Materials and Reagents
Equipment
Procedure
Representative data
Figure 2. Example data from lipid kinase assay. Recombinant VPS34/VPS15 or Class I PI3Ka was assayed as described in the presence or absence of a pan PI3K inhibitor. Following loading of samples onto a TLC plate and running, the plate was exposed to x-ray film and developed.
Notes
Recipes
Acknowledgments
This work was supported by the Medical Research Council (MRC) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer).
References
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