The lipid kinase VPS34 is an essential mediator of multiple aspects of intracellular trafficking via the formation of phosphoinositide-3-phosphate (PI(3)P) on membranes, this is critical to mediate efficient trafficking of cargo by endocytosis. In addition, VPS34 kinase activity is essential for inducing autophagy in combination with the protein kinase ULK1. Autophagy acts as a catabolic pathway that is up regulated in response to stress, but is negatively regulated by the master growth protein kinase mTOR. This study sought to identify novel control mechanisms that may mediate the regulation of VPS34 between the pathways of endocytosis and autophagy. During nutrient rich conditions a binding protein of VPS34, UVRAG, was identified to be phosphorylated. Further analysis has identified that phosphorylation is mediated by mTOR and that this occurs at two sites, S550 and S571. Multiple lines of evidence suggest that phosphorylation does not alter the stoichiometry or localisation of the complex nor does it mediate recruitment of additional factors. Phosphorylation of UVRAG acts to increase lipid kinase activity in vitro and cellular PI(3)P levels by ~ 2 fold, mutation of S550 and S571 to alanine residues abrogate this increase in activity. Examination of autophagy, receptor mediated endocytosis and recycling have demonstrated no effect of UVRAG phosphorylation upon their rate of trafficking. Mutation of UVRAG phosphorylation sites however leads to a significant lysosome abnormality that is demonstrated by a dispersed phenotype. Preliminary analysis suggests that this may occur due to abnormalities in the process of autophagic lysosome reformation, a process that is dependent upon the activity of mTOR. This suggests a previously uncharacterised role of PI(3)P in lysosomal regulation and adds to current understanding of regulation between VPS34 and mTOR. Additionally data presented here examines a novel PI3KC3 inhibitor that demonstrates profound selectivity over other PI3K isoforms and lipid kinases. This will be important to further examine the functional role of VPS34 and UVRAG.
|Date of Award||2014|
|Supervisor||Ian Ganley (Supervisor)|