Phosphoinositides regulate a wide range of essential cellular processes in almost every cellular compartment. Research conducted to better understand the diverse and widespread influence of this small family of phospholipids has uncovered unique signalling functions for each phosphoinositide however the most recently discovered phosphoinositide, PtdIns5P, remains poorly understood. Since its discovery there have been numerous enzymes implicated in the metabolism of PtdIns5P but exactly where and how PtdIns5P is made is not completely understood. The importance of PtdIns5P has been highlighted in publications that have revealed its involvement in regulating signal transduction pathways and cellular processes in various cellular compartments. The initial aim of the project was to uncover novel regulators of oxidative stress-induced PtdIns5P using small molecular weight inhibitors targeted against pathways activated in response to oxidative stress and a library of shRNA constructs targeted against phosphoinositide-related enzymes and co-enzymes. Using these two approaches several PtdIns5P regulators were identified including PTEN, the PI3K pathway and the NAD+/NADH pathway. Similarly to previously published work, PIP4K was shown to negatively regulate H2O2-induced PtdIns5P and in particular PIP4K2A was shown to be important.Numerous groups have described a role for PtdIns5P in the regulation of PKB signalling. Activated PKB is responsible for phosphorylating many downstream targets in order to initiate cell signalling pathways involved in cell survival, growth and proliferation and consequently the regulation of PKB is frequently altered in many types of cancers. The role of PtdIns5P in the regulation of PKB activation was examined using differential expression of PIP4K2A to manipulate the cellular level of PtdIns5P. It was shown that increasing the level of PtdIns5P causes enhanced PKB activation and decreasing the level of PtdIns5P suppresses PKB activation. Furthermore this is likely to be occurring through the regulation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2, two lipids that can mediate PKB activation at the plasma membrane. Interestingly, this effect seems to be specific to oxidative stress as insulin stimulation led to increased PtdIns(3,4,5)P3 and PtdIns(3,4)P2 however this did not translate to an increase in PKB activation and importantly, insulin was unable to induce an increase in PtdIns5P suggestive of a vital role for PtdIns5P in the activation of PKB. Further work suggested PtdIns5P may regulate the ubiquitination of PTEN. Finally, analysis of multiple signalling molecules that impinge on the PKB pathway showed that PtdIns5P may be able to maintain several signalling molecules in a phosphorylated and therefore active state in order to enhance PKB activation.
|Date of Award||1 Aug 2015|
- The University of Manchester
|Supervisor||Angeliki Malliri (Supervisor) & Nullin Divecha (Supervisor)|