Investigating the potential role of PIP4Ks in PI3K/Akt signalling

Abstract

Class I phosphoinositide 3-kinases (PI3Ks) generate the essential lipid second messenger PtdIns(3,4,5)P3 which plays a key role in the regulation of numerous cellular processes including cell growth and survival, gene transcription, cytoskeletal organisation and glucose metabolism through its downstream effectors and in particular the serine/threonine protein kinase Akt. Therefore, the PI3K/Akt signalling plays a critical regulatory role in diverse cellular processes and its dysregulation is implicated in the pathogenesis of many human diseases including cancer and type 2 diabetes. Overexpression of phosphatidylinositol-5-phosphate 4-kinase β (PIP4Kβ), a lipid kinase which phosphorylates the poorly understood phosphoinositide phospholipid PtdIns5P to produce PtdIns(4,5)P2, has been demonstrated to lead to the attenuation of PtdIns(3,4,5)P3 levels and decreased Akt phosphorylation in insulin-stimulated cells. Conversely, mice lacking PIP4Kβ exhibit increased insulin-induced Akt phosphorylation, suggesting a potential role of PIP4Ks in the regulation of PI3K/Akt signalling. The aim of this project was to investigate the potential role of PIP4Kalpha, the most active isoform among PIP4Ks, in the regulation of PI3K/Akt signalling and whether its substrate, PtdIns5P, is involved in this regulation.While YM201636, an inhibitor of PIKfyve an enzyme believed to be involved in PtdIns5P production, markedly reduced Akt phosphorylation in PDGF-stimulated NIH/3T3 cells, contrary to expectations, overexpression of PIP4Kalpha in NIH/3T3 and HeLa S3 cells had no effect on PtdIns(3,4,5)P3 levels or Akt phosphorylation upon stimulation with PDGF and IGF-1 respectively. However, PtdIns(3,4,5)P3 levels were significantly decreased in insulin-stimulated L6 myotubes overexpressing PIP4Kalpha, indicating a possible cell type specific modulation of PI3K signalling by PIP4Kalpha. Interestingly, despite the decreased PtdIns(3,4,5)P3 levels, insulin-stimulated Akt phosphorylation remained unaffected in PIP4Kalpha expressing L6 myotubes. PIP4Kalpha overexpression also resulted in increased levels of PtdIns(3,4)P2. This suggests an increased 5-dephosphorylation of PtdIns(3,4,5)P3 through the action of one or more 5-phosphatases. Although the precise 5-phosphatase(s) are not known, the data indicate that this cannot be SHIP2 which has previously been implicated in the regulation of PtdIns(3,4,5)P3 levels in insulin signalling. Taken together, the data presented in this thesis indicate a role for PIP4Kalpha in PI3K signalling in a cell type specific manner. This might have important physiological implications.

Date of Award	1 Aug 2013
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Katherine Hinchliffe (Supervisor)