Name: Anna Jadwiga Kielkowska

Dissertation title: The role of PTEN as a PI(3,4)P2 lipid phosphatase in Class I phosphoinositide 3-kinase signalling

Abstract

Class I phosphoinositide 3-kinases (Class I PI3Ks) are essential players involved in the signalling events in the cell and are critical promoters of cellular growth, survival and metabolism. Once activated by environmental stimuli such as growth factors, cytokines or antigens, they exert their catalytic activity by phosphorylating phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to yield a second messenger - PI(3,4,5)P3. Unrestrained PI(3,4,5)P3 signalling has been classically associated with hyperactivation of the Class I PI3K/AKT pathway and has been shown to be a molecular trigger of many pathophysiologies in humans, including autoimmune disorders, respiratory diseases and cancer. To date, two classes of lipid phosphatases SHIP1/2 and PTEN have been reported, which dephosphorylate PI(3,4,5)P3 on positions 5’ and 3’ of the inositol ring to generate PI(3,4)P2 and PI(4,5)P2 respectively, and thus quench Class I PI3K signalling. Moreover, PI(3,4)P2 levels in the cell are regulated by two important lipid 4-phosphatases - INPP4A/B. While the role of PTEN as a tumour suppressor is well established, functions of SHIP1/2 and INPP4A/B are just starting to emerge. A major barrier to progress in this field has been the lack of high quality measurements of PI(3,4)P2, to assess the impact it may have on shaping cellular behaviour. This dissertation summarises the work performed to develop a novel, HPLC-ESI MS/MS based method, in order to measure the product of PI(3,4,5)P3 5-dephosphorylation, PI(3,4)P2, separated from its more abundant regioisomer in cells - PI(4,5)P2. This and an existing HPLC-ESI MS/MS method for measuring PI(3,4,5)P3, have enabled us to describe the fluxes through Class I PI3K-controlled PI(3,4,5)P3 generation and its subsequent 3- and 5- dephosphorylation pathways in human mammary epithelial cells (Mcf10a) stimulated with epidermal growth factor (EGF). By means of genetic suppression of PTEN and INPP4B, we revealed an unexpectedly high level of PI(3,4)P2 that accumulates in EGF-stimulated PTEN-INPP4B-KO Mcf10a cells. Further, an in vitro biochemical assay suggested a novel role for PTEN as a direct PI(3,4)P2 3-phosphatase in Mcf10a cells. This important observation was supported by in sillico phosphatidylinositol lipid modelling of the relevant pathways. In an effort to understand its potential physiological significance, we demonstrated that PI(3,4)P2 accumulation correlates with the ability of genetically modified Mcf10a cells to form gelatin-degrading invadopodia. Finally, we used a mouse prostate cancer model to show PTEN’s importance in controlling PI(3,4)P2 levels in vivo, pointing to a potential role for PI(3,4)P2 in PTEN-dependent tumourigenesis. I hope that the work described in this dissertation will contribute to the current knowledge of phosphatidylinositol lipid biology in the context of Class I PI3K signalling and will simulate future efforts to gain an in-depth understanding of the roles of PTEN and PI(3,4)P2 in cellular physiology.