Influencing Factors of Phosphatidylinositol 3-Kinase Membrane Binding and Catalysis

Abstract

The phosphatidylinositol 3-kinase (PI3K) signalling pathway is one of the most frequently genetically altered pathways in human cancers (Samuels et al., 2004). Class I PI3Ks are lipid kinases that bind to the cell membrane and phosphorylate the lipid substrate, phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), in order to produce the second messenger, PIP3. In turn, this regulates several biological signalling pathways involved in cell growth, proliferation, differentiation, and survival (Qiu et al., 1998; Roche, Koegl, & Courtneidge, 1994; Yao & Cooper, 1995). The work in this thesis explores how different PI(4,5)P2 fatty acyl chain arrangements and specific PI3K amino acids can affect membrane binding interactions and catalysis events for both wild-type (WT) and oncogenic class I PI3Ks. Firstly, the effects of different PI(4,5)P2 lipid species were investigated on PI3K lipid kinase activity using biochemical methods, and on PI3K membrane binding using biophysical methods. The influences of the same PI(4,5)P2 lipid species on WT and oncogenic PI3Ka enzyme membrane binding interactions, and on the physical properties of the membrane alone, were then explored at an atomic level of representation using computational methods in the form of molecular dynamics simulations. Finally, a major known hydrophobic component to PI3Ka WT membrane interactions was explored in a common oncogenic PI3Ka H1047R mutant enzyme, to assess if oncogenic forms of the PI3Ka enzyme rely on the same interaction mechanisms as the WT enzyme. The findings from this study showed that the ability of class I WT and oncogenic PI3K enzymes to phosphorylate PI(4,5)P2 is regulated by different PI(4,5)P2 fatty acyl chains, but PI3K membrane binding interactions are not. Furthermore, it was shown that the presence of a major hydrophobic determinant of the PI3Ka structure is crucial for oncogenic PI3Ka H1047R catalysis, and is required, but not essential, for oncogenic PI3Ka H1047R membrane binding events. These findings ultimately provide insight into the regulation of the class I WT and oncogenic PI3K signalling pathways, and may assist in the future development of tumourselective interventions that target this pathway.