Biological Roles and Regulation of Phosphatidylinositol 3-Kinase (PI3K) Isoforms
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Class IA PI3Ks are signaling molecules that control cell survival, growth, proliferation and metabolism. Dysregulated PI3K signaling is found in patients with diseases such as diabetes and cancer. The two aims of my thesis research were (1) to determine the distinct roles of the PI3K isoforms p110Α and p110Β in regulating hepatic lipid and glucose metabolism and (2) to investigate the regulation of p110Α and p110Β by the heterotrimeric G protein GΑ<sub>q</sub>. For aim 1, mice with liver-specific gene deletion of p110Α or p110Β were generated. My studies found that mice lacking hepatic p110Α were largely protected from high-fat diet-induced liver steatosis, whereas p110Β ablation did not attenuate triglyceride accumulation in the liver. The protective effect of p110Α ablation is probably due to decreased liver uptake of long chain fatty acids. High-fat diet-induced increases in mRNA and protein levels of liver fatty acid binding protein were blunted in the p110Α-null liver. On the other hand, mice lacking hepatic p110Β developed glucose intolerance and hyperinsulinemia. Higher cAMP levels and increased expression of adenylyl cyclase 5 correlated with increased gluconeogenesis and glycogenolysis in p110Β<super>-/-</super> hepatocytes. Mice with p110Α-null liver did not exhibit glucose intolerance or hyperinsulinemia. Furthermore, ablation of p110Α decreased insulin signaling in the liver, whereas deletion of p110Β had relatively minor effects on this signaling pathway. Aim 2 of my thesis research investigated the mechanism by which GΑ<sub>q</sub> inhibits PI3Ks. My studies used purified recombinant proteins and fluorescence spectroscopy to demonstrate that GΑ<sub>q</sub> directly binds to p110Α and blocks Ras binding to p110Α. In addition, in vitro PI3K activity assays revealed that GΑ<sub>q</sub> inhibits the four PI3K enzyme complexes p110Α/p85Α, p110Α/p85Β, p110Β/p85Α and p110Β/p85Β. It was determined that GΑ<sub>q</sub> binds to the p85-binding domain of p110Α and does not appear to directly interact with the catalytic domain. Further, I found that GΑ<sub>q</sub> can bind to free p85 in the iSH2 region independently of p110 binding. In summary, findings from my thesis research indicate that p110Α and p110Β have differential effects on hepatic lipid and glucose metabolism and that activated GΑ<sub>q</sub> can directly bind and inhibit PI3K complexes.