The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3'-hydroxyl group of phosphatidylinositol (PIs) and phosphoinositides. The generated phospholipids are critical signaling molecules. Based on substrate specificity and sequence homology, PI3Ks are grouped into three classes: Class I, Class II, and Class III. In vivo, Class I PI3Ks are believed to preferentially phosphorylate PtdIns(4,5)P2 to generate PI(3,4,5)P3, a pivotal signaling molecule that activates multiple downstream signaling cascades, including the Akt/TOR pathway. Class III PI3K is composed of a sole member, Vps34, that converts PtdIns to PI(3)P. Vps34 is the only PI3K reported to be evolutionarily conserved from yeast to mammals. An important cellular process controlled by PI3Ks is autophagy, which is involved in many physiological and pathological conditions. The current dogma is that in metazoans, autophagy requires PI(3)P, the product of Class III PI3K Vps34. On the contrary, autophagy is inhibited by PI(3,4,5)P3, the product of Class IA PI3Ks, that mediates activation of the Akt/mTOR pathway. However, the direct role of PI3Ks, especially that of the Class IA PI3Ks, in autophagy remains unclear. Using p110a and p110 conditional knockout mice, we have recently shown that the Class IA p110 isoform is a positive regulator of autophagy, both in cell culture and in vivo. p110 promotes autophagy by activating Vps34 kinase activity and the generation of the autophagy- essential phospholipid PI(3)P. This autophagy-promoting function of p110 is independent of its catalytic activity. These findings prompt us to propose the central hypothesis that the Class IA p110 subunit positively regulates autophagy acting as a molecular scaffold. In this proposal, we plan to study the molecular mechanisms underlying the autophagy-promoting function of p110, and to explore its biological roles. Based on our preliminary data, we propose that p110 may promote autophagy by activating the small GTPase Rab5, which has been recently shown to activate Vps34 and promote autophagy. We also hypothesize that p110 changes its subcellular localization and autophagy-promoting activity in response to trophic factor deprivation. Moreover, although it is well recognized that the Class III PI3K Vps34 plays an essential role in autophagy in yeast, its role in mammals remains elusive. Surprisingly, a recent report showed that autophagosomes still form in Vps34-null sensory neurons, suggesting that the molecular and physiological role of Vps34 in mammalian autophagy needs to be re-examined. Our recent study indicates a molecular connection between p110 and Vps34. Hence in this proposal, we will also use tissue-specific Vps34 knockout mice to study Vps34 and its interplay with p110 in regulating autophagy. Completion of this project will uncover the novel function of p110 as a molecular scaffold to promote autophagy both at basal state and in response to trophic factor availability, and define the role of Vps34 in autophagy in mammals. This will help our understanding to the roles of PI3Ks in regulating cellular homeostasis, metabolism, and their involvement in human diseases such as cancer. PUBLIC HEALTH RELEVANCE: The phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases generating various phospholipids that are critical signaling molecules and control a wide variety of biological processes including autophagy. Both PI3Ks and autophagy are involved in many pathological conditions including degenerative diseases and malignancies. Our study is designed to uncover the physiological roles and molecular mechanisms of PI3Ks in autophagy regulation, and will help with the understanding of the etiology of numerous human diseases as well as with the development of approaches targeting PI3Ks in treating human diseases such as cancer.