Phosphatidylinositol 3-kinases and autophagy

描述（由申请人提供）：磷脂酰肌醇3-激酶（PI3Ks）是磷酸化磷脂酰肌醇（pi）和磷酸肌醇3'-羟基的脂质激酶。生成的磷脂是关键的信号分子。基于底物特异性和序列同源性，pi3k被分为三类：I类、II类和III类。在体内，I类pi3k被认为优先磷酸化PtdIns(4,5)P2以产生PI(3,4,5)P3，这是一种激活多种下游信号级联反应的关键信号分子，包括Akt/TOR通路。III类PI3K由唯一的成员Vps34组成，Vps34将ptdin转换为PI(3)P。Vps34是唯一报道的从酵母到哺乳动物进化保守的PI3K。pi3k控制的一个重要细胞过程是自噬，它参与了许多生理和病理条件。目前的理论认为，在后生动物中，自噬需要PI(3)P，这是III类PI3K Vps34的产物。相反，IA类pi3k的产物PI(3,4,5)P3介导Akt/mTOR通路的激活，可抑制自噬。然而，pi3k，尤其是IA类pi3k在自噬中的直接作用尚不清楚。利用p110a和p110¿条件敲除小鼠，我们最近发现IA类p110¿异构体在细胞培养和体内都是自噬的积极调节因子。p110¿通过激活Vps34激酶活性和产生自噬必需磷脂PI(3)P来促进自噬。p110¿的自噬促进功能独立于其催化活性。这些发现促使我们提出了IA类p110¿亚基作为分子支架积极调节自噬的中心假设。在本研究中，我们计划研究p110¿促进自噬功能的分子机制，并探讨其生物学作用。根据我们的初步数据，我们提出p110¿可能通过激活小GTPase Rab5来促进自噬，而最近的研究表明，小GTPase Rab5可以激活Vps34并促进自噬。我们还假设p110¿改变其亚细胞定位和自噬促进活性，以响应营养因子剥夺。此外，尽管III类PI3K Vps34在酵母的自噬中起重要作用，但其在哺乳动物中的作用尚不清楚。令人惊讶的是，最近的一份报告显示，在Vps34缺失的感觉神经元中仍然可以形成自噬体，这表明Vps34在哺乳动物自噬中的分子和生理作用需要重新研究。我们最近的研究表明p110¿和Vps34之间存在分子联系。因此，在本提案中，我们还将使用组织特异性Vps34敲除小鼠来研究Vps34及其与p110¿在调节自噬中的相互作用。该项目的完成将揭示p110¿作为分子支架在基础状态和营养因子可用性响应下促进自噬的新功能，并确定Vps34在哺乳动物自噬中的作用。这将有助于我们理解pi3k在调节细胞稳态、代谢中的作用，以及它们在人类疾病（如癌症）中的作用。

DESCRIPTION (provided by applicant): 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.