Phosphatidylinositol 3-kinases and autophagy

描述(申请人提供)：磷脂酰肌醇3-激酶(PI3K)是使磷脂酰肌醇(PI)和磷脂酰肌醇的3‘-羟基磷酸化的脂酶。生成的磷脂是关键的信号分子。根据底物的特异性和序列的同源性，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来产生PI(3，4，5)P3，PI(3，4，5)P3是一个关键的信号分子，激活包括Akt/TOR通路在内的多个下游信号级联反应。第三类PI3K由一个唯一的成员Vps34组成，它将PtdIns转化为PI(3)P。Vps34是唯一被报道从酵母到哺乳动物在进化上保守的PI3K。PI3Ks控制的一个重要的细胞过程是自噬，它参与了许多生理和病理条件。目前的教条是，在后生动物中，自噬需要PI(3)P，即III类PI3K Vps34的乘积。相反，自噬被IA类PI3Ks的产物PI(3，4，5)P3抑制，PI(3，4，5)P3介导Akt/mTOR途径的激活。然而，PI3K，尤其是IA类PI3K在自噬中的直接作用仍不清楚。利用p110a和p110条件基因敲除小鼠，我们最近证明，无论是在细胞培养中还是在体内，IA类p110异构体都是自噬的正调控因子。P110？通过激活Vps34激酶活性和产生自噬必需的磷脂PI(3)P来促进自噬。这种自噬促进功能不依赖于其催化活性。这些发现促使我们提出了中心假设，即IA类p110？亚基作为分子支架积极调节自噬。在这个方案中，我们计划研究p110促进自噬功能的分子机制，并探索其生物学作用。根据我们的初步数据，我们认为p110可能通过激活小的GTP酶Rab5来促进自噬，该酶最近被证明激活Vps34并促进自噬。我们还假设，在营养因子剥夺的情况下，p110会改变其亚细胞定位和自噬促进活性。此外，尽管众所周知，III类PI3K Vps34在酵母的自噬中起着重要的作用，但它在哺乳动物中的作用仍然难以捉摸。令人惊讶的是，最近的一份报告显示，Vps34缺失的感觉神经元中仍然形成自噬小体，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，在这项提案中，我们还将使用组织特异性Vps34基因敲除小鼠来研究Vps34及其与p110在调节自噬方面的相互作用。该项目的完成将揭示P110作为分子支架的新功能，以促进基础状态下的自噬和响应营养因子的可获得性，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在调节细胞内稳态、新陈代谢中的作用，以及它们参与人类疾病(如癌症)的过程。

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.