Physiology of Class III PI 3-kinase Signaling 2

描述（由申请人提供）：自噬是细胞对营养胁迫的反应，其中双壁膜结构的形成将细胞质成分和细胞器隔离并将其传递给溶酶体降解。这释放出的营养物质可用于新的生物合成活动。自噬对小鼠围产期存活至关重要，在神经元、肝细胞和胰腺β细胞的正常组织维持中发挥作用，并有助于对病原体的先天免疫反应。它在病理状态下的激活导致肌肉萎缩等疾病状态，自噬活性的降低可能导致衰老和神经退行性疾病中的神经功能衰退。因此，自噬的药理调节可能具有显著的临床益处。调节自噬的一种方法是通过III类PI 3-激酶hVps34，它是酵母、苍蝇和哺乳动物细胞自噬所必需的。hVps34存在于含有自噬特异性成分（Atg14L）或囊泡运输（UVRAG）的多蛋白复合物中，以及在这两种途径中都起作用的成分（hVps15, beclin-1）。调控hVps34活性的机制、其对这些复合物的招募以及它们的亚细胞定位尚不清楚。本提案通过对哺乳动物细胞和斑马鱼中hVps34调控的重点分析来解决这些问题。目的1使用化学遗传学方法检测hVps15蛋白激酶对hVps34的调控。这是基于我们令人兴奋的数据显示，hVps15与hVps34的结合，以前认为需要hVps15的活性，实际上是激酶独立的，只需要hVps15与ATP结合。利用能够利用ATP类似物的hVps15突变体，我们将定义hVps15的激酶依赖性和独立信号传导，并鉴定hVps15底物。目的2检测hVps34复合物形成的动力学，在全细胞水平上，并使用光漂白后荧光恢复（FRAP），在自噬体膜上。我们将确定营养饥饿是否调节hVps34复合物之间的亚基交换，并询问hVps34相关蛋白是否串联或单独募集到自噬体膜上。Aim 3使用荧光波动光谱（一种能够确定活细胞内胞质hVps34复合物化学计量的方法）来测量营养物质对复合物形成的调节。最后，Aim 4使用新的突变体选择性地破坏hVps34与钙调素的结合以及hVps15与Rab5和Rab7的结合，阻断hVps34的降解，并在保留其蛋白激酶活性的同时消除hVps34的脂质激酶活性。这些突变体将在培养细胞和斑马鱼中用于敲除/拯救方法，以确定体内调节hVps34信号的机制。综上所述，这些研究将为hVps34如何受营养应激调节以及它如何靶向自噬体膜提供重要的新信息。鉴于hVps34是参与自噬的关键激酶之一，更好地了解其调控将导致对这一关键细胞过程的新见解。

DESCRIPTION (provided by applicant): Autophagy is a cellular response to nutrient stress, in which the formation of a double-walled membrane structure sequesters cytosolic components and organelles and delivers them to the lysosome for degradation. This liberates nutrients for use in new biosynthetic activity. Autophagy is critical for perinatal survival in mice, plays a role in normal tissue maintenance in neurons, hepatocytes, and pancreatic beta cells, and contributes to innate immune responses to pathogens. Its activation under pathological conditions leads to disease states such as muscle wasting, and decreases in autophagic activity may contribute to neurological decline in aging and in neurodegenerative disease. Thus, pharmacological modulation of autophagy may have significant clinical benefit. One approach to modulating autophagy would be through the Class III PI 3-kinase, hVps34, which is required for autophagy in yeast, flies and mammalian cells. hVps34 exists in multi-protein complexes containing components specific for autophagy (Atg14L) or vesicular trafficking (UVRAG), as well as components that function in both these pathways (hVps15, beclin-1). The mechanisms that regulate hVps34 activity, its recruitment to these complexes, and their subcellular localization, are not well understood. This proposal addresses these questions through a focused analysis of hVps34 regulation in mammalian cells and in zebrafish. Aim 1 uses a chemical genetic approach to examine the regulation of hVps34 by the hVps15 protein kinase. It is based on our exciting data showing that the binding of hVps15 to hVps34, previously thought to require hVps15 activity, is in fact kinase independent and only requires hVps15 binding to ATP. Using hVps15 mutants that are able to utilize ATP analogues, we will define kinase dependent and independent signaling by hVps15, and identify hVps15 substrates. Aim 2 examines the dynamics of hVps34 complex formation, both at the whole cell level and, using fluorescence recovery after photobleaching (FRAP), on the autophagosomal membrane. We will determine whether nutrient starvation regulates subunit exchange between hVps34 complexes, and ask whether hVps34-associated proteins are recruited in tandem, or individually, to autophagosomal membranes. Aim 3 uses fluorescence fluctuation spectroscopy, a method able to define the stoichiometry of cytosolic hVps34 complexes in living cells, to measure the regulation of complex formation by nutrients. Finally, Aim 4 uses novel mutants that selectively disrupt hVps34 binding to calmodulin and hVps15 binding to Rab5 and Rab7, block hVps34 degradation, and abolish hVps34 lipid kinase activity while preserving its protein kinase activity. These mutants will be used in a knockdown/rescue approach in cultured cells and in zebrafish, to define mechanisms that regulate hVps34 signaling in vivo. Taken together, these studies will provide important new information on how hVps34 is regulated by nutrient stress, and how it is targeted to autophagosomal membranes. Given that hVps34 is one of the key kinases involved in autophagy, a better understanding of its regulation will lead to new insights into this critical cellular process.