Physiology of Class III PI 3-kinase Signaling 2

描述(申请人提供)：自噬是一种细胞对营养胁迫的反应，在这种情况下，形成的双壁膜结构隔离了细胞液成分和细胞器，并将它们运送到溶酶体进行降解。这释放了营养物质，用于新的生物合成活动。自噬对小鼠的围产期存活至关重要，在神经元、肝细胞和胰岛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。在病理条件下，它的激活会导致肌肉萎缩等疾病状态，自噬活动的减少可能会导致衰老和神经退行性疾病的神经功能衰退。因此，药物调节自噬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，这是酵母、苍蝇和哺乳动物细胞自噬所必需的。HVps34存在于含有自噬(Atg14L)或囊泡运输(UVRAG)特异性成分的多蛋白复合体中，以及在这两条途径中发挥作用的成分(hVps15，beclin-1)。调节hVps34活性的机制、它在这些复合体中的募集以及它们的亚细胞定位还不是很清楚。这项建议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利用化学遗传学方法研究hVps15蛋白激酶对hVps34的调节作用。它是基于我们令人兴奋的数据表明，hVps15与hVps34的结合，以前被认为需要hVps15活性，实际上是不依赖于激酶的，只需要hVps15与ATP结合。使用能够利用ATP类似物的hVps15突变体，我们将通过hVps15定义依赖和非依赖的信号转导，并鉴定hVps15底物。目的2研究hVps34复合体在整个细胞水平和自噬体膜上的光漂白后荧光恢复(FRAP)形成的动力学。我们将确定营养饥饿是否调节hVps34复合体之间的亚基交换，并询问hVps34相关蛋白是串联还是单独招募到自噬体膜上。目的3利用荧光涨落光谱，一种能够确定活细胞内hVps34复合体的化学计量比的方法，来测量营养物质对复合体形成的调节。最后，Aim 4使用了新的突变体，选择性地破坏了hVps34与钙调蛋白的结合以及hVps15与Rab5和Rab7的结合，阻止了hVps34的降解，并在保持其蛋白激酶活性的同时取消了hVps34的脂激酶活性。这些突变体将在培养细胞和斑马鱼中用于击倒/挽救方法，以确定体内调节hVps34信号的机制。综上所述，这些研究将提供关于hVps34如何受营养胁迫调节以及它如何靶向自噬体膜的重要新信息。鉴于hVps34是参与自噬的关键激酶之一，对其调控的更好理解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.