Physiology of Class III PI 3-kinase Signaling 2

描述（由申请人提供）：自噬是对营养胁迫的细胞反应，其中双壁膜结构的形成隔离细胞溶质组分和细胞器并将其递送至溶酶体进行降解。这释放出营养素用于新的生物合成活动。自噬对于小鼠的围产期存活至关重要，在神经元、肝细胞和胰腺β细胞中的正常组织维持中起作用，并且有助于对病原体的先天免疫应答。其在病理条件下的激活导致疾病状态，如肌肉萎缩，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下降。因此，自噬的药理学调节可能具有显着的临床益处。调节自噬的一种方法是通过III类PI 3-激酶hVps 34，其是酵母、苍蝇和哺乳动物细胞中自噬所需的。hVps 34存在于多蛋白复合物中，其含有对自噬（Atg 14 L）或囊泡运输（UVRAG）特异性的组分，以及在这两种途径中起作用的组分（hVps 15，beclin-1）。调节hVps 34活性的机制、其向这些复合物的募集及其亚细胞定位尚不清楚。该提案通过重点分析哺乳动物细胞和斑马鱼中hVps 34的调节来解决这些问题。目的1采用化学遗传学方法研究hVps 15蛋白激酶对hVps 34的调控。这是基于我们令人兴奋的数据，表明hVps 15与hVps 34的结合，以前认为需要hVps 15活性，实际上是激酶独立的，只需要hVps 15与ATP结合。使用能够利用ATP类似物的hVps 15突变体，我们将定义hVps 15的激酶依赖性和非依赖性信号传导，并鉴定hVps 15底物。目的2检查hVps 34复合物形成的动力学，无论是在整个细胞水平，并使用荧光恢复后光漂白（FRAP），在自噬体膜上。我们将确定营养饥饿是否调节hVps 34复合物之间的亚基交换，并询问hVps 34相关蛋白是否被串联或单独招募到自噬体膜。目的3使用荧光波动光谱，一种能够定义活细胞中胞质hVps 34复合物的化学计量的方法，以测量营养素对复合物形成的调节。最后，目标4使用新的突变体，选择性地破坏hVps 34与钙调蛋白的结合和hVps 15与Rab 5和Rab 7的结合，阻断hVps 34降解，并消除hVps 34脂质激酶活性，同时保留其蛋白激酶活性。这些突变体将用于在培养的细胞和斑马鱼中的敲低/拯救方法，以定义在体内调节hVps 34信号传导的机制。总之，这些研究将提供重要的新信息hVps 34是如何调节营养胁迫，以及它是如何针对自噬体膜。鉴于hVps 34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.