Autophagy Regulation by Hippo Kinases STK3/STK4

项目总结 自噬是一个复杂的分解代谢过程，它降解溶酶体中的胞浆成分并发挥作用。 在维持细胞动态平衡方面的突出作用。虽然被广泛地牵连在脑血管疾病的病理生理学 尽管有许多疾病，但对自噬过程的各个步骤的详细调控仍然存在 开发不足。为了深入了解这一重要问题，我们建议描述一种新的自噬-- 我们最近发现的调控途径，涉及哺乳动物保守的河马蛋白激酶STK3/STK4 和必需的Atg8自噬蛋白家族。 我们已经报道，河马激酶的哺乳动物同源基因STK3和STK4是河马蛋白的重要组成部分。 多种生物中的自噬，两者都能使Atg8家族成员LC3B在氨基酸上磷酸化 到50岁。STK3/4介导的磷酸化是自噬小体与溶酶体融合的关键，这是 自噬过程，以及细胞清除细胞内细菌的能力，是 自噬。我们发现了一种新的自噬调控方式，涉及到LC3B的直接磷酸化 通过STK3/4提供了一个独特的机会，以获得对自噬调控的新的机械性见解 过程，包括Atg8蛋白质家族。 为此，我们将探讨自噬调节的分子和细胞回路。 STK3/STK4，利用蛋白质组、分子和细胞学技术的强大组合。具体来说，我们 将使用生化方法来表征STK3/4与Atg8家族其他成员的相互作用， 除了LC3B，由于STK3/4结合了额外的Atg8蛋白，但尚不清楚它们是否也可以作为 底物。在目标2中，我们将使用细胞学和蛋白质组学方法来剖析细胞内-和 STK3/4磷酸化Atg8蛋白的分子后果。最后，在目标3中，我们将设计 结合STK4蛋白质组学研究筛选靶向RNAi以确定对该小说至关重要的调控因子 STK3/4-LC3B/Atg8轴调节自噬。 这些研究具有创新性，因为我们将使用尖端技术来阐明分子 河马STK3/4通过ATG8中的关键自噬蛋白调节自噬的机制 一家人。此外，这些研究可能会产生重大影响，因为它们将提供重要的 关于STK3/4激酶和Atg8家族如何调控哺乳动物自噬的信息；这样的见解可能 事实证明，这不仅对我们对自噬过程的基本理解很重要，而且可能有助于 未来针对自噬相关疾病的治疗。

PROJECT SUMMARY Autophagy is a complex catabolic process that degrades cytosolic components within the lysosome and plays a prominent role in maintaining cellular homeostasis. Although widely implicated in the pathophysiology of numerous diseases, the detailed regulation of the separate steps of the autophagy process remains underexplored. To gain insight into this important question, we propose to characterize a novel autophagy- regulatory pathway that we recently identified, involving the conserved mammalian Hippo kinases STK3/STK4 and the essential Atg8 autophagy protein family. We have reported that STK3 and STK4, the mammalian orthologs of Hippo kinase, are essential for autophagy in diverse organisms, and both can phosphorylate the Atg8 family member LC3B on amino acid Thr50. STK3/4-mediated phosphorylation is critical for fusion of autophagosomes with lysosomes, a late step in the autophagy process, as well as the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Our discovery of this novel mode of autophagy regulation involving direct phosphorylation of LC3B by STK3/4 provide a unique opportunity to gain new mechanistic insight into the regulation of the autophagy process, including the Atg8 family of proteins. To this end, we will address the molecular and cellular circuitry of autophagy regulation mediated by STK3/STK4 by using a strong combination of proteomic, molecular and cytological techniques. Specifically, we will use biochemical approaches to characterize STK3/4 interactions with other members of the Atg8 family, besides LC3B, since STK3/4 bind additional Atg8 proteins yet it is not clear if they also can serve as substrates. In Aim 2, we will use cytological and proteomic approaches to dissect the intracellular- and molecular consequences of phosphorylation of Atg8 proteins by STK3/4. Finally, in Aim 3, we will devise targeted RNAi screens in conjunction with STK4 proteomic studies to identify regulators critical for the novel STK3/4-LC3B/Atg8 axis to regulate autophagy. These studies are innovative because we will use cutting-edge techniques to elucidate the molecular mechanism by which the Hippo kinases STK3/4 regulate autophagy via the key autophagy proteins in the Atg8 family. Moreover, these studies are likely to have significant impact because they will provide important information on how STK3/4 kinases and the Atg8 family regulate autophagy in mammals; such insights could prove important not only to our basic understanding of the autophagy process, but may also help develop future treatments against autophagy-linked diseases.