Autophagy Regulation by Hippo Kinases STK3/STK4

Hippo 激酶 STK3/STK4 的自噬调节

• 批准号: 9336318
• 负责人: Malene Hansen
• 金额: $ 38.03万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9336318
• 关键词: Address; Affect; Alzheimer' s Disease; Amino Acids; Autophagocytosis; Autophagosome; Bacteria; Bacterial Infections; Binding; Biochemical; Caenorhabditis elegans; Catabolic Process; Cell physiology; Cells; Complex; Cytological Techniques; Cytology; Defect; Disease; Embryo; Event; Family; Family member; Fibroblasts; Functional disorder; Future; Genes; Grant; Homeostasis; Immune System Diseases; In Vitro; Infection; Intracellular Transport; Knowledge; Lead; Link; Lysosomes; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Microtubules; Molecular; Mus; Organism; Orthologous Gene; Pathway interactions; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protein Family; Proteins; Proteomics; Public Health; RNA interference screen; Recruitment Activity; Recycling; Regulation; Regulatory Pathway; Reporting; Role; Signal Transduction; Small Interfering RNA; Starvation; Structure; Techniques; Testing; Transport Vesicles; Vesicle Transport Pathway; Work; Yeasts; experimental study; genetic approach; in vivo; innovation; insight; live cell imaging; member; novel; protein function; screening; trafficking

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.

项目摘要 自噬是一个复杂的分解代谢过程，可降解溶酶体内的胞质成分并发挥作用 在维持细胞稳态方面的重要作用。尽管广泛涉及 许多疾病，自噬过程的各个步骤的详细调节仍然存在 尚未得到充实的。为了深入了解这个重要问题，我们建议表征一种新型的自噬 - 我们最近确定的调节途径，涉及保守的哺乳动物河马激酶STK3/STK4 以及必需的ATG8自噬蛋白家族。 我们报告说，河马激酶的哺乳动物直系同源物stk3和stk4对于 在各种生物体中自噬，并且都可以在氨基酸上磷酸化ATG8家族成员LC3B THR50。 STK3/4介导的磷酸化对于自体融合与溶酶体的融合至关重要 自噬过程以及细胞清除细胞内细菌的能力，这是一种已建立的货物 自噬。我们发现了这种新型自噬调节模式，涉及LC3B的直接磷酸化 由STK3/4提供了一个独特的机会，可以获得对自噬调节的新机械洞察力 过程，包括ATG8蛋白质家族。 为此，我们将解决由自噬调节的分子和细胞电路 STK3/STK4通过使用蛋白质组学，分子和细胞学技术的强组合。具体来说，我们 将使用生化方法来表征与ATG8家族其他成员的STK3/4相互作用， 除LC3B外，由于STK3/4绑定了其他ATG8蛋白，但尚不清楚它们是否也可以用作 基材。在AIM 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对ATG8蛋白磷酸化的分子后果。最后，在AIM 3中，我们将设计 靶向RNAi筛选与STK4蛋白质组学研究结合使用，以鉴定对新型的调节剂 STK3/4-LC3B/ATG8轴以调节自噬。 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 河马激酶STK3/4通过ATG8中的关键自噬蛋白调节自噬的机制 家庭。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 有关STK3/4激酶和ATG8家族如何调节哺乳动物自噬的信息；这样的见解可以 证明不仅对我们对自噬过程的基本理解很重要，还可以帮助发展 未来针对自噬疾病的治疗方法。