Protein Phosphatase Control of AMPK Function

AMPK 功能的蛋白磷酸酶控制

• 批准号: 10547739
• 负责人: MEI KONG
• 金额: $ 30.17万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10547739
• 关键词: AMP-activated protein kinase kinase; Address; Back; Binding; Biological Process; Body Weight; Catalytic Domain; Cells; Chemicals; Complex; Data; Desire for food; Diabetes Mellitus; Family; Fatty Liver; Fatty acid glycerol esters; Genes; Glucose; Goals; Hepatic; High Fat Diet; Holoenzymes; Homeostasis; In Vitro; Investigation; Knock-out; Knockout Mice; Lipids; Liver; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator; Metabolic; Metabolic stress; Metabolic syndrome; Metabolism; Modeling; Molecular; Mus; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Primary carcinoma of the liver cells; Protein Dephosphorylation; Protein Family; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Recovery; Regulation; Reporting; Repression; Research; Role; Series; Signal Transduction; Solid; Specificity; System; Tissues; Weight; Whole Organism; Xenograft Model; adenoviral mediated; cancer type; comparison control; conditional knockout; energy balance; experimental study; fatty acid oxidation; glucose tolerance; glucose uptake; improved; in vivo; knock-down; liver cancer model; liver function; mouse model; novel; novel therapeutic intervention; novel therapeutics; prevent; protein phosphatase 6; recruit; restoration; small hairpin RNA; tumor; tumor growth; tumorigenesis

PROJECT SUMMARY The goal of this proposed research is to reveal the negative regulator of AMPK and eventually target this pathway to generate an efficient AMPK activator for treatment of metabolic syndromes. AMPK senses metabolic stress and is a central mediator in maintaining metabolic homeostasis and energy balance. Thus, AMPK activation has become an attractive target for treating metabolic syndromes, including diabetes and cancer. While it has been demonstrated that AMPK activity is tightly regulated by reversible protein phosphorylation, and despite many efforts to identify AMPK kinases, it is still unclear how AMPK is dephosphorylated or inactivated upon recovery from metabolic stress. One of the main reasons for the limited progress in identifying an AMPK phosphatase is because of the promiscuous activity of serine-threonine phosphatases and its specificity is governed by associated proteins. We and others previously identified that PP2A family protein phosphatases are involved in AMPK dephosphorylation. However, the PP2A phosphatase family contains hundreds of possible different complexes. To identify a specific complex that directly dephosphorylates AMPK, using protein mass- spectrometry analysis, we found that protein phosphatase 6 (PP6, a PP2A family phosphatase) regulatory subunit SAPS3 is associated with AMPK. Furthermore, our preliminary data demonstrated that SAPS3/AMPK binding is glucose responsive and required for AMPK dephosphorylation. To evaluate the role of SAPS3 in vivo, we have generated a novel mouse model by flanking the gene encoding SAPS3, ppp6r3, with a loxP sequence that allow us to develop tissue specific knock out of SAPS3. Using recently developed SAPS3 liver-specific knockout mice, we found that deletion of SAPS3 increases AMPK phosphorylation in liver and displays phenotypes similar to AMPK activation in regulation of metabolism and tumorigenesis. Therefore, we propose a series of experiments in this application to address our central hypothesis that SAPS3-containing PP6 phosphatase complex dephosphorylates and inhibits AMPK activity, thereby regulating AMPK-mediated functions. Three specific aims are proposed as follows: 1) elucidating molecular mechanisms underlying AMPK inhibition by SAPS3-containing PP6 complex, 2) determining the biological function of SAPS3 in metabolic/energy homeostasis in vivo via regulation of AMPK using SAPS3 liver specific knockout mice, 3) examining the role of SAPS3 in tumorigenesis via regulation of AMPK in two mouse tumor models. These studies will provide a solid mechanistic basis for AMPK signaling regulated by protein phosphatase and in vivo evidence that AMPK-mediated biological functions are tightly controlled by protein phosphatase. Results from the proposed research will also advance new therapeutic directions by targeting the SAPS3/AMPK interaction, which could be an effective approach to activate AMPK for treating metabolic syndromes.

项目摘要 这项研究的目的是揭示AMPK的负调节因子，并最终靶向这一通路 以产生用于治疗代谢综合征的有效AMPK激活剂。AMPK感知代谢应激 并且是维持代谢稳态和能量平衡的中心介质。因此，AMPK激活具有 成为治疗代谢综合征，包括糖尿病和癌症的有吸引力的目标。虽然已经 表明AMPK活性受到可逆蛋白磷酸化的严格调节，尽管许多 尽管人们努力鉴定AMPK激酶，但仍不清楚AMPK是如何在恢复后去磷酸化或失活的 代谢压力在鉴定AMPK磷酸酶方面进展有限的主要原因之一是 由于丝氨酸-苏氨酸磷酸酶的混杂活性， 相关蛋白质我们和其他人先前发现PP2A家族蛋白磷酸酶参与了 AMPK去磷酸化。然而，PP2A磷酸酶家族包含数百种可能的不同的 配合物为了鉴定直接使AMPK去磷酸化的特定复合物，使用蛋白质质量- 光谱分析，我们发现蛋白磷酸酶6（PP6，一个PP2A家族磷酸酶）调节 亚基SAPS3与AMPK相关。此外，我们的初步数据表明，SAP 3/AMPK 结合是葡萄糖响应性的，并且是AMPK去磷酸化所需的。为了评估SAP 3在体内的作用， 我们通过在编码SAP 3的基因ppp6r3的侧翼插入loxP序列， 这使得我们能够开发出组织特异性敲除SAP 3。使用最近开发的SAP 3肝脏特异性 在敲除小鼠中，我们发现SAPS3的缺失增加了肝脏中AMPK的磷酸化， 在代谢和肿瘤发生的调节中与AMPK活化相似的表型。因此，我们建议 在本申请中进行了一系列实验，以解决我们的中心假设，即含有SAP 3的PP 6 磷酸酶复合物去磷酸化并抑制AMPK活性，从而调节AMPK介导的 功能协调发展的本研究的主要目的是：1）阐明AMPK的分子机制 通过含SAP 3的PP 6复合物的抑制，2）确定SAP 3在 使用SAP 3肝特异性敲除小鼠通过调节AMPK体内代谢/能量稳态，3） 在两种小鼠肿瘤模型中通过AMPK的调节检查SAPS3在肿瘤发生中的作用。这些研究 将为蛋白磷酸酶调节AMPK信号传导提供坚实的机制基础和体内证据 AMPK介导的生物学功能受到蛋白磷酸酶的严格控制。结果 拟议的研究还将通过靶向SAPS3/AMPK相互作用推进新的治疗方向， 可能是激活AMPK治疗代谢综合征的有效途径。