Multisite phosphorylated S6K1 directs a regulatory module determining adipocyte lipid metabolism

多位点磷酸化 S6K1 指导决定脂肪细胞脂质代谢的调节模块

基本信息

批准号：
10349543
负责人：
PAUL L FOX
金额：
$ 46.75万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-09 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10349543
关键词：
Address Adipocytes Adipose tissue Affinity Amino Acyl Transfer RNA Area Binding Binding Proteins Body Weight CRISPR/Cas technology Cell membrane Cells Code Coenzyme A Complementary DNA Complex Cyclin-Dependent Kinase 5 Development Docking EMS1 gene Epidemic Exhibits FRAP1 gene Family Fatty acid glycerol esters Genes Health Care Costs High Fat Diet Human In Vitro Insulin Insulin Resistance Kinetics Knock-in Knock-in Mouse LCN2 gene Lipids Mass Spectrum Analysis Mediating Messenger RNA Metabolic Pathway Metabolism Mitochondria Modeling Molecular Conformation Mus Mutation Mutation Analysis Obesity Pathologic Pathway interactions Phenotype Phosphorylation Phosphorylation Site Phosphotransferases Point Mutation Raptors Ribosomal Protein S6 Ribosomal Protein S6 Kinase Role Signal Pathway Signal Transduction Site Specificity Surface Plasmon Resonance Testing Triglycerides Vesicle Wild Type Mouse combinatorial deletion analysis diet-induced obesity economic cost experimental study extracellular fatty acid metabolism fatty acid oxidation fatty acid transport fatty acid-transport protein glutamyl-prolyl-tRNA synthetase improved in vivo insight insulin sensitivity knock-down lipid metabolism long chain fatty acid member mimetics mouse model new therapeutic target novel novel therapeutics obesity prevention oxidation prevent therapeutic target uptake

项目摘要

Project Summary/Abstract Obesity is an epidemic-scale problem in the U.S. and worldwide with enormous health and economic costs. The mTORC1-S6 kinase 1 (S6K1) axis drives anabolic pathways determining obesity. We recently identified glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to mouse adiposity. Insulin-stimulated EPRS phosphorylation at Ser999 by S6K1 in adipocytes induces its binding to fatty acid transport protein 1 (FATP1) and translocation to the plasma membrane to increase long-chain fatty acid (LCFA) uptake. Recent studies reveal that phosphorylation of S6K1 by cyclin-dependent kinase 5 (Cdk5) at Ser424 and Ser429 in the S6K1 C-terminus are required for phosphorylation of EPRS, but not for canonical substrates such as RPS6. This unexpected finding indicates that embedded in S6K1 is a target-selective phospho-code in which combinatorial phospho-site phosphorylation determines kinase targets. To identify additional targets of multi-phosphorylated S6K1 (termed S6K1*) but not mTORC1-activated S6K1, we transfected HEK cells with S6K1 bearing phospho-mimetic mutations at the 3 phospho-sites, or wild-type S6K1 cDNA. Three new S6K1* targets were identified by mass spectrometry and validated in adipocytes – coenzyme A synthase (COASY), cortactin, and lipocalin 2. Importantly, all are implicated in adipocyte lipid metabolism: P-EPRS transports FATP1 to the plasma membrane for increased LCFA uptake; COASY catalyzes the final two steps of synthesis of coenzyme A, required for LCFA activation; lipocalin 2 increases LCFA β-oxidation and insulin resistance; and cortactin is required for insulin-stimulated transport of Glut4-containing vesicles to plasma membranes. We propose that S6K1* directs an adipocyte lipid metabolon, and is a major contributor to obesity-related phenotypes driven by the mTORC1-S6K1 axis. We will test this hypothesis by pursuit of 3 Specific Aims: In Aim 1 we determine S6K1*/target docking domains. By mass spectrometry and site-directed mutation analysis, we will determine specific S6K1*-directed phosphorylation sites in the targets. In Aim 2 we determine the function of phosphorylated S6K1* targets in adipocyte lipid metabolism. We will determine the mechanism of insulin-stimulated transport and binding of P-EPRS to the adipocyte plasma membrane; the role of phosphorylation in COASY catalytic activity and localization; whether P-cortactin transports P-EPRS/FATP1-containing vesicles to the plasma membrane; and extracellular secretion and intracellular localization of P-lipocalin 2, and its role in LCFA oxidation. In Aim 3 we elucidate In vivo role of S6K1* in lipid metabolism and obesity. We will determine the effect of diet-induced obesity on the S6K1* activation pathway and on target phosphorylation in mice. Taking advantage of our new mouse model (generated by Crispr-Cas9 technology) bearing a Ser429-to-Ala mutation in Rps6kb1 (mouse gene encoding S6K1) that lack S6K1* activity, while retaining canonical S6K1 activity, we will test the role of S6K1* in target phosphorylation in vivo, in lipid metabolism, and in diet-induced obesity.

项目概要/摘要肥胖是美国和全世界范围内的一个流行病，造成了巨大的健康和经济损失。我们最近发现 mTORC1-S6 激酶 1 (S6K1) 轴驱动决定肥胖的合成代谢途径。谷氨酰脯氨酰 tRNA 合成酶 (EPRS) 作为 mTORC1-S6K1 靶标，导致小鼠肥胖。脂肪细胞中胰岛素刺激的 EPRS Ser999 被 S6K1 磷酸化，诱导其与脂肪酸结合转运蛋白 1 (FATP1) 并易位至质膜以增加长链脂肪酸 (LCFA) 最近的研究表明，S6K1 被细胞周期蛋白依赖性激酶 5 (Cdk5) 在 Ser424 和 Ser424 处磷酸化。 S6K1 C 末端的 Ser429 是 EPRS 磷酸化所必需的，但对于经典底物如这一意外发现表明 S6K1 中嵌入了一个目标选择性磷酸化代码，其中组合磷酸位点磷酸化确定激酶靶标，以确定多磷酸化的其他靶标。 S6K1（称为 S6K1*），但不是 mTORC1 激活的 S6K1，我们用 S6K1 转染 HEK 细胞在 3 个磷酸位点上带有磷酸模拟突变，或野生型 S6K1 cDNA 三个新的 S6K1* 靶标。通过质谱法鉴定并在脂肪细胞中进行验证 - 辅酶 A 合酶 (COASY)、cortactin、和脂质运载蛋白 2。重要的是，所有这些都与脂肪细胞脂质代谢有关：P-EPRS 将 FATP1 转运至质膜增加 LCFA 吸收；COASY 催化辅酶合成的最后两个步骤； A、脂运载蛋白 2 增加 LCFA β-氧化和胰岛素抵抗；我们认为，胰岛素刺激将含有 Glut4 的囊泡转运至质膜是必需的。 S6K1* 指导脂肪细胞脂质代谢，并且是肥胖相关表型的主要贡献者我们将通过追求 3 个具体目标来检验该假设：在目标 1 中，我们确定通过质谱和定点突变分析，我们将确定S6K1*/目标对接域。在目标 2 中，我们确定了特定 S6K1* 定向磷酸化位点的功能。脂肪细胞脂质代谢中磷酸化的 S6K1* 靶标我们将确定胰岛素刺激的机制。 P-EPRS 与脂肪细胞质膜的转运和结合；磷酸化的作用； COASY 催化活性和定位；P-cortactin 是否将含有 P-EPRS/FATP1 的囊泡转运至质膜；P-脂质运载蛋白 2 的细胞外分泌和细胞内定位及其作用 LCFA 氧化。在目标 3 中，我们阐明了 S6K1* 在脂质代谢和肥胖中的体内作用。饮食诱导的肥胖对小鼠 S6K1* 激活途径和靶点磷酸化的影响。利用我们带有 Ser429-to-Ala 的新小鼠模型（通过 Crispr-Cas9 技术生成） Rps6kb1（编码 S6K1 的小鼠基因）发生突变，缺乏 S6K1* 活性，同时保留规范的 S6K1 活性，我们将测试 S6K1* 在体内靶标磷酸化、脂质代谢和饮食诱导中的作用肥胖。