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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.

项目总结/文摘