Multisite phosphorylated S6K1 directs a regulatory module determining adipocyte lipid metabolism

项目总结/摘要 肥胖在美国和全世界都是一个巨大的健康和经济成本的问题。的 mTORC 1-S6激酶1（S6 K1）轴驱动决定肥胖的合成代谢途径。我们最近发现 谷氨酰-脯氨酰tRNA合成酶（EPRS）作为mTORC 1-S6 K1靶标，导致小鼠肥胖。 胰岛素刺激的脂肪细胞中S6 K1对EPRS Ser 999的磷酸化诱导其与脂肪酸的结合 转运蛋白1（FATP 1）和转运到质膜以增加长链脂肪酸（LCFA） 摄取。最近的研究表明，细胞周期蛋白依赖性激酶5（Cdk 5）在Ser 424和Ser 426位点对S6 K1的磷酸化作用， S6 K1 C-末端的Ser 429是EPRS磷酸化所需的，但不是典型底物如 RPS6这个意想不到的发现表明，嵌入S6 K1中的是靶选择性磷酸化密码，其中 组合磷酸化位点磷酸化决定激酶靶。为了鉴定多磷酸化的其他靶点， S6 K1（称为S6 K1 *）而不是mTORC 1激活的S6 K1，我们用S6 K1转染HEK细胞， 在3个磷酸化位点带有磷酸化模拟突变，或野生型S6 K1 cDNA。三个新的S6 K1 * 目标 通过质谱法鉴定并在脂肪细胞中验证-辅酶A合酶（COASY），corpine， 和脂质运载蛋白2。重要的是，所有这些都与脂肪细胞的脂质代谢有关：P-EPRS将FATP 1转运到 质膜增加LCFA摄取; COASY催化辅酶合成的最后两步 A，LCFA激活所需;脂质运载蛋白2增加LCFA β-氧化和胰岛素抵抗; Coronin是 胰岛素刺激的含Glut 4的囊泡转运到质膜所需。我们建议 S6 K1 * 指导脂肪细胞脂质代谢子，并且是肥胖相关表型的主要贡献者， mTORC 1-S6 K1轴。我们将通过追求3个具体目标来测试这个假设：在目标1中，我们确定 S6 K1 */靶对接结构域。通过质谱和定点突变分析，我们将确定 靶点中特异性S6 K1 *-定向磷酸化位点。在目标2中，我们确定了 磷酸化S6 K1 * 靶向脂肪细胞脂质代谢。我们将确定胰岛素刺激 P-EPRS向脂肪细胞质膜的转运和结合;磷酸化在 COASY催化活性和定位; P-coronin是否将含有P-EPRS/FATP 1的囊泡转运到 质膜;以及P-脂质运载蛋白2的细胞外分泌和细胞内定位，及其在 LCFA氧化。在目标3中，我们阐明了S6 K1 * 在脂质代谢和肥胖中的体内作用。我们将确定 饮食诱导的肥胖对小鼠S6 K1 * 活化途径和靶磷酸化的影响。 利用我们的新小鼠模型（由Crispr-Cas9技术产生）， Rps 6 kb 1（编码S6 K1的小鼠基因）中的突变，缺乏S6 K1 * 活性，同时保留典型的S6 K1 活性，我们将测试S6 K1 * 在体内靶磷酸化，脂质代谢和饮食诱导的磷酸化中的作用。 肥胖

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.