Multisite phosphorylated S6K1 directs a regulatory module determining adipocyte lipid metabolism

项目摘要/摘要 肥胖症在美国和世界范围内是一个流行病规模的问题，给健康和经济带来了巨大的代价。这个 MTORC1-S6激酶1(S6K1)轴驱动决定肥胖的合成代谢途径。我们最近确认了 谷氨酰脯氨基tRNA合成酶(Eprs)作为mTORC1-S6K1的靶标导致小鼠肥胖。 胰岛素刺激脂肪细胞S6K1激活Ser999位Eprs磷酸化诱导其与脂肪酸结合 运输蛋白1(FATP1)和质膜转位增加长链脂肪酸(LCFA) 领悟。最近的研究表明，S6K1在Ser424和Ser424处被细胞周期蛋白依赖性激酶5(CDK5)磷酸化。 S6K1 C末端的Ser429是Eprs磷酸化所必需的，但不是典型的底物如 作为RPS6。这一意想不到的发现表明，在S6K1中嵌入了一种靶向选择性磷酸码，其中 组合的磷酸化位点的磷酸化决定了激酶的靶标。识别多个磷酸化的额外靶点 S6K1(称为S6K1*)，但不是mTORC1激活的S6K1，我们用S6K1转染HEK细胞 携带3个磷酸化位点的模拟磷酸化突变，即野生型S6K1基因。三个新的S6K1*目标 经质谱学鉴定，并在脂肪细胞辅酶A合成酶(COASY)、皮质酮、 和Lipocalin 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FATP1转运到 增加LCFA摄取的质膜；COASY催化辅酶合成的最后两步 LCFA激活所必需的；Lipocalin 2增加LCFAβ氧化和胰岛素抵抗；而皮质酮是 在胰岛素刺激下将含GLUT4的囊泡运输到质膜所需的。我们建议 S6K1*指导脂肪细胞脂代谢，是肥胖相关表型的主要贡献者，其驱动因素 MTORC1-S6K1轴。我们将通过追求三个具体目标来检验这一假设：在目标1中，我们确定 S6K1*/目标插接域。通过质谱学和定点突变分析，我们将确定 靶标中特定的S6K1*导向的磷酸化位点。在目标2中，我们确定了 脂肪细胞脂代谢中的磷酸化S6K1*靶标。我们将确定胰岛素刺激的机制 P-Eprs转运和结合到脂肪细胞质膜；磷酸化在脂肪细胞中的作用 COASY催化活性和定位；P-Cortactin是否将含P-EPRS/FATP1的囊泡转运到 质膜；P-Lipocalin 2的细胞外分泌和细胞内定位，以及它在 LCFA氧化。在目的3中，我们在体内阐明了S6K1*在脂代谢和肥胖中的作用。我们将决定 饮食诱导肥胖对小鼠S6K1*激活通路和靶向磷酸化的影响。 利用我们的新鼠标模型(由Crispr-Cas9技术生成)，带有Ser429-to-Ala Rps6kb1(编码S6K1的小鼠基因)的突变，缺乏S6K1*活性，但保留规范的S6K1 活性，我们将测试S6K1*在体内靶向磷酸化、脂代谢和饮食诱导中的作用 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.