Role of Tuberin S1365 Phosphorylation in mTORC1 Regulation

马铃薯蛋白 S1365 磷酸化在 mTORC1 调节中的作用

基本信息

批准号：
9769515
负责人：
Brittany Dunkerly-Eyring
金额：
$ 4.5万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-23 至 2021-07-22
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9769515
关键词：
5&apos -AMP-activated protein kinase Acrylates Address Affect Autophagocytosis Binding Biological Assay Cardiovascular Diseases Cause of Death Cellular Metabolic Process Chronic Clinical Complex Crosslinker Cultured Cells Cyclic GMP Cyclic GMP-Dependent Protein Kinases Cysteine Data Disease Elements Engineering Excision Extracellular Signal Regulated Kinases FRAP1 gene Fibrinogen Food Functional disorder GTP Binding GTP-Binding Protein alpha Subunits, Gs GTPase-Activating Proteins Genetic Grant Growth Heart Heart Abnormalities Heart Diseases Immunofluorescence Immunologic In Vitro Insulin Ischemia Knock-in Knock-in Mouse Laboratories Left Ventricular Hypertrophy Lysosomes MAPK1 gene Malignant Neoplasms Mass Spectrum Analysis Metabolic Metabolic Control Methods Modeling Modification Molecular Mus Muscle Muscle Cells Mutate Mutation Myocardial Ischemia Natriuretic Peptides Neurons Nitric Oxide Normal Cell Nutrient Nutrient Depletion Organ Patients Peptide Signal Sequences Phosphorylation Phosphorylation Site Phosphotransferases Play Prevalence Proteins Regulation Reperfusion Therapy Rest Role Serine Signal Transduction Sirolimus Site Somatotropin Stimulus Stress T-Lymphocyte TSC2 gene Testing Therapeutic Transducers Translating Withdrawal analog base cardiovascular disorder risk cell growth cell type hemodynamics high risk improved in vivo in vivo Model inhibitor/antagonist loss of function loss of function mutation mimetics mortality mutant novel pressure prevent protein activation proteostasis response tool

项目摘要

Project Summary Cardiovascular disease (CVD) is a leading cause of death worldwide, with approximately 17.7 million people dying from CVD in 2015. Patients with abnormal left ventricular hypertrophy are at higher risk for CVD. This type of muscle growth is stimulated by multiple factors, but one with a particularly central role is the protein cluster - mechanistic target of rapamycin complex 1 (mTORC1). Tuberin (TSC2), a GTPase-activating protein (GAP), is an intrinsic negative regulator of mTORC1. TSC2 is phosphorylated by many kinases, including Akt, p90RSK, AMP activated kinase (AMPK), and extracellular signaling related kinase (ERK1/2). These transduce metabolic and growth signaling to impact mTORC1 activation in one or the other direction. We recently found that cGMP-activated protein kinase G (PKG) also suppresses mTORC1 activation, and identified S1365 on TSC2 as the critical site modified for this regulation. New data with gain and loss of function S1365 phospho- mutations in vitro and in vivo support this signaling. However, many questions remain. It is unknown if PKG directly phosphorylates TSC2 and/or if other kinases are involved. While preliminary data shows S1365 modification is a potent modifier (in either direction) with growth hormone and hemodynamic stress, whether this indeed serves as a central command switch over all mTORC1 input signaling, and/or if it alters TSC2 translocation to or from the lysosome, a putative key mTORC1 control mechanism, are both unknown. Lastly, S1365 is near multiple phosphorylation sites on TSC2 targeted by AMPK, that also stimulate its GAP activity. This raises potential crosstalk between a metabolic control input to mTORC1 and that via S1365 targeting. In this project, I will address each of these questions. In Aim 1, I use two assays developed by Kevan Shokat that detect if a selective kinase modifies TSC2 directly, or if other kinases are involved. These use a mutated kinase that can accept a bulky ATP, or an ATP-acrylate crosslinker that binds a mutated TSC2 substrate (with serine-cysteine substitution at S1365). Mutated TSC2 S1365A or S1365E KI mice or cultured cells are used to test its impact over alternative kinase inputs into TSC2 control of mTORC1. In Aim 2, I determine if these mutations impact TSC2 translocation to the lysosome upon activation, a key element of its control over mTORC1. Studies use immunofluorescence colocalization with the various TSC2 mutations and mTORC1 stimuli. Aim 3 is translational, and uses an in vivo model to test if a global knock-in S1365A and S1365E mouse has altered responsiveness to AMPK-related modulation of mTOR. This is performed with short term (12 or 24 hrs) food withdrawal or ischemia-reperfusion in the heart. Together, these studies will greatly advance our discovery of a novel tool to modulate mTORC1 signaling and its potential to treat cardiac disease.

项目概要心血管疾病 (CVD) 是全球主要死亡原因，约有 1,770 万人患有心血管疾病 2015年死于CVD。左心室异常肥厚的患者患CVD的风险较高。这肌肉生长的类型受到多种因素的刺激，但其中一个具有特别核心作用的是蛋白质簇 - 雷帕霉素复合物 1 (mTORC1) 的机械靶标。马铃薯蛋白 (TSC2)，一种 GTP 酶激活蛋白 (GAP) 是 mTORC1 的内在负调节因子。 TSC2 被许多激酶磷酸化，包括 Akt、 p90RSK、AMP 激活激酶 (AMPK) 和细胞外信号传导相关激酶 (ERK1/2)。这些转导代谢和生长信号以一个或另一个方向影响 mTORC1 激活。我们最近发现 cGMP 激活的蛋白激酶 G (PKG) 也抑制 mTORC1 激活，并在 TSC2 作为针对此法规进行修改的关键站点。具有功能获得和丧失的新数据 S1365 磷酸化体外和体内的突变支持这种信号传导。然而，仍然存在许多问题。不知道是否PKG 直接磷酸化 TSC2 和/或如果涉及其他激酶。虽然初步数据显示S1365 修饰是一种有效的调节剂（在任一方向），具有生长激素和血流动力学压力，无论是这确实充当所有 mTORC1 输入信号的中央命令开关，和/或如果它改变了 TSC2 与溶酶体的易位（一种假定的关键 mTORC1 控制机制）都是未知的。最后， S1365 靠近 AMPK 靶向的 TSC2 上的多个磷酸化位点，这也会刺激其 GAP 活性。这增加了 mTORC1 的代谢控制输入与通过 S1365 靶向的代谢控制输入之间的潜在串扰。在在这个项目中，我将解决这些问题。在目标 1 中，我使用了 Kevan Shokat 开发的两种检测方法检测选择性激酶是否直接修饰 TSC2，或者是否涉及其他激酶。这些使用突变的可以接受大体积 ATP 的激酶，或结合突变 TSC2 底物的 ATP-丙烯酸酯交联剂（具有 S1365 处的丝氨酸-半胱氨酸取代）。突变的 TSC2 S1365A 或 S1365E KI 小鼠或培养细胞用于测试其对 TSC2 对 mTORC1 控制的替代激酶输入的影响。在目标 2 中，我确定这些是否突变影响 TSC2 在激活后易位至溶酶体，这是其控制的关键因素 mTORC1。研究使用免疫荧光与各种 TSC2 突变和 mTORC1 共定位刺激。 Aim 3是翻译性的，并使用体内模型来测试是否全局敲入S1365A和S1365E 小鼠对 mTOR 的 AMPK 相关调节的反应性发生了改变。这是短期执行的（12或24小时）食物戒断或心脏缺血再灌注。总之，这些研究将极大地推进我们对调节 mTORC1 信号传导的新工具及其治疗心脏病潜力的发现。