Role of Tuberin S1365 Phosphorylation in mTORC1 Regulation

项目摘要 心血管疾病(CVD)是全球主要的死亡原因，约有1770万人 2015年死于心血管疾病。有异常左心室肥厚的患者发生心血管疾病的风险更高。这 肌肉生长的类型受到多种因素的刺激，但其中具有特别核心作用的是蛋白质 雷帕霉素复合体1的簇-机械靶点(MTORC1)。GTP酶激活蛋白Tuberin(TSC2) (GAP)，是mTORC1的内在负性调节因子。TSC2被许多激酶磷酸化，包括Akt， P90RSK、AMP激活的激酶(AMPK)和细胞外信号相关激酶(ERK1/2)。这些传感器 代谢和生长信号在一个或另一个方向影响mTORC1的激活。我们最近发现 CGMP激活的蛋白激酶G(PKG)也抑制mTORC1的激活，并发现S1365 TSC2作为针对这一规定修改的关键位点。具有功能得失的新数据S1365磷酸- 体外和体内的突变支持这一信号。然而，许多问题仍然存在。目前尚不清楚PKG是否 直接磷酸化TSC2和/或如果涉及其他激酶。而初步数据显示S1365 修饰是生长激素和血流动力学压力的有效调节剂(无论是从哪个方向)，无论是 这确实充当了所有mTORC1输入信令的中央命令开关，并且/或者如果它改变了TSC2 移位到溶酶体或移位出溶酶体，这是一种可能的关键mTORC1控制机制，这两种机制都是未知的。最后， S1365位于AMPK靶向TSC2上的多个磷酸化位点附近，这也刺激了其GAP活性。 这增加了代谢控制输入到mTORC1和通过S1365靶向输入之间的潜在串扰。在……里面 在这个项目中，我将逐一回答这些问题。在目标1中，我使用了由Kevin Shokat开发的两种分析方法 检测是否有选择性的激酶直接修饰TSC2，或者是否涉及其他的激酶。它们使用的是突变的 可以接受笨重的ATP的激酶，或结合突变的TSC2底物的ATP-丙烯酸酯交联剂(与 S1365处的丝氨酸-半胱氨酸替代)。突变的TSC2 S1365A或S1365E Ki小鼠或培养细胞用于 测试其对TSC2控制mTORC1的替代激酶输入的影响。在目标2中，我确定这些 突变影响TSC2在激活时移位到溶酶体，这是其控制 MTORC1.研究使用免疫荧光共定位与不同的TSC2突变和mTORC1 刺激物。AIM 3是翻译的，并使用体内模型来测试全局敲入S1365A和S1365E 小鼠已经改变了对AMPK相关的mTOR调节的反应性。这是在短期内进行的 (12或24小时)停食或心脏缺血再灌流。总之，这些研究将极大地 推进我们发现一种新的工具来调节mTORC1信号及其治疗心脏病的潜力。

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.