Phosphorylation of TSC2 (S1365) as a novel Regulator of mTORC1 Signaling in T Cells

项目摘要 mTOR在整合来自免疫微环境的信号以调节T细胞中起关键作用。 激活、分化和功能。我们已经能够证明， 复合物2（TSC 2）蛋白在调节T细胞中mTORC 1活化中起重要作用。TSC 2是一个 RasGap蛋白，抑制Rheb GT3的活性，进而激活mTORC 1。我们已经表明 T细胞中TSC 2的基因缺失导致mTORC 1活性增强， CD 8 + T细胞效应子功能。然而，尽管TSC 2-/- T细胞对病毒和肿瘤有强烈的反应，但它们的 持续的mTORC 1活性导致记忆CD 8 + T细胞生成减少。最近，卡斯实验室 已经确定了一个新的磷酸化位点TSC 2调节心肌细胞mTORC 1活性。 该位点（S1365）的磷酸化导致mTORC 1信号传导的抑制。突变该位点（S A） 导致mTORC 1活性增加，心脏病恶化， 压力过载（PO）应力。或者，在该位点产生磷酸化模拟物（S β E）减轻了 mTORC 1活性，并在压力超负荷时提供心力衰竭保护。我们假设 TSC 2（S1365）位点可能在调节mTORC 1活性中起重要作用。我们 初步研究表明，在TCR接合时，该位点确实被磷酸化。T细胞 在非刺激条件下具有未改变的mTORC 1活性（不同于TSC 2-/- T细胞），但在TCR接合时显示出显著增加的活性。具有SE突变的T细胞表现出 相反此外，TSC 2（S1365）的磷酸化显著地被缺氧、低pH和缺氧诱导。 活性氧表明，这一途径在整合应激信号中起着关键作用， 以调节T细胞分化和功能。在这个项目中，我们试图定义和理解一部小说， 和T细胞中mTORC 1调节的选择性机制。这项提案的总体目标是剖析 TSC 2在S1365处的磷酸化调节T细胞中mTORC 1活化的机制，以及 从而选择性地调节T细胞活化、分化和功能。在此完成后， 我们的研究结果将有助于阐明新的和关键的mTORC 1调节信号机制，在T 细胞，并对开发疫苗和工程化更强大的T细胞进行免疫治疗具有意义。 细胞疗法这可能反过来导致改善的治疗策略，用于预防和治疗 感染以及癌症。

PROJECT SUMMARY mTOR plays a critical role in integrating signals from the immune microenvironment to regulate T cell activation, differentiation and function. We have been able to demonstrate that the Tuberous Sclerosis Complex 2 (TSC2) protein plays an important role in regulating mTORC1 activation in T cells. TSC2 is a RasGap protein that inhibits the activity of Rheb GTPase that in turn activates mTORC1. We have shown that genetic deletion of TSC2 in T cells leads to enhanced mTORC1 activity and a marked increase in CD8+ T cell effector function. However, while TSC2-/- T cells respond robustly to viruses and tumors, their persistent mTORC1 activity leads to a decrease in memory CD8+ T cell generation. Recently, the Kass lab has identified a novel phosphorylation site on TSC2 that regulates mTORC1 activity in cardiac myocytes. Phosphorylation of this site (S1365) leads to the inhibition of mTORC1 signaling. Mutating this site(SA) leads to an increase mTORC1 activity and the development of worse heart disease and mortality from pressure-overload (PO) stress. Alternatively, creating a phosphomimetic (SE) at this site mitigates mTORC1 activity and imparts protection from heart failure upon pressure overload. We hypothesized that the TSC2 (S1365) site might play an important role in regulating mTORC1 activity in T cells. Our preliminary studies demonstrate that upon TCR engagement this site is indeed phosphorylated. T cells harboring the SA mutation have unaltered mTORC1 activity in the non-stimulated condition (unlike TSC2-/- T cells), but show markedly increased activity upon TCR engagement. T cells with the SE mutation exhibit the opposite. Furthermore, phosphorylation of TSC2 (S1365) is markedly induced by hypoxia, low pH and reactive oxygen species suggesting that this pathway plays a critical role in integrating stress signals in order to regulate T cell differentiation and function. In this project we seek to define and understand a novel and selective mechanism of mTORC1 regulation in T cells. The overall goal of this proposal is to dissect the mechanisms by which phosphorylation of TSC2 at S1365 regulates mTORC1 activation in T cells, and consequently selectively regulates T cell activation, differentiation and function. Upon the completion of this proposal our findings will help elucidate novel and critical mTORC1 regulatory signaling mechanisms in T cells, and have implications for developing vaccines and engineering more robust T cells for Adoptive Cellular Therapy. This may in turn result in improved treatment strategies for preventing and treating infections as well as cancer.