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

项目总结 MTOR在整合来自免疫微环境的信号以调节T细胞方面起着关键作用 激活、分化和功能。我们已经能够证明结节性硬化症 复合体2(TSC2)蛋白在调节T细胞mTORC1活化中起重要作用。TSC2是一种 RasGap蛋白，抑制Rheb GTP酶的活性，进而激活mTORC1。我们已经展示了 T细胞中TSC2的基因缺失导致mTORC1活性增强，并显著增加 CD8+T细胞效应器功能。然而，尽管TSC2-/-T细胞对病毒和肿瘤有很强的反应，但他们的 持续的mTORC1活性会导致记忆性CD8+T细胞生成减少。最近，卡斯实验室 已经在TSC2上发现了一个新的磷酸化位点，它调节心肌细胞中mTORC1的活性。 该位点(S1365)的磷酸化导致mTORC1信号的抑制。突变这个位点(SA) 导致mTORC1活性增加，并导致更严重的心脏病和死亡率 压力-过载(PO)应力。或者，在此站点创建仿磷酸盐(SE)可缓解 MTORC1的活动，并提供保护，防止心力衰竭的压力超负荷。我们假设 TSC2(S1365)位点可能在调节T细胞mTORC1活性中起重要作用。我们的 初步研究表明，在TCR参与时，这个位点确实被磷酸化了。T细胞 携带SA突变的人在非刺激条件下mTORC1活性没有改变(与TSC2-/-不同 T细胞)，但在TCR参与时活性显著增加。带有SE突变的T细胞表现出 恰恰相反。此外，TSC2(S1365)的磷酸化在低氧、低pH和 活性氧物种表明，这一途径在整合胁迫信号方面发挥着关键作用 以调节T细胞的分化和功能。在这个项目中，我们试图定义和理解一部小说 T细胞mTORC1调控的选择性机制。这项提案的总体目标是剖析 S1365处TSC2的磷酸化调节T细胞mTORC1激活的机制，以及 从而选择性地调节T细胞的激活、分化和功能。在完成这项工作后 我们的发现将有助于阐明T细胞中新的和关键的mTORC1调控信号机制 细胞，并对开发疫苗和设计更强大的T细胞用于收养具有重要意义 细胞疗法。这反过来可能导致改进预防和治疗的治疗策略。 感染和癌症一样重要。

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.