Unexpected role for AMPK and mTORC1 in cellular adaptation to nutrient stress

项目摘要 以保守蛋白激酶AMPK（AMP活化蛋白激酶）为中心的信号网络 和mTOR（雷帕霉素的机械靶点）使细胞能够感知和适当地响应动态的 各种系统和局部线索的波动。能量应激激活AMPK以促进分解代谢， 抑制合成代谢途径以平衡能量供给和需求。AMPK还促进葡萄糖和脂质 体内平衡事实上，AMPK激活药物二甲双胍（又名GlucoPhage）代表了最广泛的 2型糖尿病的治疗方法奇怪的是，AMPK矛盾地作为肿瘤抑制因子或 取决于细胞环境的肿瘤促进剂。营养素（即氨基酸）和生长因子（即IGF/胰岛素） 协同激活mTOR复合物1（mTORC 1），其驱动合成代谢细胞代谢（即蛋白质和脂质 合成）并抑制自噬。在生理学上，异常的mTORC 1信号转导有助于多种 包括癌症和II型糖尿病的疾病，并且mTOR抑制剂被FDA批准用于肾细胞癌， 结节性硬化综合征（TSC）和血管成形术后支架再狭窄。尽管生理和 AMPK和mTORC1的治疗重要性，我们对这些分子水平的理解存在重大差距 细胞信号网络在先前的工作中，我们发现mTOR的磷酸化（在S1261上）促进mTORC 1 信号传导和细胞生长/大小。出乎意料的是，激酶组筛选将AMPK鉴定为mTOR S1261激酶。 AMPK磷酸化mTOR的mTORC1激活位点的发现提出了一个明显的矛盾， 然而，AMPK典型地抑制mTORC 1响应于严重的能量应激。我们的初步结果 调和这一矛盾的发现，并暴露出我们对AMPK及其与 mTORC 1。我们发现，在诱导营养胁迫，特别是氨基酸饥饿后， 通过再摄食或自噬激活AMPK并增加mTOR S1261磷酸化， mTORC1信号传导并以AMPK依赖性方式促进细胞存活。此外，mTOR S1261 磷酸化需要Rheb，一种受TSC负控制的上游mTORC 1激活剂。本提案中 我们研究了AMPK和mTORC 1在细胞适应营养应激中的意想不到的作用。我们 假设AMPK-mTORC1轴的功能是在长期的代谢过程中维持代谢的存活水平， 营养压力或促进代谢恢复时，压力消退。这项研究采用了培养的 细胞和缺乏mTOR S1261磷酸化的CRISPR工程小鼠模型，将提供概念上的 它将改变我们对AMPK在代谢控制中的作用及其与mTORC1的关系的看法。 阐明AMPK对mTORC1的反常激活将促进我们对mTOR和AMPK的理解， 并且可以确定用于治疗相关病症（例如癌症）的新的治疗策略 和代谢性疾病。

Project Summary Signaling networks centered on the conserved protein kinases AMPK (AMP-activated protein kinase) and mTOR (mechanistic target of rapamycin) enable cells to sense and respond appropriately to dynamic fluctuations in a variety of systemic and local cues. Energetic stress activates AMPK to promote catabolic and suppress anabolic pathways to balance energy supply and demand. AMPK also promotes glucose and lipid homeostasis. In fact, the AMPK-activating drug metformin (aka, GlucoPhage) represents the most widely prescribed treatment for type II diabetes. Curiously, AMPK paradoxically functions as a tumor suppressor or tumor promoter depending on cellular context. Nutrients (i.e. amino acids) and growth factors (i.e. IGF/insulin) cooperate to activate mTOR complex 1 (mTORC1), which drives anabolic cell metabolism (i.e. protein and lipid synthesis) and suppresses autophagy. Physiologically, aberrant mTORC1 signaling contributes to diverse disorders including cancer and type II diabetes, and mTOR inhibitors are FDA-approved for renal cell carcinoma, tuberous sclerosis complex (TSC), and stent restenosis following angioplasty. Despite the physiologic and therapeutic importance of AMPK and mTORC1, major gaps exist in our molecular-level understanding of these cell signaling networks. In prior work we showed that phosphorylation of mTOR (on S1261) promotes mTORC1 signaling and cell growth/size. Unexpectedly, a kinome screen identified AMPK as an mTOR S1261 kinase. Finding that AMPK phosphorylates mTOR on an mTORC1-activating site presented an apparent paradox, however, as AMPK canonically inhibits mTORC1 in response to severe energetic stress. Our preliminary results reconcile this paradoxical finding and expose a major gap in our understanding of AMPK and its relationship with mTORC1. We find that after induction of nutrient stress, specifically amino acid starvation, amino acids provided either by re-feeding or autophagy activate AMPK and increase mTOR S1261 phosphorylation, which re-activates mTORC1 signaling and promotes cell survival in an AMPK dependent manner. Moreover, mTOR S1261 phosphorylation requires Rheb, an upstream mTORC1 activator under negative control by TSC. In this proposal we investigate an unexpected role for AMPK and mTORC1 in cellular adaptation to nutrient stress. We hypothesize that the AMPK-mTORC1 axis functions to maintain a survival level of metabolism during prolonged nutrient stress or facilitates metabolic recovery when the stress subsides. This research, which employs cultured cells and a CRISPR-engineered mouse model lacking mTOR S1261 phosphorylation, will provide conceptual advance as it will shift how we think about AMPK in metabolic control and its relationship with mTORC1. Elucidating paradoxical activation of mTORC1 by AMPK will advance our understanding of mTOR and AMPK in health and disease and may identify new therapeutic strategies for treatment of linked disorders, such as cancer and metabolic diseases.