Regulation of cellular growth in adverse environmental conditions

目的：我的NSERC资助的研究计划的长期目标是获得在不利环境中调节细胞增殖的潜在机制的基本理解。本研究的总体目标是确定由缺氧控制的O2传感机制以及它们如何影响IGF依赖性细胞增殖。背景：雷帕霉素的机制靶点（mTOR）整合来自O2的信号以正向调节细胞生长。缺氧通过抑制mTOR信号传导降低细胞增殖。胰岛素样生长因子I（IGF-I）是一种促进细胞增殖的肽。缺氧和IGF-I信号之间存在联系，但IGF-I信号与mTOR的联系尚不清楚。缺氧诱导IGF结合蛋白-1（IGFBP-1），IGF-I作用的关键负调节因子。磷酸化将IGFBP-1转化为IGF-I作用的生物抑制剂。在缺氧条件下调节IGFBP-1磷酸化和抑制IGF作用的机制在很大程度上还不清楚。进展和理由：我的NSERC资助的研究结果提出了一个新的概念，即缺氧触发位点特异性IGFBP-1过度磷酸化以抑制细胞增殖。我们已经证明mTOR是IGF-I作用的重要调节剂。我2014年NSREC资助的数据表明，缺氧诱导的IGFBP-1磷酸化可能与mTOR有关。我假设缺氧抑制mTOR，mTOR激活蛋白激酶CK 2，用于缺氧诱导的IGFBP-1磷酸化和IGF-I作用的抑制。我将使用HepG 2细胞和技术，例如，RNAi/免疫印迹/MRM-MS和功能蛋白质组学来验证我的假设，3个目标。*目标1. 确定mTORC 1是调节缺氧诱导的IGFBP-1磷酸化所必需的。我们将通过siRNA沉默来鉴定mTORC 1信号传导的上游激活剂，以显示在IGFBP-1磷酸化中mTORC 1抑制的需要以及通过IGF-I作用减少细胞生长。目标2. 确定CK 2在IGFBP-1磷酸化中的作用。我们将使用多种策略，如免疫共沉淀，磷酸化位点定位和CK 2抑制剂抗性突变体，以确定IGFBP-1是真正的CK 2底物。目标3。 确定缺氧时CK 2受mTORC 1调节。我们将在常氧或缺氧条件下沉默CK 2（有/无mTOR 1/C2沉默），以确定mTOR是否调节缺氧条件下CK 2的表达/活性和IGF-I的生物活性。*重要性：这项研究将提供涉及mTOR和CK 2的相互作用细胞机制的基础知识，这些机制控制IGF在缺氧中的作用，对发育，生理和异常过程至关重要。HQP：我实验室的学员将获得蛋白质生物化学，功能蛋白质组学和分子细胞生物学基础方面的强大背景，以获得学术界/工业界/政府实验室的认可/奖学金和职业生涯。

OBJECTIVE: The Long Term Goal of my NSERC-funded research program is to gain a basic understanding of the underlying mechanisms that regulate cellular proliferation in adverse environments. The overall objectives of this research are to determine the O2 sensing mechanism/s controlled by hypoxia and how they affect IGF-dependent cell proliferation.****BACKGROUND: The mechanistic target of rapamycin (mTOR) integrates signals from O2 to positively regulate cell growth. Hypoxia decreases cell proliferation by repressing mTOR signaling. Insulin-like growth factor I (IGF-I) is a peptide that promotes cell proliferation. A link between hypoxia and IGF-I signaling exists, but a link of IGF-I signaling with mTOR is unclear. Hypoxia induces IGF binding protein-1 (IGFBP-1), a key negative regulator of IGF-I action. Phosphorylation converts IGFBP-1 into a bio-inhibitor of IGF-I action. The mechanisms that regulate IGFBP-1 phosphorylation in hypoxia and inhibit IGF action are largely uncharacterized.****PROGRESS AND JUSTIFICATION: Findings from my NSERC-funded research have put-forth a novel concept that hypoxia triggers site-specific IGFBP-1 hyperphosphorylation to inhibit cell proliferation. We have shown that mTOR is an important regulator of IGF-I action. My 2014 NSREC-funded data suggest that hypoxia induced IGFBP-1 phosphorylation may be linked with mTOR. I hypothesize that hypoxia inhibits mTOR, which activates protein kinase CK2 for hypoxia-induced IGFBP-1 phosphorylation and inhibition of IGF-I-action. I will use HepG2 cells and techniques e.g., RNAi/immunoblotting/MRM-MS and functional proteomics to test my hypothesis in 3 Aims.*******Aim1. Determine that mTORC1 is required in regulating hypoxia-induced IGFBP-1 phosphorylation. We will identify upstream activators of mTORC1 signaling using by siRNA silencing to show the requirement of mTORC1 inhibition in IGFBP-1 phosphorylation and reduction of cell growth via IGF-I action.*******Aim 2. Determine the role of CK2 in IGFBP-1 phosphorylation. We will use multiple strategies such as co-immunoprecipitation, phospho-site mapping and a CK2 inhibitor resistant mutant to determine that IGFBP-1 is bona fide CK2 substrate.*******Aim 3. Determine that CK2 is regulated by mTORC1 in hypoxia. We will silence CK2 with/without mTORC1/C2 silencing in normoxia or hypoxia to determine whether mTOR regulates CK2 expression/activity and IGF-I bioactivity in hypoxia.*******SIGNIFICANCE: This research will provide fundamental knowledge of the interactive cellular mechanisms involving mTOR and CK2 that control IGF action in hypoxia, critical for developmental, physiological, and aberrant processes.*******HQP: Trainees in my laboratory will receive a strong background in the fundamentals of protein biochemistry, functional-proteomics, and molecular-cell biology to achieve recognition/scholarships and a career in academia/industry/governmental laboratories.***