Cell Cycle Regulation of IRP2 Phosphorylation During Hematopoiesis

摘要 铁是细胞生长所必需的，因为它是参与DNA合成的蛋白质中的辅因子， 线粒体呼吸和血红蛋白的产生。脊椎动物的铁代谢在后期受到控制 由铁调节蛋白2(Irp2)转录。IRP2与mRNAs中的铁反应元件(IRES)结合 与铁摄取(转铁蛋白受体1)、隔离(铁蛋白)和输出(铁蛋白)有关的蛋白质，以及 调节这些mRNAs的翻译或稳定性。缺铁时，Irp2结合IRES调节mRNA 翻译或稳定性，而在铁充足时，Irp2被FBXL5-SCF泛素连接酶降解。 在这里，我们表明Irp2是由G2/M期Ser157的铁非依赖性磷酸化来调节的，这种磷酸化破坏了 它在有丝分裂过程中与铁蛋白ire mRNA相互作用，并抑制铁蛋白翻译。IRP2-S157A的表达 在Irp2KO-MEFS中，会导致G2/M期延迟并减缓增殖。S157蛋白磷酸化的意义在于 在Ser157突变为Ala157(Irp2A/A)的小鼠中进行了研究。IRP2A/A小鼠表现为巨细胞性贫血， 红系终末分化缺陷、脾肿大和全身铁代谢失调。我们 提出铁蛋白和其他IRE-mRNAs对细胞周期的调节可能提供了一种机制来调节 细胞周期中不稳定的铁池。我们的总体目标是确定S157磷酸化的丢失 扰乱红血球的生成并导致贫血。我们的目标是1)确定IRP2的细胞自主角色- S157蛋白在骨髓移植红细胞生成中的磷酸化及流式细胞术分析 细胞周期状态、增殖、铁和其他参数，2)识别异常调节的机制 WT和Irp2A/A BM体外诱导分化小鼠红细胞在Irp2A/A小鼠体内的应用 细胞，并对红细胞群体进行蛋白质组分析以确定WT和Irp2A/A之间的蛋白质变化 3)建立红系特异性Irp2A/A敲打小鼠模型，用于研究红系终末 差异化。我们预计，我们的研究将为正常和失控提供新的见解 红血球生成。

ABSTRACT Iron is essential for cellular growth due to its role as a cofactor in proteins involved in DNA synthesis, mitochondrial respiration and hemoglobin production. Vertebrate iron metabolism is controlled post- transcriptionally by iron-regulatory protein 2 (Irp2). Irp2 binds to iron-responsive elements (IREs) in the mRNAs of proteins involved in iron uptake (transferrin receptor 1), sequestration (ferritin) and export (ferroportin), and regulates the translation or stability of these mRNAs. During iron deficiency, Irp2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency, Irp2 is degraded by the FBXL5-SCF ubiquitin ligase. Here we show that Irp2 is regulated by iron-independent phosphorylation of Ser157 during G2/M that disrupts its interaction with ferritin IRE mRNA during mitosis and depresses ferritin translation. Expression of Irp2-S157A in Irp2KO-MEFs causes a G2/M delay and slows proliferation. The significance of S157 phosphorylation was investigated in mice where Ser157 was mutated to Ala157 (Irp2A/A)). Irp2A/A mice exhibit macrocytic anemia, defective erythroid terminal differentiation, splenomegaly, and dysregulated systemic iron metabolism. We propose that cell-cycle regulation of ferritin and other IRE-mRNAs may provide a mechanism to modulate the cellular labile iron pool during the cell cycle. Our overall goal is to determine how loss of S157 phosphorylation perturbs erythropoiesis and causes anemia. Our aims are to 1) determine the cell-autonomous role for Irp2- S157 phosphorylation in erythropoiesis by bone marrow (BM) transplantation and flow cytometric analysis of cell-cycle status, proliferation, iron and other parameters, 2) identify mechanisms underlying dysregulated erythropoiesis in Irp2A/A mice using a mouse erythroblast in vitro differentiation system with WT and Irp2A/A BM cells, and proteomic analysis of erythroblast populations to identify protein changes between WT and Irp2A/A cells, and 3) generate an erythroid-specific Irp2A/A knockin mouse model to study erythroid terminal differentiation. We anticipate that our studies will provide new insights of normal and dysregulated erythropoiesis.