IRON METABOLISM AND PHOSPHORYLATION OF THE IRE-BP BY PKC

铁是几乎所有人都必须的但具有潜在毒性的营养物质。 活着的有机体。缺铁是最常见的营养不良 人类的缺乏症。与此同时，过多的铁储备一直是 与心血管疾病和某些疾病的发生有关 癌症。铁通过一种细胞因子的作用调节自身的代谢命运 铁反应元件结合的调节性RNA结合蛋白 蛋白质或IRE-BP。IRE-BP与铁响应元件(IRE)结合 在铁蛋白(铁储存蛋白)和TFR(铁吸收蛋白)的mRNAs中 并分别调节它们的翻译或稳定性。IRE-BP 似乎是迄今为止特性不佳的胞浆乌头酸酶。 关于其他因素的作用机制，我们知之甚少 铁可能对IRE-BP功能有调节作用。 我们的总体目标是确定细胞如何在 铁的摄取或代谢命运对其状态变化的反应 分化或扩散的，特别是关于 IRE-BP的磷酸调节作用。我们新奇的观察表明 纯化的IRE-BP和合成肽结合片段 蛋白质在体外可被蛋白激酶C(PKC)磷酸化。二 在IRE-BP中已经确定了可能的PKC磷酸化位点。 佛波醇酯激活PKC导致快速和持续的 对大鼠成纤维细胞中IRE-BP磷酸化的刺激。当HL 60 诱导细胞分化为单核/巨噬细胞 佛波酯PMA观察到IRE RNA的快速和持久的激活 不依赖于IRE-BP从头合成的结合活性 蛋白。我们建议：1)表征磷酸化的影响 用PKC对IRE-BP功能测定中磷酸化改变RNA 铁调节的IRE-BP结合活性和/或能力 2)进行PKC磷酸化的结构-功能研究 站点在IRE-BP中的目标是定义站点定向的效果 PKC第1位和第2位突变对IRE-BP功能的影响；3)用途 这些突变体的生理学相关性研究 蛋白激酶C在调节IRE-BP活性和/或转铁蛋白受体mRNA中的磷酸化 佛波酯对RF2和HL-60细胞的蓄积作用 强调定义铁和铁的监管的程度 改变IRE-BP功能中的磷酸化重叠。由于PKC有一个 我们的研究开始在许多信号转导途径中发挥重要作用 定义一种新的、可能不依赖铁的监管机制 细胞铁新陈代谢。这些研究代表了一个全面的 分子和细胞方法：1)揭示一种新的机制 细胞铁代谢的调节；2)进一步定义模型系统 受调控的RNA-蛋白质相互作用如何影响基因表达；3) 评价磷酸化对铁-S功能或组装的影响 以及4)确定FE-S蛋白和 基因调控中的代谢酶。

Iron is an essential but potentially toxic nutrient for virtually all living organisms. Iron deficiency is the most common nutritional deficiency in humans. At the same time excessive iron stores have been associated with the occurrence of cardiovascular disease and certain cancers. Iron regulates its own metabolic fate through the action of a regulatory RNA binding protein, The Iron Responsive Element Binding Protein or IRE-BP. The IRE-BP binds to Iron Responsive Elements (IRE) in ferritin (iron storage Protein) and TfR (iron uptake protein) mRNAs and regulates their translation or stability, respectively. The IRE-BP appears to be the heretofore poorly characterized cytosolic aconitase. Very little is known concerning the mechanisms by which factors other than iron may modulate IRE-BP function. Our overall goal is to determine how cells program alterations in the uptake or metabolic fate of iron in response to changes in their state of differentiation or proliferation, particularly with regard to phosphoregulation of the IRE-BP. Our novel observations demonstrate that the purified IRE-BP and synthetic peptide fragments of the binding protein can be phosphorylated by protein kinase C (PKC) in vitro. Two putative PKC phosphorylation sites have been identified in the IRE-BP. Activation of PKC by phorbol esters leads to a rapid and sustained stimulation of IRE-BP phosphorylation in rat fibroblasts. When HL 60 cells are induced to differentiate into monocytes/macrophages by the phorbol ester PMA we observe a rapid and prolonged activation of IRE RNA binding activity which is not dependent on de novo synthesis of IRE-BP protein. We propose to: 1) characterize the effects of phosphorylation by PKC on IRE-BP function to determine in phosphorylation alters the RNA binding activity and/or ability of the IRE-BP to be iron-regulated in vitro; 2) perform a structure-function study of the PKC phosphorylation sites in the IRE-BP with the goal of defining the effect of site-directed mutation of PKC site 1 and site 2 on IRE-BP function in vitro; and 3) use these mutants to investigate the physiological relevance of phosphorylation by PKC in regulating IRE-BP activity and/or TfR mRNA accumulation in RF2 and HL 60 cells exposed to phorbol esters with emphasis on defining the extent that regulation by iron and phosphorylation overlap in altering IRE-Bp function. Since PKC has an essential role in many signal transduction pathways our studies begin to define a novel, potentially iron-independent mechanism for the regulation of cellular iron metabolism. These studies represent a comprehensive molecular and cellular approach to: 1) unravel a novel mechanism for the regulation of cellular iron metabolism; 2) further define a model system of how regulated RNA-protein interaction affects gene expression; 3) evaluate how phosphorylation may affect function or assembly of Fe-S clusters; and 4) define the provocative roes of FE-S proteins and metabolic enzymes in gene regulation.