IRON METABOLISM AND PHOSPHORYLATION OF THE IRE-BP BY PKC

铁是一种基本的，但潜在的有毒营养素，几乎所有 活的有机体 缺铁是最常见的营养不良。 人类的缺陷。 与此同时，过量的铁储存已被 与心血管疾病的发生有关， 癌的 铁调节自己的代谢命运，通过行动， 铁反应元件结合蛋白 蛋白质或IRE-BP。 IRE-BP结合铁响应元素（IRE） 铁蛋白（铁储存蛋白）和TfR（铁摄取蛋白）mRNA 并分别调节它们的翻译或稳定性。 IRE-BP 似乎是迄今为止特征不佳的胞质乌头酸酶。 关于其他因素的机制知之甚少。 铁可能调节IRE-BP功能。 我们的总体目标是确定细胞是如何在细胞内编程改变的。 铁的吸收或代谢命运，以响应其状态的变化 分化或增殖，特别是关于 IRE-BP的磷酸调节。 我们的新观察表明， 纯化的IRE-BP和结合的合成肽片段 蛋白质在体外可被蛋白激酶C（PKC）磷酸化。 两 已经在IRE-BP中鉴定了推定的PKC磷酸化位点。 佛波醇酯激活PKC导致快速和持续的 刺激大鼠成纤维细胞中IRE-BP磷酸化。 当HL 60 细胞被诱导分化成单核细胞/巨噬细胞， 我们观察到佛波酯PMA快速和长期激活IRE RNA 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 通过PKC对IRE-BP功能的影响来确定在磷酸化改变RNA IRE-BP的结合活性和/或被铁调节的能力， 2）进行PKC磷酸化的结构-功能研究 IRE-BP中的站点，目的是定义站点定向 PKC位点1和位点2突变对IRE-BP功能的体外影响;和3）应用 研究这些突变体的生理相关性， 通过PKC磷酸化调节IRE-BP活性和/或TfR mRNA 在暴露于佛波酯的RF 2和HL 60细胞中积累， 强调确定铁和 磷酸化重叠改变IRE-Bp功能。 由于PKC具有 我们的研究开始， 定义一种新的，潜在的铁独立的机制， 细胞铁代谢。 这些研究代表了一个全面的 分子和细胞的方法：1）解开一个新的机制， 调节细胞铁代谢; 2）进一步确定模型系统 调节RNA-蛋白质相互作用如何影响基因表达; 3） 评估磷酸化如何影响Fe-S的功能或组装 簇;和4）定义FE-S蛋白的刺激性作用， 代谢酶在基因调控中的作用

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.