IRON METABOLISM AND PHOSPHORYLATION OF THE IRE-BP BY PKC

铁对几乎所有人来说都是必不可少的，但也有潜在的毒性

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.