| Iron regulatory proteins (IRP) are principal participants in a sensory and regulatory system that post-transcriptionally regulates the uptake, storage and utilization of iron in vertebrates. IRP1 is a bifunctional protein that exists either as the cytosolic aconitase (c-aconitase), the holo-protein containing a [4Fe-4S] cluster or as the genetically active IRP1 apoprotein when it lacks the cluster. The sequence- and structure-specific binding of IRP1 to iron responsive elements (IREs), found in the untranslated regions of mRNA encoding proteins involved in cellular iron metabolism, regulates mRNA translation or stability. Cellular iron status is the best established determinant of IRP1 activity, regulating RNA binding by the assembly and disassembly of the [4Fe-4S] cluster. Many other factors, including reactive oxygen and nitrogen species, can also regulate the stability of the [4Fe-4S] cluster, thus altering IRP1 protein function. However in the absence of appropriate cluster assembly or stability, as likely occurs in oxidative stress, diseases affecting Fe-S cluster biogenesis, or in response to serine 138 (Ser 138) phosphorylation, there is a requirement for cluster-independent regulation of IRP1 to avoid the deleterious consequences of excess accumulation of IRE RNA binding activity.;We investigated the mechanism through which Ser138 phosphorylation, heme and non-heme iron control the degradation of IRP1. IRP1, but not c-aconitase, was the preferred substrate for Ser 138 phosphorylation in cells. Iron deficiency promoted phosphorylation of IRP1 at Ser138 by increasing availability of the IRP1 apoprotein. Ser138 phosphorylated IRP1 was shown to be polyubiquitinated even in iron deficiency in cells suggesting that phosphorylation alone initiates proteasomal degradation of IRP1. Hemin stimulates polyubiquitination of Ser138 phosphorylated IRP1. A structure function analysis identified key cysteine (Cys) residues that are required for IRP1 degradation. Mutation of these Cys to alanine revealed they are required for regulation of IRP1 stability by heme but not non-heme iron, suggesting that multiple pathways control the degradation of the IRP1. Heme was shown to bind IRP1 in a saturable manner and mutations of specific Cys in IRP1 abrogated heme binding. Taken together, these studies indicate that IRP1 is regulated by multiple iron-dependent pathways to control accumulation of RNA binding activity. |
