RESUMO
Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5' or 3' untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe-4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe-4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe-S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe-4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.
Assuntos
Aconitato Hidratase/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Serina , Aconitato Hidratase/química , Aerobiose , Alanina , Substituição de Aminoácidos , Anaerobiose , Ácido Aspártico , Sítios de Ligação , Citosol/enzimologia , Escherichia coli/metabolismo , Ácido Glutâmico , Proteína 1 Reguladora do Ferro , Proteínas Reguladoras de Ferro , Isoenzimas/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5' or 3' untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação ao Ferro , Proteínas Ferro-Enxofre/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores de Superfície Celular/genética , Receptores da Transferrina/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Proteína 1 Reguladora do Ferro , Proteína 2 Reguladora do Ferro , Proteínas Reguladoras de Ferro , Proteínas Ferro-Enxofre/biossíntese , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/biossínteseRESUMO
Regulation of p53 gene expression at the post-transcriptional level was investigated during growth induction of human peripheral blood mononuclear cells (PBMCs). Freshly isolated PBMCs, which are in the Go phase of the cell cycle, were shown to express low levels of p53 mRNA that was rapidly degraded with a half life of 1 h. The rapid decay of p53 mRNA in quiescent PBMCs was dependent on global protein synthesis as treatment with cycloheximide resulted in stabilization of the p53 message. PBMCs were stimulated to enter the cell cycle by treatment with a combination of the mitogenic lectin phytohemagglutinin (PHA) and phorbol ester (TPA). Progressive stabilization of the p53 message occurred in PBMCs during growth induction. By 24 h of incubation in the presence of PHA and TPA, the half life of p53 mRNA was 6 h and p53 mRNA steady state levels were increased 4.5 to 5.0-fold. p53 protein was not detected in quiescent PBMCs, but was readily detected in PBMCs stimulated for 24 h with PHA and TPA. Stabilization of p53 mRNA was observed in PBMCs treated with either PHA or TPA, but to a lesser degree than when PHA and TPA were used as co-stimulants. These results indicate that differential degradation of p53 messenger RNA occurs in quiescent vs mitogen stimulated PBMCs and suggest that post-transcriptional regulation importantly contributes to increased p53 mRNA steady state levels as PBMCs enter the cell cycle.