Phosphorylation and Activation of both Iron Regulatory Proteins 1 and 2 in HL-60 Cells

Schalinske, Kevin L.; Eisenstein, Richard S.

doi:10.1074/jbc.271.12.7168

Cited by 122 publications

(135 citation statements)

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“…Our work described here supports the hypothesis that phosphorylation of S711 preferentially affects the aconitase function of IRP1. In contrast, previous work from our laboratory (9)(10)(11)(12)(13) indicates that although S138 phosphorylation greatly destabilizes the Fe-S cluster in IRP1 and consequently indirectly alters c-acon activity, the primary effect of S138 phosphorylation is to alter the set point and mechanism of iron regulation of the RNA-binding activity. Hence, it appears that phosphorylation of S138 or S711 by PKC or other kinases may serve to dictate the use of IRP1͞c-acon in cellular metabolism and regulation.…”

Section: Discussioncontrasting

confidence: 68%

“…Lysate protein (5 g) was blotted as described in ref. 10. Antibodies produced against either IRP1 or the c-myc epitope at the C terminus of IRP1, horseradish peroxidase-coupled second antibody, and a chemiluminescence kit (SuperSignal, Pierce) were used.…”

Section: Methodsmentioning

confidence: 99%

“…Treatment of HL-60 cells with a PKC activator increases IRP1 phosphorylation and RNA-binding activity, with a concurrent increase in transferrin receptor mRNA level (9,10). Two PKC phosphorylation sites, S138 and S711, have been proposed in IRP1 (9).…”

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Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Pitula

Deck

Clarke

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Iron-regulatory protein 1 (IRP1) is a dual-function protein with mutually exclusive roles as a posttranscriptional regulator of animal-cell iron metabolism or as the cytosolic isoform of the ironsulfur enzyme aconitase (c-acon). Much effort has focused on the role of IRP1 in posttranscriptional gene regulation and in factors that influence its interconversion with c-acon, but little is known about the metabolic function and regulation of c-acon. The role of PKC-dependent phosphorylation of S711 on IRP1͞c-acon function was examined. Phosphorylation state-specific antibodies revealed that S711 is phosphorylated by PKC in vitro and in human embryonic kidney cells treated with a PKC activator. In aco1 yeast, the phosphomimetic mutants S711D and S711E exhibited severely impaired aconitase function, whereas S711A and S711T were unaffected relative to the WT protein. Aconitase activity in yeast extracts displayed a similar pattern when assayed for capacity to convert citrate to isocitrate: WT, S711A, and S711T were active, but S711D and S711E activity was undetectable. In contrast, when measured by the conversion of isocitrate to cis-aconitate, S711D and S711E displayed substantial activity, indicating that phosphorylation impairs the citrate but not isocitrate mode of aconitase function. This possibility was confirmed in vivo by demonstrating that S711D and S711E specifically antagonized the requirement for isocitrate in two metabolic scenarios. Iron-responsive element RNA-binding affinity was unaffected by S711 mutations. Our results show that S711 is a target of phosphorylation capable of conferring distinct effects on c-acon function potentially dictating changes in cytosolic citrate͞isocitrate metabolism. I t is critical that organisms balance the metabolic requirement for iron with its potential toxicity. Vertebrates possess an elegant system to sense cellular iron status, regulate the uptake and metabolic fate of iron, and thereby maintain iron homeostasis. Iron-regulatory protein 1 (IRP1) and IRP2 are sensory components of this critical regulatory network that promote increased iron uptake when cells are iron depleted and storage of iron when cells are iron overloaded (1, 2). IRPs control mRNA fate by binding to iron-responsive elements (IRE) in the untranslated regions of mRNA encoding proteins that control iron homeostasis. The RNA-binding activity of IRP can be regulated by multiple factors. Iron promotes the loss of IRP1 RNA-binding activity through insertion of a [4Fe-4S] cluster into the protein, converting it to the cytosolic isoform of the tricarboxylic acid (TCA) cycle enzyme aconitase (c-acon). Solvent accessibility of the Fe-S cluster in aconitases allows for perturbants to promote loss of the cluster from c-acon, converting it to IRP1 (1, 2). Consequently, reactive species such as NO and H 2 O 2 are able to promote removal of the Fe-S cluster, favoring generation of IRP1 from c-acon, although H 2 O 2 may do so through direct and indirect mechanisms (reviewed in refs. 1 and 2).Whereas much is known a...

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Section: Discussioncontrasting

confidence: 68%

Section: Methodsmentioning

confidence: 99%

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Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Pitula

Deck

Clarke

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…IRP-1 can therefore directly sense and respond to perturbations of iron levels and redox potential. Moreover, phosphorylation of IRP-1 may also be involved in the regulation of its two functions because it preferentially occurs in the apoprotein (25,26). Unlike other enzymes that recognize mRNA (1), the functional implication of the cytoplasmic aconitase activity of IRP-1 is not well understood.…”

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confidence: 99%

Ligand-induced Structural Alterations in Human Iron Regulatory Protein-1 Revealed by Protein Footprinting

Gegout

Schlegl

Schläger³

et al. 1999

Journal of Biological Chemistry

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Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.Intracellular iron homeostasis in mammals and many other eukaryotes is controlled at the post-transcriptional level by two related cytoplasmic proteins, the iron regulatory proteins (IRP-1 and IRP-2) 1 (for reviews see Refs. 1 and 2). These two proteins bind to a conserved RNA stem-loop structure, the so-called iron-responsive element. An increasing number of mRNAs have been shown to carry one IRE motif in the 5Ј-untranslated regions of mRNAs encoding ferritin (L-and Hchains), erythroid 5-aminolevulinate synthase, mitochondrial aconitase, and succinate dehydrogenase, as well as in the 3Ј-untranslated region of transferrin receptor (TfR) and Nramp2 mRNAs, thus modulating their expression according to iron availability (3-12). For instance, the interaction of IRP with a single IRE in the 5Ј-untranslated region of ferritin mRNA inhibits translation by preventing ribosome binding (13-15). On the other hand, IRP binding to the five IREs present in the 3Ј-untranslated region of TfR mRNA stabilizes the message against degradation (10, 16). Therefore, the specific interaction between IRP and mRNA ensures a coordinated regulation of the expression of proteins involved in the uptake (TfR), storage (ferritin), and utilization (erythroid 5-aminolevulinate synthase) of iron (2). Because the IRPs appear to regulate two enzymes of the citric acid cycle, they may also play an important role in mediating iron regulation of mitochondrial energy production (9, 10).IRP-1 and IRP-2 are affected by iron in different manners. Whereas iron increases the rate of degradation of IRP-2 (17, 18), i...

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“…Both IRP1 and IRP2 are phosphorylated in vivo, apparently through the action of protein kinase C (PKC) (17,18). Treatment of cells with PKC stimulators results in an increase in IRE binding activity and TfR mRNA abundance concomitant with an increase in IRP phosphorylation (17,18).…”

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confidence: 99%

The Aconitase Function of Iron Regulatory Protein 1

Narahari¹,

Wang

et al. 2000

Journal of Biological Chemistry

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Iron regulatory proteins (IRP) are sequence-specific RNA-binding proteins that mediate iron-responsive gene regulation in animals. IRP1 is also the cytosolic isoform of aconitase (c-aconitase). This latter activity could complement a mitochondrial aconitase mutation (aco1) in Saccharomyces cerevisiae to restore glutamate prototrophy. In yeast, the c-aconitase activity of IRP1 was responsive to iron availability in the growth medium. Although IRP1 expression rescued aco1 yeast from glutamate auxotrophy, cells remained growth-limited by glutamate, displaying a slow-growth phenotype on glutamate-free media. Second site mutations conferring enhanced cytosolic aconitase-dependent (ECA) growth were recovered. Relative c-aconitase activity was increased in extracts of strains harboring these mutations. One of the ECA mutations was found to be in the gene encoding cytosolic NADP ؉ -dependent isocitrate dehydrogenase (IDP2). This mutation, an insertion of a Ty delta element into the 5 region of IDP2, markedly elevates expression of Idp2p in glucose media. Our results demonstrate the physiological significance of the aconitase activity of IRP1 and provide insight into the role of c-aconitase with respect to iron and cytoplasmic redox regulation. The iron regulatory proteins (IRP)1 are a small family of sequence-specific RNA-binding proteins that mediate gene regulation by binding to iron-responsive elements (IRE) located in either the 5Ј-or 3Ј-untranslated region of a variety of animal cell mRNAs. IRE-containing mRNAs encode proteins of iron storage and transport as well as proteins involved in iron utilization or intermediary metabolism (for review see Refs. 1 and 2). IRPs exert their effect through translational regulation (for ferritin, m-aconitase, and erythroid aminolevulinate synthase (eALAS) mRNAs) or by controlling mRNA stability (for transferrin receptor (TfR) mRNA), depending on the location of the IRE (1-3). Two IRPs have been identified to date, called IRP1 and IRP2 (4 -7). Both bind similar IRE sequences and appear to be capable of mediating iron-responsive gene regulation, although each has a distinct IRE preference (8 -10). IRP1 is a bifunctional protein, having activity as an IREbinding protein (IRE-BP) or as the cytosolic isoform of aconitase (c-aconitase) (1, 11). The two activities of IRP1 are mutually exclusive (1, 11-13). Interconversion of IRP1 between an IRE-BP and c-aconitase is itself regulated by iron, through the assembly/disassembly of a [4Fe-4S] cluster (1, 11-13). Fe-S cluster assembly occurs under conditions of excess iron, converting IRP1 to c-aconitase and stimulating synthesis of ferritin, m-aconitase, and eALAS, while TfR synthesis is depressed. Iron depletion promotes cluster disassembly, conversion of caconitase to IRE-BP, and repression of ferritin, m-aconitase, and eALAS synthesis, while stimulating TfR expression. In contrast to IRP1, IRP2 has only the IRE binding activity and is regulated by iron through protein degradation via the proteasome pathway (1, 14 -16).The control of IRP ...

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Phosphorylation and Activation of both Iron Regulatory Proteins 1 and 2 in HL-60 Cells

Cited by 122 publications

References 50 publications

Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Selective inhibition of the citrate-to-isocitrate reaction of cytosolic aconitase by phosphomimetic mutation of serine-711

Ligand-induced Structural Alterations in Human Iron Regulatory Protein-1 Revealed by Protein Footprinting

The Aconitase Function of Iron Regulatory Protein 1

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