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

Abstract

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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