A Phosphomimetic Mutation at Ser-138 Renders Iron Regulatory Protein 1 Sensitive to Iron-Dependent Degradation

Abstract

Proc. Natl. Acad. Sci. USA 95:15235-15240, 1998). Along these lines, we show here that a highly purified preparation of recombinant human IRP1 bearing a phosphomimetic S138E substitution (IRP1 S138E ) lacks aconitase activity, which is a hallmark of [4Fe-4S] cluster integrity. Similarly, IRP1 S138E expressed in mammalian cells fails to function as aconitase. Furthermore, we demonstrate that the impairment of [4Fe-4S] cluster assembly in mammalian cells sensitizes IRP1 S138E to iron-dependent degradation. This effect can be completely blocked by the iron chelator desferrioxamine or by the proteasome inhibitors MG132 and lactacystin. As expected, the stability of wild-type or phosphorylation-deficient IRP1 S138A is not affected by iron manipulations. Ser-138 and flanking sequences appear to be highly conserved in the IRP1s of vertebrates, whereas insect IRP1 orthologues and nonvertebrate IRP1-like molecules contain an S138A substitution. Our data suggest that phosphorylation of Ser-138 may provide a basis for an additional mechanism for the control of vertebrate IRP1 activity at the level of protein stability.The iron regulatory proteins IRP1 and IRP2 are involved in the coordinate posttranscriptional regulation of cellular iron metabolism by binding to mRNA iron-responsive elements (IREs). These are hairpin structures within the 5Ј or 3Ј untranslated regions of a growing family of mRNAs that encode proteins of iron uptake, storage, utilization, and transport, as well as energy metabolism (8,18). Among the best-characterized IRE-containing mRNAs are those encoding the transferrin receptor, which plays a critical role in cellular iron uptake, and ferritin, a protein for intracellular iron storage. IRE-IRP interactions largely account for the reciprocal control of the transferrin receptor and ferritin expression in response to iron perturbations (25).IRP1 and IRP2 are homologous to mitochondrial aconitase (5,8,23), an enzyme of the citric acid cycle, and in fact, IRP1 is its cytosolic counterpart. The active site of aconitase contains a cubane [4Fe-4S] cluster (1). In IRP1, this cluster is assembled within iron-replete cells and prevents IRE binding. However, iron starvation, nitrogen monoxide (NO), and extracellular H 2 O 2 (5, 23) trigger the cluster's destabilization. The resulting switch to apo-IRP1 is associated with the loss of aconitase and acquisition of IRE-binding activity. The mechanism for IRP2 regulation is distinct and does not involve iron-sulfur cluster biochemistry. While IRP2 is stable in iron-starved and hypoxic cells, it undergoes proteasomal degradation in the presence of iron, oxygen (16), or the nitrosonium cation (NO ϩ ) (19). It has been proposed that the activities of IRP1 and IRP2 may also be regulated by phosphorylation (8). The physiological significance of this finding remains elusive. Both IRP1 and IRP2 can be subjected to phosphorylation by protein kinase C (PKC) (9, 27). The rate of IRP1 phosphorylation by PKC is negatively affected by the presence of the iron-sulfur cluster (26). Ex...

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation

“…The fact that only a small portion of c-acon is recruited to IRP1 even in severe iron deficiency (3) suggests that unregulated conversion of this large pool of c-acon into the RNA-binding form would have negative consequences unless a compensatory mechanism limits IRP1 activation. Interestingly, IRP1 can be degraded when the Fe-S switch is inhibited, but the mechanistic links underlying this have not been fully explored (5)(6)(7)(8)(9)(10)(11). These studies suggest that the level to which IRP1 RNA-binding activity is increased in response to inhibition of Fe-S cluster biogenesis depends on the extent to which the apoprotein is degraded.…”

“…On the basis of studies with phosphomimetic mutants of Ser-138 (e.g. IRP1 S138E ), phosphorylation of IRP1 allows its conversion to c-acon (6,7,25), but the Fe-S cluster is much more unstable and, unlike IRP1 S138S , undergoes spontaneous non-oxidative demetallation, allowing its preferential accumulation in the RNA-binding form in cells (6,21). Ser-138 phosphomutants of IRP1 are more sensitive to iron-mediated protein degradation (6,7).…”

Down-regulation of Iron Regulatory Protein 1 Activities and Expression in Superoxide Dismutase 1 Knock-out Mice Is Not Associated with Alterations in Iron Metabolism

“…An interesting hypothetical mechanism of down-regulation of IRP1 in SOD1 Ϫ/Ϫ may involve stimulation of protein kinase C activity by O 2 . (37), phosphorylation of IRP1 at Ser-138, associated with destabilization of its [4Fe-4S] cluster (38), and subsequent increased sensitivity of phosphorylated apo-IRP1 to iron-dependent degradation (39). We also considered the possibility that aberrations in zinc metabolism in SOD1 knockout mice, reported previously in yeast SOD1 mutants (40), could be the cause of altered IRP1 IRE binding.…”

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

“…Finally, we (S.L.C. and R.S.E., unpublished data) and others (14) have found that S138 phosphomimetic mutants of IRP1 undergo iron-dependent degradation.Relatively less is known about the role of S711 in IRP1 phosphoregulation. S711 is located near R699, which is required for citrate͞isocitrate binding, and R728, R732, and other residues involved in RNA binding (15), suggesting that phosphorylation might target one or both of these functions.…”

“…Finally, we (S.L.C. and R.S.E., unpublished data) and others (14) have found that S138 phosphomimetic mutants of IRP1 undergo iron-dependent degradation.…”