Iron regulatory proteins (IRPs)-1 and -2 bind specific mRNA hairpin structures known as iron-responsive elements and thereby post-transcriptionally regulate proteins involved in iron uptake, storage, and utilization. In this study, we compared modulation of the RNA-binding activities of IRP-1 and IRP-2. We show that in vitro RNA-binding can be inhibited for each IRP by the alkylation of free sulfhydryl groups with N-ethylmaleimide, or by oxidation with diamide. The in vivo iron regulation of IRP-1 and IRP-2 appeared to involve different pathways. Both proteins are activated in Ltk cells following iron chelation. This induction, however, was distinguishable by the addition of translation inhibitors, which temporarily delayed activation of IRP-1 by up to 8 h, but fully blocked IRP-2 induction for up to 20 h. The activation of IRP-2 was also prevented by transcription inhibition with actinomycin D. Further analysis revealed that, while both IRPs are rapidly inactivated following iron treatment of iron-depleted cells, the repression of IRP-2 was again completely translation dependent. Immunoblot analysis suggests that iron modulation of IRP-1 activity is predominantly a post-translational process. This contrasts with IRP-2, whose activation reflected the accumulation of stable IRP-2 protein by de novo synthesis. IRP-2 inactivation/degradation occurred upon readdition of iron, but it required translation of another protein. The existence of an independent regulator of IRP-2 may help explain the differential regulation and expression of the two IRP proteins in different tissues and cell lines.

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