Differential modulation of the RNA-binding proteins IRP-1 and IRP-2 in response to iron. IRP-2 inactivation requires translation of another protein.
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 posttranslational 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.
Henderson
B R
BR
Swiss Institute for Experimental Cancer Research, Epalinges s/Lausanne.
Kühn
L C
LC
D016475:3T3 Cells; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology*; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D035925:Iron-Regulatory Proteins; D007700:Kinetics; D007739:L Cells; D008546:Melanoma, Experimental; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008961:Models, Structural; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured
D035925:Iron-Regulatory Proteins; D016601:RNA-Binding Proteins; D003609:Dactinomycin; D008623:Mercaptoethanol; D012313:RNA; D003570:Cytidine Triphosphate; D003513:Cycloheximide; D007501:Iron; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D003676:Deferoxamine