Sodium nitroprusside promotes IRP2 degradation via an increase in intracellular iron and in the absence of S nitrosylation at C178.
In iron-replete cells the posttranscriptional regulator IRP2 undergoes ubiquitination and proteasomal degradation. A similar response occurs in cells exposed to sodium nitroprusside (SNP), an NO-releasing drug. It has been proposed that nitroprusside ([Fe(CN)5NO]2-) fails to donate iron into cells and that it promotes IRP2 degradation via S nitrosylation at C178. This residue is located within a stretch of 73 amino acids, earlier proposed to define an iron-dependent degradation domain. Surprisingly, we show that IRP2 bearing a C178S mutation or a Delta73 deletion is sensitive to degradation not only by ferric ammonium citrate (FAC) but also by SNP. Moreover, FAC and SNP attenuate the RNA-binding activities of IRP2 and its homologue IRP1 with similar kinetics. Actinomycin D, cycloheximide, succinylacetone, and dimethyl-oxalylglycine antagonize IRP2 degradation in response to both FAC and SNP, suggesting a common mechanistic basis. IRP2 is not only sensitive to fresh, but also to photodegraded SNP and remains unaffected by S-nitrosoglutathione (GSNO), an established nitrosation agent. Importantly, both fresh and photodegraded SNP, but not GSNO, promote a >4-fold increase in the calcein-accessible labile iron pool. Collectively, these results suggest that IRP2 degradation by SNP does not require S nitrosylation but rather represents a response to iron loading.
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755 Cote-Ste-Catherine Rd., Montreal, Quebec H3T 1E2, Canada.
Jian
J
Wang
Carine
C
Fillebeen
Guohua
G
Chen
Bill
B
Andriopoulos
Kostas
K
Pantopoulos
D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D005290:Ferric Compounds / Q000494:pharmacology; D006801:Humans; D007501:Iron / Q000378:metabolism* / Q000494:pharmacology; D035942:Iron Regulatory Protein 2 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology*; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D005290:Ferric Compounds; D020030:Nitric Oxide Donors; D000644:Quaternary Ammonium Compounds; D009599:Nitroprusside; D007501:Iron; D035942:Iron Regulatory Protein 2; D003545:Cysteine; C013531:ferric ammonium citrate