title: IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities.
abstract: In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711-->Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1(S711E) displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1(S711E) retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1(S711E) completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1(S711E) is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.
authors.affiliation: Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 3755 Côte-Ste-Catherine Road, Montréal, Québec, H3T 1E2,Canada.
authors.forename: Carine
authors.initials: C
authors.lastname: Fillebeen
authors.forename: Annie
authors.initials: A
authors.lastname: Caltagirone
authors.forename: Alain
authors.initials: A
authors.lastname: Martelli
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Moulis
authors.forename: Kostas
authors.initials: K
authors.lastname: Pantopoulos
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007501:Iron / Q000494:pharmacology; D035941:Iron Regulatory Protein 1 / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D011485:Protein Binding; D012313:RNA; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014162:Transfection
chemical_list: D012694:Serine; D012313:RNA; D007501:Iron; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1