title: The iron-sulfur cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites.
abstract: Iron regulatory protein 1 (IRP1) modulates iron metabolism by binding to mRNAs encoding proteins involved in the uptake, storage, and metabolic utilization of iron. Iron regulates IRP1 function by promoting assembly of an iron-sulfur cluster in the apo or RNA binding form, thereby converting it to the active holo or cytoplasmic aconitase form. In continuing our studies on phosphoregulation of IRP1 by protein kinase C (PKC), we noted that the purified apoprotein was more efficiently phosphorylated than was the form partially purified from liver cytosol by chromatography on DEAE-Sepharose which had characteristics of the [3Fe-4S] form of the protein. RNA binding measurements revealed a 20-fold increase in RNA binding affinity and a 4-5-fold higher rate of phosphorylation after removal of the Fe-S cluster from the highly purified [4Fe-4S] form. Phosphorylation of apo-IRP1 by PKC was specifically inhibited by IRE-containing RNA. The RNA binding form had a more open structure as judged by its much greater sensitivity to limited cleavage by a number of proteases. N-Terminal sequencing of chymotryptic peptides of apo-IRP1 demonstrated an increased accessibility to proteolysis of sites (residues 132 and 504) near or within the putative cleft of the protein, including regions that are thought to be involved in RNA binding (residues 116-151) and phosphoregulation (Ser 138). Enhanced cleavage was also observed in the proposed hinge linker region (residue 623) on the surface of the protein opposite from the cleft. Taken together, our results indicate that significant structural changes occur in IRP1 during cluster insertion or removal that affect the accessibility to RNA binding and phosphorylation sites.
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin-Madison, 53706-1571, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Schalinske
authors.forename: S A
authors.initials: SA
authors.lastname: Anderson
authors.forename: P T
authors.initials: PT
authors.lastname: Tuazon
authors.forename: O S
authors.initials: OS
authors.lastname: Chen
authors.forename: M C
authors.initials: MC
authors.lastname: Kennedy
authors.forename: R S
authors.initials: RS
authors.lastname: Eisenstein
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D013455:Sulfur / Q000737:chemistry
chemical_list: D001059:Apoproteins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D010455:Peptides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D008623:Mercaptoethanol; D013455:Sulfur; D007501:Iron; D011493:Protein Kinase C; D002918:Chymotrypsin; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1