Inhibition of an iron-responsive element/iron regulatory protein-1 complex by ATP binding and hydrolysis.
Iron regulatory protein-1 binding to the iron-responsive element of mRNA is sensitive to iron, oxidative stress, NO, and hypoxia. Each of these agents changes the level of intracellular ATP, suggesting a link between iron levels and cellular energy metabolism. Furthermore, restoration of iron regulatory protein-1 aconitase activity after NO removal has been shown to require mitochondrial ATP. We demonstrate here that the iron-responsive element-binding activity of iron regulatory protein is ATP-dependent in HepG2 cells. Iron cannot decrease iron regulatory protein binding activity in cell extracts if they are simultaneously treated with an uncoupler of oxidative phosphorylation. Physiologic concentrations of ATP inhibit iron-responsive element/iron regulatory protein binding in cell extracts and binding of iron-responsive element to recombinant iron regulatory protein-1. ADP has the same effect, in contrast to the nonhydrolyzable analog adenosine 5'-(beta,gamma-imido)triphosphate, indicating that in order to inhibit iron regulatory protein-1 binding activity, ATP must be hydrolyzed. Indeed, recombinant iron regulatory protein-1 binds ATP with a Kd of 86+/-17 microM in a filter-binding assay, and can be photo-crosslinked to azido-ATP. Upon binding, ATP is hydrolyzed. The kinetic parameters [Km=5.3 microM, Vmax=3.4 nmol.min(-1).(mg protein)(-1)] are consistent with those of a number of other ATP-hydrolyzing proteins, including the RNA-binding helicases. Although the iron-responsive element does not itself hydrolyze ATP, its presence enhances iron regulatory protein-1's ATPase activity, and ATP hydrolysis results in loss of the complex in gel shift assays.
Popovic
Zvezdana
Z
Laboratory Medicine and Pathobiology, University of Toronto, Canada.
Templeton
Douglas M
DM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007501:Iron / Q000378:metabolism*; D035941:Iron Regulatory Protein 1 / Q000378:metabolism*; D010085:Oxidative Phosphorylation; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020218:Response Elements
D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007501:Iron; D035941:Iron Regulatory Protein 1