Phosphorylation and activation of both iron regulatory proteins 1 and 2 in HL-60 cells.
Iron regulatory proteins (IRPs) are RNA-binding proteins that post-transcriptionally regulate synthesis of iron uptake (transferrin receptor) and storage (ferritin) proteins. Our previous work demonstrating that IRP1 is phosphorylated by protein kinase C supported the hypothesis that factors in addition to iron modulate IRP function. We have investigated changes in activity and expression of both IRP1 and IRP2 during phorbol 12-myristate 13-acetate (PMA)-induced differentiation of HL-60 cells. In contrast to IRP1, IRP2 was highly phosphorylated in untreated cells. PMA stimulated phosphorylation of IRP1 and IRP2 by at least 2-3-fold without affecting incorporation of [35S]methionine into the proteins. IRP1 and IRP2 isolated from PMA-treated cells displayed different phosphopeptides. Phosphorylation of IRPs was associated with a 2-fold increase in high affinity RNA binding activity without altering KD, and this was accompanied by a 50% increase in transferrin receptor mRNA abundance. PMA acted on a latent pool of binding activity that is present in a nonaconitase oxidized form and is largely composed of a stable but inactive species of IRP2. Desferal and hemin modulated iron-responsive element binding activity in HL-60 cells without affecting the phosphorylation state of IRP1. Hemin appeared to reduce the abundance of phosphorylated IRP2. Thus, multiple factors affect the function of both IRPs and indicate that extracellular agents may program changes in cellular iron metabolism by altering the phosphorylation state of these regulatory RNA-binding proteins.
Schalinske
K L
KL
Department of Nutritional Sciences, University of Wisconsin, Madison, 53706-1571, USA.
Eisenstein
R S
RS
D000154:Aconitate Hydratase / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D004789:Enzyme Activation; D018922:HL-60 Cells; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000187:drug effects / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011990:Receptors, Transferrin; D006427:Hemin; D011494:Protein Kinases; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1; D035942:Iron Regulatory Protein 2; D003676:Deferoxamine; D013755:Tetradecanoylphorbol Acetate