PHOSPHORYLATION AND ACTIVATION OF BOTH IRON REGULATORY PROTEIN-1 AND PROTEIN-2 IN HL-60 CELLS

Iron regulatory proteins (IRPs) are RNA-binding proteins that post-tra nscriptionally regulate synthesis of iron uptake (transferrin receptor ) and storage (ferritin) proteins. Our previous work demonstrating tha t IRP1 is phosphorylated by protein kinase C supported the hypothesis that factors in addition to iron modulate IRP function. We have invest igated changes in activity and expression of both IRP1 and IRP2 during phorbol 12-myristate 13 acetate (PMA)-induced differentiation of HL-6 0 cells. In contrast to IRP1, IRP2 was highly phosphorylated in untrea ted cells. PMA stimulated phosphorylation of IRP1 and IRP2 by at least 2-3-fold without affecting incorporation of [S-35]methionine into the proteins. IRP1 and IRP2 isolated from PMA-treated cells displayed dif ferent phosphopeptides. Phosphorylation of IRPs was associated with a 2-fold increase in high affinity RNA binding activity without altering K-D, and this was accompanied by a 50% increase in transferrin recept or 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 i ron-responsive element binding activity in HL-60 cells without affecti ng the phosphorylation state of IRP1. Hemin appeared to reduce the abu ndance of phosphorylated IRP2. Thus, multiple factors affect the funct ion of both IRPs and indicate that extracellular agents may program ch anges in cellular iron metabolism by altering the phosphorylation stat e of these regulatory RNA-binding proteins.