IRON-RESPONSIVE ELEMENT-BINDING PROTEIN - PHOSPHORYLATION BY PROTEIN-KINASE-C

The iron-responsive element-binding protein (IRE-BP) is a cytosolic RN A-binding protein that functions in the maintenance of iron homeostasi s by post-transcriptionally regulating transferrin receptor and ferrit in synthesis. Little is known concerning how factors other than iron m ay modulate the activity of this central regulator of cellular iron ut ilization. We present evidence indicating that phosphorylation of the IRE-BP by protein kinase C (PKC) could provide a mechanism for regulat ion of IRE-BP function. Purified rat liver IRE-BP was phosphorylated b y PKC up to 1.3 mol of phosphate/mol of protein with Ser the modified amino acid. Ser was also the phosphoacceptor in the IRE-BP in intact c ells. The K(m) of PKC for the IRE-BP was 0.4 muM. Tryptic phosphopepti de mapping identified one major phosphopeptide plus several other pept ides with lesser amounts of phosphate. Synthetic peptides of the IRE-B P containing Ser 138 (site A) and Ser 711 (site B) were phosphorylated by PKC. In HL 60 cells, addition of phorbol 12-myristate 13-acetate ( PMA) stimulated IRE-BP phosphorylation within 30 min and increased hig h affinity IRE RNA binding activity 2-fold. After 90 min, the level of phosphorylation had increased further, and high affinity IRE RNA bind ing activity had increased 3-fold above the control. Incorporation of [S-35]Met into immunoprecipitable IRE-BP was not altered in cells trea ted with PMA for 30 or 90 min. PMA also stimulated IRE-BP phosphorylat ion in rat fibroblasts. Taken together, our studies begin to define a novel mechanism by which hormones, growth factors, and other agents ma y regulate cellular iron utilization through specific phosphoregulatio n of the IRE-BP.