Iron-responsive element-binding protein. Phosphorylation by protein kinase C.
The iron-responsive element-binding protein (IRE-BP) is a cytosolic RNA-binding protein that functions in the maintenance of iron homeostasis by post-transcriptionally regulating transferrin receptor and ferritin synthesis. Little is known concerning how factors other than iron may modulate the activity of this central regulator of cellular iron utilization. We present evidence indicating that phosphorylation of the IRE-BP by protein kinase C (PKC) could provide a mechanism for regulation of IRE-BP function. Purified rat liver IRE-BP was phosphorylated by 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 cells. The Km of PKC for the IRE-BP was 0.4 microM. Tryptic phosphopeptide mapping identified one major phosphopeptide plus several other peptides with lesser amounts of phosphate. Synthetic peptides of the IRE-BP 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 high affinity IRE RNA binding activity 2-fold. After 90 min, the level of phosphorylation had increased further, and high affinity IRE RNA binding activity had increased 3-fold above the control. Incorporation of [35S]Met into immunoprecipitable IRE-BP was not altered in cells treated with PMA for 30 or 90 min. PMA also stimulated IRE-BP phosphorylation in rat fibroblasts. Taken together, our studies begin to define a novel mechanism by which hormones, growth factors, and other agents may regulate cellular iron utilization through specific phosphoregulation of the IRE-BP.
Eisenstein
R S
RS
Department of Nutritional Sciences, University of Wisconsin, Madison 53706-1571.
Tuazon
P T
PT
Schalinske
K L
KL
Anderson
S A
SA
Traugh
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D035925:Iron-Regulatory Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D035925:Iron-Regulatory Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011990:Receptors, Transferrin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate