Rs. Eisenstein et al., IRON-RESPONSIVE ELEMENT-BINDING PROTEIN - PHOSPHORYLATION BY PROTEIN-KINASE-C, The Journal of biological chemistry, 268(36), 1993, pp. 27363-27370
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.