Abstract
Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.
Highlights
Iron is essential for cellular proliferation, excess iron can be toxic due to its ability to generate reactive oxygen species
Ferri- compared with the mock-transfected control (Fig. 5B). These tin-H and ferritin-L mRNA amounts were not significantly results indicate that the Iron regulatory protein 2 (IRP2) Ser-157 Cdk1 phosphorylation altered during cell cycle progression (Fig. 4B), indicating that site is antagonized by Cdc14A and identify a novel in vivo the increase in ferritin abundance during G2/M was due to Cdc14A substrate
G1/S (47), less is known about the regulation of iron homeostasis during the cell cycle
Summary
Plasmids and Site-directed Mutagenesis—The WT rat IRP2 cDNA was C-terminally tagged with 5ϫ-Myc and cloned into pcDNA3.1/Zeo(ϩ) (Invitrogen) with NheI and XbaI. Beads were washed four times with PBS containing 0.05% Nonidet P-40, boiled in SDS-loading buffer, and loaded onto an 8% SDS-PAGE or 4 –12% NuPAGE Bis-Tris gel (Invitrogen). Cells were harvested with Nonidet P-40 lysis buffer, and IRP2-Myc was immunoprecipitated from 2 mg of cellular extract with 10 g of anti-Myc antibody and protein G-agarose. [35S]Methionine/Cysteine Labeling and Immunoprecipitations—HeLa cells synchronized by a double thymidine block were washed with PBS and cultured for the last 1.5 h in Dulbecco’s modified Eagle’s medium (5% fetal bovine serum, Cys-, and Met-free) containing 100 Ci/ml (1175 Ci/mmol) Tran35S-Label (MP Biomedicals, Inc.). Beads were washed four times with PBS containing 0.05% Nonidet P-40, boiled in SDS-loading buffer, and resolved on 8% or 15% SDS-PAGE gels.
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