Iron-independent phosphorylation of iron regulatory protein 2 regulates ferritin during the cell cycle

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.

FIGURE 1.

Identification of an in vivo iron-independent IRP2 phosphorylation site. A, HEK293T cells transfected with IRP2-Myc were treated with DFO or FAC and labeled with 2.5 mCi/ml [³²P]orthophosphate. Immunoprecipitated IRP2-Myc was separated by SDS-PAGE, excised from the dried gel, eluted, digested with trypsin, and analyzed by two-dimensional phosphopeptide mapping. The results are representative of four independent experiments. B, phosphopeptide 1 from FAC-treated cells was eluted, hydrolyzed, and electrophoresed in two-dimensions with phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) standards that were visualized by ninhydrin staining. C, phosphopeptide 1 from trypsin or Lys-C digestion was subjected to solid-phase Edman degradation. Radioactive phosphate release was detected by Cerenkov counting. D, sequence analysis of IRP2 yielded three conserved candidate phosphopeptides with a serine (S) in position 5 of a complete (▾) or incomplete (▿) digest. E, HEK293T cells transfected with WT, S503A, S407A, or S157A IRP2-Myc were labeled with 1 mCi/ml [³²P]orthophosphate. Immunoprecipitated IRP2-Myc was analyzed by SDS-PAGE. The results in B, C, and E are representative of two independent experiments.

FIGURE 2.

IRP2 Ser-157 is phosphorylated by Cdk1 during mitosis. Stably expressed WT or S157A FLAG-IRP2 was immunoprecipitated from Flp-In T-REx-293 cells and incubated with recombinant Cdk1/cyclin B1 (A) or Cdk2/cyclin A (B) and [γ-³²P]ATP. Phosphorylated FLAG-IRP2 was separated by SDS-PAGE and analyzed by phosphorimaging and anti-FLAG Western blot. C, HeLa cells were grown asynchronously (Asyn) or treated with nocodazole for 18 h. Nocodazole was removed, and cells were released and harvested at the indicated times for Western blot analysis with anti-IRP2-pS157, anti-IRP2, anti-H3-pS10, anti-cyclin B1, and anti-β-tubulin antibodies. D, HEK293 cells were grown asynchronously (Asyn) or arrested in mitosis by treatment with nocodazole (Noc) for 18 h. The Cdk inhibitor purvalanol A (PA) (10 μm) was added for the last 2 h of treatment. Lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-IRP2-pS157, anti-IRP2, anti-Cdk1-pY15, anti-Cdk1, anti-cyclin B1, and anti-β-tubulin antibodies. E, HEK293 cells were treated with 10 μm purvalanol A for the last 30, 60, or 120 min of an 18-h nocodazole treatment. Lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-IRP2-pS157, anti-IRP2, and anti-β-tubulin antibodies. All results are representative of at least two independent experiments.

FIGURE 3.

IRP2 Ser-157 phosphorylation during mitosis is associated with reduced RNA-binding activity and altered ferritin and TfR1 expression. HeLa cells were synchronized at the G₁/S border by a double-thymidine block or left asynchronous (Asyn). A, cells were released for the indicated times and fixed for flow cytometric analysis of DNA content. G₁ phase is indicated by the 2N peak (black), S phase is indicated by the cross-hatched peak (gray), and G₂/M phases are indicated by the 4N peak (black). B, duplicate plates of cells were harvested for Western blot analysis with anti-IRP2-pS157, anti-IRP2, anti-cyclin B1, anti-H3-pS10, anti-ferritin, anti-TfR1, and anti-β-tubulin antibodies. The results in A and B are representative of five independent experiments. A cross-reacting band is indicated by an asterisk. C, extracts were used in an electrophoretic mobility shift assay with a ³²P-labeled ferritin-L IRE probe and supershifted with anti-IRP2 antibody. RNA complexes were separated by non-denaturing PAGE and visualized by phosphorimaging. A representative gel from three independent experiments is shown. D, IRP2 RNA-binding activity was normalized to IRP2 protein, which was normalized to β-tubulin protein, and plotted as the percentage of RNA binding in G₁ (0 h). The average Ft-L IRP2 RNA-binding activity was 83.1% (±7.1%) during S phase (6 h) and 41.3% (±6.6%) during G₂/M (10 h). The bar graph is the result of three independent experiments ± S.D. *, p < 0.01; **, p < 0.02, as determined by unpaired two-tailed Students t test.

FIGURE 4.

Regulation of IRE-containing mRNAs during the cell cycle. Ferritin (A) and TfR1 (C) synthesis were measured throughout the cell cycle by labeling cells with ³⁵S-Met/Cys for the last 1.5 h of double thymidine release. FAC is cells treated (50 μg/ml) for 3 h and labeled with ³⁵S-Met/Cys for the last 1.5 h. Immunoprecipitated ferritin or TfR1 was separated by SDS-PAGE and analyzed by phosphorimaging. Control immunoprecipitation lacking antibody is indicated by (–). FTH and FTL (B) and TfR1 (D) mRNA levels during the cell cycle were determined by real-time qRT-PCR. Triplicate reactions were performed for each double thymidine release time point and data were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. The graphs were generated from three independent experiments and represent fold increase relative to 0 h after double thymidine release. Data are presented as mean ± S.E. Statistical significance of the differences between time points within an individual transcript was determined by a one-way analysis of variance: *, p ≤ 0.0027.

FIGURE 5.

Cdc14A associates with and dephosphorylates IRP2. A, Flp-In T-REx-293 cells stably expressing WT or S157A FLAG-IRP2 were transiently transfected with catalytically inactive (CI) Myc-Cdc14A-CI. Cells were treated with nocodazole for 18 h, and lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates and lysates were resolved by SDS-PAGE and analyzed by Western blotting with anti-FLAG or anti-Myc antibodies. The results are representative of two independent experiments. B, HEK293 cells were transiently transfected with Myc-Cdc14A-WT or Myc-Cdc14A-CI and treated with nocodazole for 18 h. Lysates were separated by SDS-PAGE and analyzed by Western blotting with anti-IRP2-pS157, anti-IRP2, anti-Myc, and anti-β-tubulin antibodies. The results are representative of three independent experiments. C, Cdc14A IRE and non-IRE mRNA levels during the cell cycle were determined by real-time qRT-PCR as described in Fig. 4.