Mitogen-activated protein kinase phosphatase 2 regulates histone H3 phosphorylation via interaction with vaccinia-related kinase 1

Abstract

Mitogen-activated protein kinase phosphatase 2 (MKP2) is a member of the dual-specificity MKPs that regulate MAP kinase signaling. However, MKP2 functions are still largely unknown. In this study, we showed that MKP2 could regulate histone H3 phosphorylation under oxidative stress conditions. We found that MKP2 inhibited histone H3 phosphorylation by suppressing vaccinia-related kinase 1 (VRK1) activity. Moreover, this regulation was dependent on the selective interaction with VRK1, regardless of its phosphatase activity. The interaction between MKP2 and VRK1 mainly occurred in the chromatin, where histones are abundant. We also observed that the protein level of MKP2 and its interaction with histone H3 increased from G1 to M phase during the cell cycle, which is similar to the VRK1 profile. Furthermore, MKP2 specifically regulated the VRK1-mediated histone H3 phosphorylation at M phase. Taken together, these data suggest a novel function of MKP2 as a negative regulator of VRK1-mediated histone H3 phosphorylation.

FIGURE 1:

MKP2 is involved in histone H3 dephosphorylation in oxidative stress conditions. (A) SH-SY5Y cells were treated with 200 μM H₂O₂ for indicated times and harvested. The cell lysates were subjected to immunoblotting with the indicated antibodies. p53 was used as an indicator of oxidative stress. (B) SH-SY5Y cells were transfected with siRNA targeting MKP2 or scrambled control siRNA and cultured for 48 h. After treatment with 200 μM H₂O₂ for the indicated times, cells were harvested and lysed. The cell lysates were then subjected to immunoblotting with the indicated antibodies.

FIGURE 2:

MKP2 inhibits VRK1-mediated histone H3 phosphorylation. (A) GST-MKP2 or GST-CI-MKP2 inhibits phosphorylation of histone H3 in a VRK1 kinase reaction in a concentration-dependent manner. GST-VRK1, purified core histone, and MKP2 or CI-MKP2 proteins were coincubated in a kinase reaction for 30 min as indicated. Purified MKP2 or CI-MKP2 proteins were used, with the following concentrations: 250 ng for lanes 3 and 8; 500 ng for lanes 4 and 9. Immunoblotting was performed using the indicated antibodies to analyze the phosphorylation of histone H3. (B and C) MKP2 does not regulate histone H3 phosphorylation by other mitotic histone kinases, such as haspin (B) or Aurora B (C). Histone kinase, core histone, and MKP2 or CI-MKP2 proteins were coincubated in a kinase reaction for 30 min as indicated. Purified MKP2 or CI-MKP2 proteins were used, with the following concentrations: 250 ng for lanes 4 and 9; 500 ng for lanes 4 and 9. Immunoblotting was performed using indicated antibodies to analyze phosphorylation of histone H3. (D) Purified GST, GST-VRK1, GST-MKP2, or GST-CI-MKP2 proteins were coincubated with histone H3 as a substrate for VRK1 in a kinase reaction containing [γ-³²P] ATP for 30 min as indicated. Purified MKP2 or CI-MKP2 proteins were used, with the following concentrations: 100 ng for lanes 2 and 5; 250 ng for lanes 3 and 6; 500 ng for lanes 4 and 7. Phosphorylations were determined by autoradiography. The protein quantities were determined by SYPRO-Ruby staining.

FIGURE 3:

MKP2 regulates VRK1-mediated histone H3 phosphorylation regardless of its phosphatase activity. (A) A549 cells were transfected with EGFP or EGFP-MKP2 or EGFP-CI-MKP2 for 24 h. Immunoblotting was performed with indicated antibodies. (B) A549 cells were transfected with HA or HA-VRK1 and EGFP-MKP2 or EGFP-CI-MKP2 for 24 h. Immunoblotting was performed with indicated antibodies. (C) A549 cells were transfected with siRNA targeting VRK1 or scrambled control siRNA and EGFP or EGFP-MKP2, and then were harvested 48 h after transfection. Cell lysates were immunoblotted with indicated antibodies.

FIGURE 4:

MKP2 cannot directly dephosphorylate histone H3. (A) GST-VRK1 and core histone were coincubated in a kinase reaction for 30 min. After 30 min, MKP2 or CI-MKP2 was added to the reaction, and the kinase reaction was continuously performed for 30 min, as indicated. The amounts of phosphorylation were determined by autoradiography. The protein quantities were determined by SYPRO-Ruby staining. (B–D) Phosphorylated histone H3 was obtained by coincubating histone H3 with the indicated histone kinases (B, VRK1; C, haspin; D, Aurora B) in the kinase reaction, and histone H3 was pulled down by immunoprecipitation using a ChIP-formulated histone H3 antibody. The phosphorylated histone H3 was coincubated with MKP2 or CI-MKP2 in the phosphatase reaction. Immunoblotting was performed using indicated antibodies to analyze the dephosphorylation of phosphorylated histone H3. Unphosphorylated histone H3 was used as a control for each kinase reaction.

FIGURE 5:

MKP2 interacts with VRK1 in the nucleus. (A and B) HeLa cells were transfected with Flag-VRK1 and EGFP-MKP2 or Flag-VRK1 and EGFP-CI-MKP2. After 24 h of expression, HeLa cells were harvested, and the cell lysates were subjected to immunoprecipitation with control IgG or FLAG antibody and sequential immunoblotting with the indicated antibodies. (C) EGFP-MKP2 or EGFP-CI-MKP2 was transfected into HeLa cells with pDsRed-Monomer-VRK1 (i). After 24 h of expression, immunocytochemistry was performed with nuclear staining using Hoechst (blue) and analyzed by fluorescence microscopy. Endogenous VRK1 and MKP2 were applied to immunocytochemistry by using antibodies against VRK1 and MKP2 (ii).

FIGURE 6:

Interaction between MKP2 and VRK1 occurs in the chromatin region. (A) EGFP-MKP2 was transfected into HeLa cells. After 24 h, subcellular and subnuclear fractionations were performed, and each fraction was subjected to immunoblotting with indicated antibodies. Antibodies against GAPDH, lamin B, or histone H3 were used as markers for the various fractions. (B) EGFP-HP1γ and pDsRed-Monomer-MKP2 were transfected into HeLa cells for 24 h. Immunocytochemistry was performed and analyzed by fluorescence microscopy. (C) Subcellular fractionation was performed on harvested HeLa cells, and a crude chromatin fraction was obtained from the pellet after nucleoplasm extraction by incubation with micrococcal nuclease. Nucleoplasm not containing chromatin fractions and crude chromatin fractions was pulled down with GST or GST-VRK1. The interaction was analyzed by immunoblotting with indicated antibodies. C.P., cytoplasm; N.P., nucleoplasm; E.C., euchromatin; N.M., nuclear matrix; H.C., heterochromatin; C.C., crude chromatin.

FIGURE 7:

Differential interaction between MKP2 and VRK1 during the cell cycle. (A) A549 cells were arrested in G1, S, and M phases with 50 μM mimosine, 100 μM thymidine, and 50 ng/μl nocodazole, respectively. After 16 h, cells were harvested and subjected to flow cytometry. Propidium iodide–stained DNA content was analyzed for verification of cell cycle synchronization by flow cytometry. (B) Immunoblotting was performed using indicated antibodies to analyze the profile of MKP2 and VRK1 following cell cycle progression. (C) Synchronized cells were fractionated to crude chromatin fractions. Those chromatin fractions were subjected to immunoprecipitation with the ChIP-formulated histone H3 antibody and sequential immunoblotting with the indicated antibodies. (D) Indicated siRNAs were transfected into A549 cells. After 36 h, cells were treated with 50 ng/μl nocodazole for 12 h. A549 cells were harvested, and the cell lysates were subjected to immunoblotting with indicated antibodies.