Characterization of a Monoclonal Antibody, HTA28, Recognizing a Histone H3 Phosphorylation Site as a Useful Marker of M-phase Cells

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.

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Differential regulation of CENP-A and histone H3 phosphorylation in G2/M

Journal of Cell Science ◽

10.1242/jcs.114.4.653 ◽

2001 ◽

Vol 114 (4) ◽

pp. 653-661 ◽

Cited By ~ 3

Author(s):

S.G. Zeitlin ◽

C.M. Barber ◽

C.D. Allis ◽

K. Sullivan

Keyword(s):

Protein A ◽

Phosphorylation Site ◽

Histone H3 ◽

Condensation Process ◽

Sequence Motif ◽

H3 Phosphorylation ◽

Centromere Protein A ◽

Putative Phosphorylation Site ◽

Mitotic Phosphorylation ◽

Histone H3 Phosphorylation

After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.

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