Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo

Purification and identification of nucleosomal histone kinase-1, NHK-1

We fractionated nuclear extract derived from Drosophila embryos and found a novel kinase activity. A kinase assay with free core histones as substrates using fractions from Q Sepharose chromatography is shown in Figure 1A. The results suggest that several kinases phosphorylate free core histones. However, when using chromatin as a substrate, it was shown that there was only one kinase acting in the flow-through fraction as indicated by phosphorylation of histone H2A–H2B (Fig. 1A, right panel, indicated by asterisk). The kinase activity that phosphorylated H2A–H2B in nucleosome form did not phosphorylate solubilized free core histones (Fig. 1A, left panel, indicated by asterisk). Further purification was undertaken to identify this unique kinase and to elucidate the role of this phosphorylation (the steps in purification are depicted in Fig. 1B).

We examined this unique activity through purification monitoring by kinase assay using chromatin. Fractions from the final HiTrap Blue chromatography step were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent staining with Coomassie Brilliant Blue R-250 (Fig. 1C). Three polypeptides with molecular masses of 70, 52, and 42 kD (indicated by asterisks in Fig. 1C) were copurified with nucleosomal histone kinase (NHK) activity. These polypeptide bands were in-gel digested with lysylendopeptidase and subjected to HPLC fractionation. Partial amino acid sequences were determined by Edman peptide analysis. Two sequences derived from these three polypeptides (EGTVFTDLAK and AANILLGLEK) were found to be identical to the segments of the Drosophila CG6386 gene product with an apparent serine/threonine kinase domain (see FlyBase report; http://flybase.bio.indiana.edu).

To exclude the possibility that another kinase is included in this fraction, we performed LC-tandem-mass analysis using the SDS gel fragments sliced from top to bottom. No other predicted kinases in Drosophila except CG6386 were present in these samples. Thus, we named this protein kinase nucleosomal histone kinase-1, NHK-1.

NHK-1 is a novel gene that encodes a protein of 599 amino acids. By Western blot analysis with anti-NHK-1 antibodies using Drosophila extract, a 70-kD band is detected throughout Drosophila development, with the highest level in early embryos (data not shown). Because its calculated molecular mass is 65.993 kD, two of the three bands observed during purification might be proteolyzed products.

This kinase is substrate-specific. Highly purified NHK-1 did not phosphorylate purified H2A–H2B dimer in the presence or absence of H3–H4 tetramer. However, NHK-1 phosphorylated nucleosomal H2A–H2B dramatically. NHK-1 phosphorylated H2A–H2B in the presence of DNA, but to a much lesser extent than in the nucleosome (Fig. 1D).

Purified recombinant NHK-1 has nucleosomal histone kinase activity

To conclude that NHK-1 is really a nucleosomal histone kinase, we undertook synthesis and purification of the recombinant proteins. To this end, we constructed bacterial expression vectors for full-length NHK-1 with N-terminal tandem Flag epitope tag (termed Flag-NHK-1). Flag-NHK-1 was expressed in Escherichia coli and purified by affinity purification via the Flag epitope tag (Fig. 2A), and its kinase activity was tested. Recombinant Flag-NHK-1 also phosphorylates only nucleosomal H2A–H2B, as does native NHK-1 (Fig. 2B). These results confirm the conclusion that the purified kinase is a nucleosome-specific histone kinase.

We generated rabbit polyclonal anti-NHK-1. Whole-embryo extracts before gastrulation (0∼3 h embryo) and after gastrulation (3∼6 h embryo) were run together with Flag-NHK-1 and subjected to subsequent immunoblotting with anti-NHK-1. The molecular size of the native NHK-1 is 70 kD, based on the immunoblotting of whole-cell extract. As a control, dNLP was used for estimation of the total protein (Ito et al. 1996a). Flag-NHK-1 ran more slowly than native NHK-1 partially because of the tandem Flag tag (Fig. 2C).

Comparison of the predicted amino acid sequence of this unique NHK-1 with known proteins revealed similarities to other known kinases, such as human Vaccinia related kinase 1 (VRK1; Nezu et al. 1997), mouse VRK1 (GenBank accession no. AAH16676), Xenopus VRK (GenBank accession no. AAH41230), and Caenorhabditis elegans VRK (2G213), with conservation of 44%, 43%, 41%, and 37% of this kinase domain, respectively (Fig. 2D). In addition to a conserved kinase domain, there is common structure, namely, an acidic amino acid region between the basic amino acid regions. This basic-acidic-basic amino acid motif (BAB motif) is conserved among species, as illustrated in Figure 2E. This motif may prove interesting for future crystallographic studies.

Concerning the functions of VRK1, it has been reported that human VRK1 phosphorylates Thr 18 of p53, the binding site of mdm2. However, the biological role of human VRK1, like that of NHK-1, is not known (Lopez-Borges and Lazo 2000). C. elegans VRK was characterized in a systematic analysis of the C. elegans genome using RNAi. Inactivation of C. elegans VRK by RNAi revealed embryonic lethality, with large cytoplasmic granules and failure to form a pronucleus (Piano et al. 2002; Kamath et al. 2003). These reports suggest that VRK has important functions for viability.

NHK-1 can phosphorylate Thr 119 in nucleosomal H2A but not in free H2A

We next examined which amino acid residues are phosphorylated by NHK-1. First, nucleosomal histones were phosphorylated by native NHK-1 in the presence of [γ-³²P]ATP and run on SDS gels. Labeled bands were digested with lysylendopeptidase followed by fractionation by HPLC. More than 90% of ³²P labeling is included in one fragment. Peptide sequencing analysis indicated that H2A C-terminal fragment residues were phosphorylated (LLSGVTIAQGGVLPNIQAVLLPKKTIEK in Fig. 3A). In addition, phosphopeptide mapping analysis revealed that threonine was phosphorylated (data not shown). These data suggest that Thr 100 and/or Thr 119 of the H2A C terminus is phosphorylated (Fig. 3A).

We then determined the specific phosphorylation site of NHK-1 using recombinant histone proteins expressed in E. coli. Chromatin containing Thr 100 substituted with alanine was phosphorylated by NHK-1 (Fig. 3B, T100A). However, chromatin with Ala 119 substituting for Thr 119 or alanines substituting for both Thr 100 and Thr 119 were not phosphorylated by NHK-1 (Fig. 3B, T119A, and T100A & T119A). These experiments indicate that NHK-1 specifically phosphorylates threonine residue 119 of H2A.

Because H2A and H2B make a dimer and run close together on SDS-PAGE, we usually refer to H2A–H2B as if they are a signal of the same size. In the earlier characterization, we referred to H2A–H2B kinase because of the size of the signals and the band appearing to be double. However, from amino acid substitution experiments, we conclude that NHK-1 phosphorylates only Thr 119 in H2A specifically in vitro. The band appeared double partially because the high sensitivity of the assay might detect the small background.

This phosphorylation site and N-terminal proximal regions LLPKK(S/T) are highly conserved among eukaryotes from yeast to mammals (Fig. 3A). To examine this further, we raised rabbit polyclonal antibodies that recognize phospho-Thr 119 of Drosophila H2A (referred to as anti-dH2A-pT119). Phosphorylation of Thr 119 in H2A by NHK-1 was detected by immunoblotting analysis by using rabbit anti-dH2A-pT119 (Fig. 3C). It is noteworthy that Flag-tagged recombinant NHK-1 also phosphorylates Thr 119 in nucleosomal H2A (Fig. 3D).

Because chromatin, not free histones, is the substrate of NHK-1, NHK-1 might have a higher affinity to chromatin than to free histones. To examine this possibility, the interaction between recombinant Flag-tagged NHK-1 and DNA, free histones, or chromatin was analyzed by immunoprecipitation and subsequent elution with different salt concentrations. DNA and free histones did not bind to the NHK-1 affinity column (Fig. 4A,B), while chromatin remained bound to the NHK-1 column until it was eluted by 0.5 M KCl buffer (Fig. 4C). This result suggests that there is an interaction between NHK-1 and chromatin that does not occur between NHK-1 and free histones. There was no obvious difference of affinity between NHK-1 and wild-type recombinant chromatin or any mutant recombinant chromatin.

Previously, it was shown that H2A is phosphorylated in vivo (for review, see van Holde 1989). However, it is not known if Thr 119 in H2A is phosphorylated in vivo or what its physiological importance is. To examine these questions, we performed immunoblotting of Drosophila embryos using anti-dH2A-pT119. Drosophila embryos (0–1 h) were fractionated into cytoplasm and acid-soluble chromatin. Thr 119 of H2A from early-stage embryos was phosphorylated in the chromatin fraction but not in the cytoplasmic fraction (Fig. 4D). These results are consistent with the fact that NHK-1 phosphorylated Thr 119 of H2A only in chromatin form in vitro.

NHK-1 activity is conserved from yeast to humans

To examine conservation of NHK activity, we tested if Drosophila NHK-1 phosphorylates Thr 120 in human H2A (Fig. 5A). We performed a kinase assay using chromatin reconstituted with core histones derived from HeLa cells and recombinant dNHK-1. The reaction mixture was subjected to immunoblotting with anti-hH2A-pT120 (Fig. 6A). The results show that dNHK-1 can phosphorylate Thr 120 of H2A in HeLa chromatin (corresponding to Thr 119 in Drosophila). These results suggest that the basic function of phosphorylation is conserved between mammals and Drosophila.

To confirm conservation among other species, we performed a kinase assay using an NHK activity that was partially purified from Saccharomyces cerevisiae extracts. A partially purified yeast NHK phosphorylated Thr 119 of H2A in Drosophila chromatin (Fig. 5B). Thus, in S. cerevisiae, there is a kinase that phosphorylates serine/threonine of the H2A C terminus in chromatin form. These results indicate that NHK activity is conserved from yeast to humans, suggesting that this phosphorylation is important for basic cell functions.

Histone H2A is phosphorylated at Thr 119 during mitosis in Drosophila embryos

To characterize phosphorylation of H2A Thr 119 in vivo, we examined early-stage Drosophila embryos using anti-dH2A-pT119. After fertilization of the Drosophila embryo, the somatic nuclei in the syncytial egg undergo 13 consecutive and nearly synchronous divisions in the absence of G1 or G2 phase (Foe and Alberts 1983). For immunostaining, we used 0–1-h Drosophila embryos, which are at the stage prior to cellularization and gastrulation. Cycles 7–12 Drosophila embryos were observed, and typical immunostaining is shown in Figure 6. As shown in Figure 6A, there was no significant staining when phospho-peptide was used to block anti-dH2A-pT119 as control. DNA staining with AcD is shown in Figure 6C, and merged features between Figure 6A and C are shown in Figure 6B. Using anti-dH2A-pT119, there is no significant staining in the early S-phase embryo, as shown in Figure 6D. However, chromatin begins to be stained with anti-dH2A-pT119 during cell cycle progression from S phase to prophase, as shown in Figure 6G–L. Stained chromatin is more condensed compared with unstained chromatin, as shown in Figure 6K, which is an enlargement of the box in Figure 6H. Chromatin is most strongly stained during metaphase, as shown in Figure 6M–O. Anaphase-to-telophase chromatin showed decreased immunostaining, suggesting that phosphorylated histone H2A at Thr 119 is dephosphorylated after mitosis (Fig. 6P–R). Because early-stage embryos have more mitotic nuclei compared with late-stage embryos, it is reasonable to observe that it was difficult to detect phosphorylation of Thr 119 of H2A with antibody in core histones of late-stage embryos or whole-stage embryos because of the decreased proportion of mitotic cells (data not shown). Thus, core histones purified from embryos up to 12 h old are relatively hypophosphorylated for Thr 119 of H2A.

Immunostaining studies of Drosophila embryos clearly indicate that phosphorylation of Thr 119 is enhanced during mitosis and decreased during S phase (Fig. 6), suggesting that this conserved phosphorylation site may play an important role in biological processes in the cell cycle, gene replication, or transcriptional regulation. This immunostaining pattern of phosphorylation of the conserved C-terminal residue, which is shown in Figure 3A, is detected in both early-stage embryos and cultured mammalian HeLa cells during mitosis (H. Aihara and T. Ito, unpubl.). It is suggested that this phosphorylation is a conserved phenomenon among species.

Localization of NHK-1 in the Drosophila embryo

To characterize NHK-1 in vivo, we examined the localization of NHK-1 in the early Drosophila embryo. For immunostaining, we used 0–1-h Drosophila embryos, which are before cellularization and gastrulation. For immunostaining, we used rabbit anti-NHK-1, which is specific for NHK-1, in immunoblotting analysis with the native crude extract as shown in Figure 2C. Cycle 7–12 Drosophila embryos were observed, and typical immunostaining is shown in Figure 7. In early S phase, NHK-1 mainly localized in the cytoplasm, as shown in Figure 7D. Localization of NHK-1 changes during cell cycle. The nuclei that are shown in Figure 7I are going into prophase. NHK-1 became localized to more condensed chromatin, as shown in yellow in the merged image (Fig. 7H) of NHK-1 staining (Fig. 7G) and DNA staining (Fig. 7I). During metaphase, NHK-1 stays in the nuclei, as shown in yellow by the merged image (Fig. 7K,M) of NHK-1staining (Fig. 7J,M) and DNA staining (Fig. 7L,O). At the end of mitosis, NHK-1 is excluded from chromatin, as shown in red nuclei by the merged image (Fig. 7Q) of NHK-1 staining (Fig. 7P) and DNA staining (Fig. 7R). The fact that NHK-1 and DNA staining overlap at the peripheral region of the nuclei might reflect NHK-1 being pumped out at the nuclear membrane but is also partially because of the resolution of the microscope, given that the chromatin and cytoplasm are close, being separated by a thin nuclear membrane and matrix.

The cell cycle without G1 and G2 phase, which is shown in early Drosophila development, illustrates these changes of NHK-1 localization. They correlate well with the phosphorylation of H2A Thr 119 that also occurs during mitosis. These correlations between NHK-1 localization and the timing of phosphorylation, together with the strict substrate specificity of NHK-1 that only phosphorylates nucleosomal histone H2A in vitro, support the concept that H2A Thr 119 is the real substrate of NHK-1 in vivo. In addition, these immunostaining data suggest that NHK-1 plays an important role in cell cycle progression and other biological phenomena.

Purification of NHK-1

NHK-1 was purified from Drosophila embryos that were collected from 0 to 12 h after egg deposition, as shown in Figure 1B. A typical preparation of NHK-1 began with 2 kg of Drosophila embryos. NHK-1 was purified from nuclear extracts derived from embryos by ammonium sulfate precipitation and subsequent successive chromatographic steps on SP-Sepharose FF (Pharmacia), Q-Sepharose FF (Pharmacia), hydroxylapatite (Bio-Rad), Blue Sepharose FF (Pharmacia), POROS Heparin (Boehringer Mannheim), Red Toyopearl (Tosoh), Sephacryl S-100 (Pharmacia), HiTrap Blue (Pharmacia), and Sephacryl S-200 (Pharmacia). The partial amino acid sequence of NHK-1 was determined principally as follows. The protein samples were subjected to electrophoresis on 10% SDS-PAGE and stained with Coomassie Brilliant Blue. The p70, p52, and p42 bands were excised and digested with lysylendopeptidase. The resulting peptides were purified by HPLC and sequenced with an automated protein sequencer (Applied Biosystems, Model cLc). Three independent runs of purification and subsequent peptide sequencing were done to ensure reproducibility of the data.

For the detailed methodology of NHK-1 purification, see Supplemental Material.

Purification of recombinant NHK-1

Tandemly connected Flag-tagged NHK-1 (Flag-NHK-1) was produced in E. coli strain Rosseta (DE3) pLysS (Invitrogen). A lysate of E. coli in which Flag-tagged NHK-1 was expressed was immunopurified with Flag M2 agarose (Sigma). After extensive washing of the agarose, Flag-tagged NHK-1 was eluted with Flag peptides (Sigma) and dialyzed against 50 mM KCl.

Reconstitution of nucleosomes

Chromatin was reconstituted with plasmid DNA and purified Drosophila core histones by using salt dialysis techniques as described previously (Ito et al. 1997). The most highly reconstituted chromatin was then purified by 15%–40% glycerol gradient sedimentation (60,000 rpm for 2.5 h at 4°C; Beckman SW60 rotor) Recombinant histone proteins were expressed and purified as described previously (Levenstein and Kadonaga 2002).

In vitro phosphorylation

For the kinase assay, typically 1 fM native NHK-1 or 100 fM recombinant NHK-1 was incubated with 20 ng of DNA, 10 ng of H2A–H2B dimer, 20 ng of all four core histones, 30 ng of H2A–H2B/DNA, or 40 ng of chromatin as substrates in the presence of 19 kBq of [γ-³²P]ATP, 10 mM HEPES (pH 7.6), 50 mM KCl, and 5 mM MgCl for 2 h at 30°C. Reaction mixtures were resolved by 15% SDS-PAGE and subjected to autoradiography. For immunoblotting with anti-dH2A-pT119 or anti-hH2A-pT120, 1 nM cold ATP was used instead of [γ-³²P]ATP in the 50-μL reaction mixture.

In vitro interaction assay

The NHK-1 affinity column was prepared by binding Flag-tagged NHK-1 to Flag M2 agarose gel. DNA, free core histones, or chromatin was incubated with the affinity column under 50 mM KCl, 5 mM MgCl₂ conditions followed by stepwise elution with 0.25, 0.50, 0.75, and 1.0 M KCl. Eluates were precipitated with trichloroacetic acid (TCA) and subjected to 15% SDS-PAGE and subsequent immunoblotting.

Immunostaining of Drosophila embryos

Drosophila embryos (collected from 0 to 1 h after egg deposition) were dechorionized and suspended in buffer containing 10 mM potassium phosphate (pH 6.8), 15 mM NaCl, 45 mM KCl, and 2 mM MgCl₂. The embryos were then permeabilized principally as described before (Ito et al. 1996b). After blocking with 10% BSA, the embryos were incubated with the primary antibody (anti-dH2A-pT119 or anti-NHK-1) overnight at 4°C. The embryos were washed, and the secondary antibody (anti-rabit IgG conjugated with Alexa), along with 4 μg/mL Actinomycin D (for DNA staining), was incubated with the embryos overnight at 4°C. The embryos were washed and then analyzed by confocal microscopy. Confocal images were collected by using a Carl Zeiss LSM 510 META confocal scanning laser head attached to a Carl Zeiss Axiovert 200 M microscope. The data shown were representative of the consistently and reproducibly observed characteristics of many embryos.