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Department of Life Science, Division of Molecular & Life Science, Pohang University of Science and Technology (POSTECH), San-31, Hyoja-Dong, Pohang 790-784, Republic of Korea,1 X-Ray Research Group, Pohang Accelerator Laboratory, Pohang 790-784, Republic of Korea,2 Division of Structural and Computational Biology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore3
Received 4 January 2007/ Returned for modification 13 March 2007/ Accepted 25 September 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A number of studies have shown that members of the aurora kinase family are responsible for phosphorylation of histone H3 (3, 7, 17, 18). Mammals contain three isotypes of aurora kinase designated aurora A, B, and C (11). Among these, aurora B is a strong candidate phosphorylator of Ser10 in histone H3 as is evident from data obtained with hesperadin, the aurora B inhibitor (14), which suppressed Ser10 phosphorylation during mitosis (7, 17). However, residual Ser10 phosphorylation was detected, even upon depletion of aurora B in cells, suggesting the presence of an additional histone H3 kinase (29).
NIMA (never in mitosis), the histone H3 Ser10 kinase in Aspergillus nidulans (6, 34), triggers chromatin condensation in cells arrested at the interphase (28). In mammals, Nercc1, the functional ortholog of NIMA, was found to be phosphorylating histone H3 (39). Nucleosomal histone kinase 1 (NHK1) from Drosophila melanogaster is the kinase shown to phosphorylate histone protein in chromatin as a substrate. NHK1 phosphorylated H2A at Thr119 in chromatin but not with free histone as the substrate (1). Recent studies showed that NHK1 participates in mitotic progression (4) and maintenance of proper chromosomal architecture (19). These data strongly indicate that NHK1 is a bona fide mitotic histone kinase.
Vaccinia-related kinase 1 (VRK1) is the mammalian homolog of NHK1 (1). Sequence similarities between NHK1 and VRK1 are evident in the kinase domain (approximately 40% identity), and the carboxyl termini contain a characteristic basic-acidic-basic motif (1). VRK1, identified from the screening of novel genes involved in cell cycle regulation from fetal liver (31), is designated on the basis of 40% sequence identity with vaccinia virus B1 kinase, which plays a critical role in viral DNA replication (38). The kinase is highly expressed in proliferative tissues, including embryonic tissues, adult testis, and thymus, as well as in several cancer cell lines, implying a functional role in cell cycle regulation and tumorigenicity (31). VRK1 participates in cell cycle progression by means of phosphorylation of a barrier-to-autointegration factor (BAF) that plays structural roles in chromatin and the nuclear envelope and displays subcellular localization changes during the cell cycle (33) and by activating the transcription of proliferation-related proteins such as retinoblastoma, cyclin-dependent kinase 2, and survivin (40).
In this report, we demonstrate that VRK1 is a chromatin-associated protein displaying cell cycle-dependent expression and subcellular localization. VRK1 phosphorylates Thr3 and Ser10 in free and core histone H3 and in nucleosomes. Overexpression of the constitutively active form of VRK1 leads to hypercondensation of the nucleus, in similarity to data obtained in studies of NIMA, the fungal enzyme, in eukaryotic cells.
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MATERIALS AND METHODS |
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cDNAs were subcloned into pDsRed1-C1 and pEGFP-N1, respectively. For expression in Escherichia coli, VRK1 and its mutants (VRK2, VRK3, and histone H3 mutants) were subcloned into pPosKJ, pProEX, or pGEX-4T-1 (Amersham) as described previously (25). VRK antisera were prepared as described previously (23). The following antibodies were purchased from commercial sources: anti-Flag epitope (M2) from Sigma; anti-green fluorescent protein (anti-GFP; C163) from Zymed; anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) and antibromodeoxyuridine (anti-BrdU) from Calbiochem; anti-phospho-cdc2 (Tyr15) and anti-cleaved caspase3 from Cell Signaling Technology; anti-RNA polymerase II from Abcam; anti-DsRed1 and anti-aurora B (AIM-1) from BD Biosciences; anti-lamin B, anti-
-tubulin, anti-cyclin D1, anti- GFP, and anti--tubulin from Santa Cruz Biotechnology; and all antihistone antibodies used in this study as well as anti-HP1 from Upstate Biotechnology. Extraction of subcellular fractions. Cytoplasmic and nucleoplasmic extracts were prepared as described previously (23). The pellet obtained following the extraction of the nucleoplasm, which contained the subnuclear organelles and chromatins, was further extracted as follows. A euchromatin-enriched fraction was isolated as the soluble fraction following treatment of the pellet with DNase I for 1 h at room temperature. Next, the pellet remaining after the euchromatin extraction was vigorously sonicated to obtain the nuclear matrix. Finally, following the nuclear matrix extraction, the pellet was treated with 6 M urea and 2% sodium dodecyl sulfate to solubilize the heterochromatin-enriched fraction. To prepare crude soluble chromatin, the pellet from the nucleoplasmic extraction was treated with micrococcal nuclease (100 U) for 1 h at 30°C. Each fraction was evaluated by Ponceau S staining and immunoblotting.
Immunoprecipitation, immunoblotting and immunocytochemistry. Immunoprecipitation, immunoblotting, and immunocytochemistry were performed as described previously (23).
TUNEL assay. For the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, HeLa cells transfected with DsRed1-C1-VRK1 for various time periods were grown on glass chips coated with poly-D-lysine (Sigma). Cells were then fixed with 4% paraformaldehyde, the glass chips were removed, and the cells were permeabilized and stained with a DeadEnd fluorometric TUNEL system kit (Promega) according to the manufacturer's instructions.
Protein kinase assay. An in vitro kinase assay was performed with 100 ng (see Fig. 4E, G, and H) to 1 µg (see Fig. 4B to D and F) of either recombinant glutathione S-transferase-VRK1 (GST-VRK1) or VRK1 protein and 100 ng (see Fig. 4E, G, and H) or 1 µg (see Fig. 4B to D and F) of substrates, including core histones, nucleosomes, each histone protein, and BAF. The duration of the kinase assay was 30 min except for that of one assay (see Fig. 4H). The standard procedure for the in vitro kinase assay was carried out as described previously (23). The kinase activity of VRK1 in cell extracts was analyzed using immunoprecipitated VRK1 with anti-VRK1 antibody.
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Cell cycle synchronization and flow cytometry. HeLa cells were synchronized as described previously (26). Briefly, cells were arrested in mitotic phase by treatment for 12 h with 10 nM taxol (Paclitaxel; Sigma) and 0.4 µg/ml nocodazole (Sigma), in G1 by treatment for 24 h with 2 mM hydroxyurea, and in G1/S by treatment for 20 h with 200 µM mimosine. A double thymidine blocking was performed as described previously (16). Briefly, cells were treated with 2 mM thymidine for 20 h, released for 6 h in fresh DMEM containing 10% FBS, and treated again with 2 mM thymidine for 12 h. Cells arrested in G1/S were released by switching the medium to fresh DMEM containing 10% FBS. Aliquots were analyzed by flow cytometry, and the remaining cells were used for the preparation of whole-cell extracts or subcellular fractions. For flow cytometric analysis, the cells were fixed with 70% ethanol for 20 min, stained with 20 µg/ml propidium iodide, and treated with 1 µg/ml RNase A for 10 min at room temperature. Samples containing 10,000 cells were then analyzed on a FACSCalibur system (Becton Dickinson).
Purification of recombinant proteins and GST pulldown assay. Monomeric BAF protein was prepared from the insoluble fraction by tobacco etch virus protease treatment to cleave the hexameric His tag as described previously (33). The purification of GST fusion proteins and the GST pulldown assay were carried out as described previously (23, 25).
Semiquantitative RT-PCR. Total RNA was isolated using TRI reagent (Molecular Research Center) according to the manufacturer's instructions. Reverse transcription-PCR (RT-PCR) was carried out as described previously (22). Briefly, following DNase I (Promega) treatment, RNA was reverse transcribed using oligo(dT) (Promega) and monkey murine leukemia virus reverse transcriptase (Roche) in the presence of RNasin (Promega). To obtain semiquantitative results, primers for both GAPDH and VRK1 were included in each reaction mixture.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (Fig. 2E), a well-known chromatin-associated protein (15), which is consistent with results from a previous proteomic analysis showing VRK1 to be a chromatin-associated protein (45). Similarly, affinity chromatography with the DNA fragment of Cyp2b10, the phenobarbital-inducible p450 gene, was applied to identify VRK1 (48). The data strongly suggest that VRK1 is localized not only in nucleoplasm but also in chromatin.
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Finally, a dominant-negative mutant of VRK1 eliminated Ser10 phosphorylation (Fig. 4F), confirming that this process is mediated by VRK1. It is known that VRK1 phosphorylates BAF (33). Thus, we compared VRK1-mediated protein phosphorylation results using BAF and histone H3. The phosphorylation patterns of BAF and histone H3 were similar, and the intensity was in proportion to VRK1 concentration (Fig. 4G) and incubation duration (Fig. 4H), although phosphorylation of greater intensity was found when monomeric BAF was used as a substrate (Fig. 4G and H). We thought that one of the reasons that BAF showed stronger phosphorylation than histone H3 during the in vitro kinase assay might be the difference in the number of residues phosphorylated by VRK1. VRK1 phosphorylates at three different residues of BAF with identical levels of potency (33), while VRK1 phosphorylates at Thr3 similarly strongly but phosphorylates Ser10 at the weaker level seen with histone H3 in vitro.
Phosphorylation of H3 by VRK1 in vivo.
To establish whether VRK1 kinase is required for histone H3 phosphorylation in vivo, we examined the effects of overexpressing Flag-VRK1 or knocking down VRK1 expression by use of siRNA. Overexpression of wild-type VRK1 increased the level of the mitotic index protein -tubulin approximately fivefold in comparison to the results seen with the vector control or the kinase-dead mutant of VRK1 (Fig. 5A) as well as the level of H3 phosphorylation at both Thr3 and Ser10 in the heterochromatin-enriched fraction (Fig. 5B). Consistently, knockdown of endogenous VRK1 by siRNA reduced phosphorylation of H3 at both Thr3 and Ser10 (Fig. 5C). Ser10 but not Thr3 phosphorylation can be confirmed indirectly, as previous studies showed that HP1 binding to chromatin and H3 phosphorylation at Ser10 are mutually exclusive (41). HP1 is loaded on chromatin via binding to triple-methylated Lys9 of H3 (8), which suppresses Ser10 phosphorylation. However, when Ser10 is phosphorylated first, HP1 binding to chromatin is inhibited (7, 17).
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in chromatin fractions (Fig. 5D). By examining chromatin-loaded HP1, we could indirectly verify VRK1-mediated Ser10 phosphorylation of H3. Since aurora B is a known histone H3 kinase during mitosis (3, 18), we examined its relationship with VRK1 with regard to H3 phosphorylation at Ser10. HeLa cells transfected with siRNAs indicated in Fig. 5E for 24 h were further grown in the culture media for 12 h in the presence of nocodazole to be arrested at the mitotic prometaphase. Cells on the mitotic phase were collected by a shaking-off method. Expectedly, depletion of VRK1 resulted in the reduction of phosphorylation at both Thr3 and Ser10 in histone H3 but not at Thr11 and Ser28, whereas depletion of aurora B resulted in the reduction of phosphorylation at both Ser10 and Ser28 but not at Thr3 and Thr11 (Fig. 5E). Interestingly, both aurora B and VRK1 were required for phosphorylation of Ser10. Furthermore, a double knockdown of the two genes dramatically reduced Ser10 phosphorylation (Fig. 5E). When we treated siVRK1, we observed a dramatic decrease in histone H3 Thr3 and Ser10 phosphorylation even though the cells were synchronized at prometaphase in the presence of nocodazole (Fig. 5F). Phosphorylation of histone H3 on Thr3 is very prominent at the prometaphase in HeLa cells (5). The incomplete knockdown of histone H3 phosphorylation shown in Fig. 5F might have been due to the action of other mitotic histone kinases, such as haspin and aurora B, that can phosphorylate Thr3 and Ser10, respectively. VRK1 expression and H3 phosphorylation at Thr3 and Ser10 coincided in mitotic cells. Specifically, during the prophase, VRK1 and phosphorylated H3 colocalized throughout the nucleus, and during prometaphase, VRK1 was delocalized from a more restricted area containing a high level of phosphorylated H3 (Fig. 5G). To measure the intrinsic activity of VRK1 during the cell cycle, we used immunoprecipitated VRK1, which was validated to check the phosphorylation at Thr3 but not at Thr11 of histone H3 (Fig. 5H). VRK1 kinase activity and expression were increased at late G2 or early M phase (Fig. 5I). The results collectively suggest that VRK1 is a key mediator of phosphorylation of histone H3 at Thr3 and Ser10 in vivo.
Hyperphosphorylation of histone H3 by a hyperactive form of VRK1.
Among the tagged (including His, Flag, enhanced GFP, and DsRed1) VRK1 constructs, VRK1 fused to the C terminus of DsRed1 (DsRed1-C1-VRK1) displayed the strongest kinase activity (Fig. 6A) and highest protein level (Fig. 6B). While Flag-VRK1 was localized in chromatin of mitotic cells (Fig. 2D), DsRed1-C1-VRK1, which was highly expressed in chromatin, bypassed cell cycle stages determined by the levels of marker proteins such as cyclin D1 for G1/S phase and -tubulin for mitotic phase and led to the hyperphosphorylation of Thr3 and Ser10 in histone H3 (Fig. 6C) compared to Flag-VRK1 results (Fig. 2D). The mechanism underlying the strong kinase activity of DsRed1-C1-VRK1 is unclear at present and requires further structural analysis. During an in vitro kinase assay, both purified GST-VRK1 and immunoprecipitated-DsRed-VRK1 phosphorylated core histone as a substrate (Fig. 6D, left panel). However, upon the incubation with total cell extracts, while the activity of DsRed-VRK1 was reduced only 1.4-fold, that of GST-VRK1 was reduced more than 10-fold (Fig. 6D, right panel). Accordingly, we speculated that a strong inhibitory factor in total cell extracts may suppress VRK1 activity. To determine where this inhibition occurs in cells, we fractionated cell extracts into cytoplasmic, nucleoplasmic, and crude chromatin fractions followed by incubation with VRK1 to analyze its activity. Inhibition of VRK1 was evident upon incubation with the nucleoplasm fraction (Fig. 6E). However, we could not identify the inhibitory binding partner in nucleoplasm. The data collectively suggest that DsRed1-C1-VRK1 is a constitutively active form of VRK1 and is not affected by an unidentified nuclear inhibitor that strongly suppresses VRK1 activity and hence shows extraordinarily stronger kinase activity than any other tag-fused VRK1 proteins.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a well-characterized chromatin-localized protein, colocalized with VRK1. Moreover, BAF, a newly identified substrate protein of VRK1, localizes in chromatin (33). The chromatin localization of VRK1 indicates a role in the phosphorylation of core histones, which are binding proteins in the nucleus. In this report we show that VRK1 phosphorylates histone H3 at two sites, Thr3 and Ser10, both in vitro and in vivo. Earlier studies provide evidence that these sites are phosphorylated by haspin (Thr3) and aurora B (Ser10). The phosphorylation patterns of these two sites were similar with respect to elapsed time. Specifically, phosphorylation become apparent at early mitosis (from late G2 to prophase), peaked at the metaphase, and disappeared at late mitosis (telophase). Our results suggest that VRK1 plays an essential role in the mitotic phase by phosphorylating Thr3 and Ser10 of histone H3. Moreover, the contribution of VRK1 with respect to Ser10 phosphorylation was similar with aurora B. It is reported that lack of histone H3 phosphorylation at Thr3 causes accumulation of cells in prometaphase and leads to failure of metaphase chromosome alignment (5). Inhibition of Ser10 phosphorylation by inactivating aurora B with hesperadin, an inhibitor, also leads to improper attachment of kinetochore fibers to the chromosome (24), changes in binding properties of chromatin proteins such as HP1 (7, 17), and failure to recruit the condensin complex and mitotic spindle assembly (10). NHK-1, a Drosophila homolog of VRK1, phosphorylates T119 in histone H2A and plays an essential role in mitotic progression and in the formation of chromosome architecture in meiosis (1, 4, 19). Although the net level of phosphorylation of histone H2A by VRK1 was weaker than that seen with H3 during in vitro kinase assay (Fig. 4B), it is possible that VRK1 phosphorylates T119 in H2A in vivo. Hence, future study on the relationship between the distinct phosphorylation patterns of different histones will open the door to deciphering the histone code, especially with respect to maintenance and the dynamics of chromatin architecture. In accordance with these previous studies, our results suggest that VRK1-mediated phosphorylation of Thr3 and Ser10 in histone H3 is functionally linked to the formation of proper chromosome architecture.
Overexpression of DsRed1-C1-VRK1, the constitutively active form of the kinase, induced marked nuclear condensation in transfected cells regardless of the cell cycle stages, in similarity to data obtained following ectopic expression of NIMA in mammalian cells. The existence of a NIMA-like pathway in mammalian cells has been proposed previously (28). Our data provide evidence that the NIMA-like pathway in mammals is mediated by VRK1. Transient expression of DsRed1-C1-VRK1 caused hyperphosphorylation of Thr3 and Ser10 of histone H3, regardless of the cell cycle stage, and concomitantly induced dramatic nuclear condensation, supporting the theory that VRK1 is the mammalian functional counterpart of NIMA. Another possibility is that VRK1 phosphorylates alternative sites in histones or other target proteins that play critical roles in chromatin condensation.
Cell cycle-regulated expression of VRK1. Recently, BAF was revealed as a bona fide substrate of VRK1 (33). VRK1 phosphorylates BAF following a short incubation period during an in vitro kinase assay. Subcellular localization of BAF is regulated during cell cycle progression (42). During interphase, BAF is present in the nucleus as a complex with chromatin formed via direct interactions with DNA and with the nuclear envelope via association with components of a nuclear lamin complex such as LAP2ß, emerin, and MAN1. In contrast, BAF localization becomes dynamic during mitotic progression. From the metaphase to the anaphase, BAF is dissociated from chromatin and localizes to the cytoplasm. In the telophase, BAF relocalizes to chromatin. Such dynamic subcellular localization of BAF is possibly regulated by its phosphorylation status (2), which in turn is modulated by VRK1 (33). Our present findings demonstrate that VRK1 expression and activity are regulated in a cell cycle-dependent manner. A gradual increase in VRK1 expression was observed from the G1 to the mitotic phase. Moreover, VRK1 activity correlated with expression. Thus, the expression and activity profiles of VRK1 may affect phosphorylation status and trigger dynamic localization of BAF during the cell cycle.
Interestingly, expression of Flag-tagged VRK1 under control of the cytomegalovirus promoter was variable in transfected cells, suggesting the existence of another mechanism to regulate the amount of VRK1 protein. Protein degradation mediated by APC/C (anaphase promoting complex-cyclosome) is critical for modulating the amount of cell cycle regulators and mitotic progression (35). APC/C is a major ubiquitin E3 enzyme responsible for the degradation of mitotic kinases, including aurora A and B, Nek2A, and Plk1, via a proteasome-dependent pathway (9, 35). The VRK1 protein level was also altered during the cell cycle. Following nocodazole washout to induce cell cycle progression, MG132 treatment blocked the degradation of VRK1 and inhibited exit from the mitotic phase, as evaluated by assays of the phospho-H3 S10 level (see Fig. S3 in the supplemental material). Thus, while the nature of the precise molecular mechanism involved in VRK1 degradation awaits further investigation, regulation of VRK1 stability by the proteasome is one of the potential mechanisms for its expression profile during the cell cycle.
In summary, our results provide evidence for the function and expression of chromatin-associated VRK1 and establish the mitotic histone kinase activity profile during cell cycle progression. We propose that VRK1 phosphorylates core histones mainly at the G2/M transition to trigger chromosome condensation.
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ACKNOWLEDGMENTS |
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This study was supported by a grant from the National R&D Program for Cancer Control (0520250-2), by the Ministry of Health & Welfare, by a Biotech grant supported by POSCO, and the Brain Korea 21 program of the Ministry of Education, Republic of Korea.
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FOOTNOTES |
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Published ahead of print on 15 October 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
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IVK) was prepared as follows: GST-VRK1 was initially preincubated with total cell extracts overnight at 4°C and collected by affinity pulldown (GPD) using glutathione beads. GST-VRK1 on beads was released by treatment with reduced glutathione. (E) Nucleoplasmic inhibition of VRK1. An in vitro kinase assay of GST-VRK1 was carried out as described for panel D except with preincubation with extracts from cytoplasm (cyto), nucleoplasm (nuc), and chromatin (chro).