Survivin Reads Phosphorylated Histone H3 Threonine 3 to Activate the Mitotic Kinase Aurora B

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Science  08 Oct 2010:
Vol. 330, Issue 6001, pp. 235-239
DOI: 10.1126/science.1189505


A hallmark of mitosis is the appearance of high levels of histone phosphorylation, yet the roles of these modifications remain largely unknown. Here, we demonstrate that histone H3 phosphorylated at threonine 3 is directly recognized by an evolutionarily conserved binding pocket in the BIR domain of Survivin, which is a member of the chromosomal passenger complex (CPC). This binding mediates recruitment of the CPC to chromosomes and the resulting activation of its kinase subunit Aurora B. Consistently, modulation of the kinase activity of Haspin, which phosphorylates H3T3, leads to defects in the Aurora B–dependent processes of spindle assembly and inhibition of nuclear reformation. These findings establish a direct cellular role for mitotic histone H3T3 phosphorylation, which is read and translated by the CPC to ensure accurate cell division.

The accurate segregation of chromosomes during mitosis depends on a complex signaling network, whose spatiotemporal coordination is achieved in part by restricting the activity of kinases to distinct subcellular structures (1). The kinase Aurora B, together with INCENP, Dasra (also known as Borealin), and Survivin, form the chromosomal passenger complex (CPC), which plays multiple roles during mitosis and meiosis by changing its localization (2). At the beginning of M phase, the CPC is localized to chromosomes, where it controls chromatin-dependent spindle assembly (3, 4) and processes at the centromere (2). During anaphase, the complex dissociates from chromosomes and relocalizes to the spindle midzone to stimulate cytokinesis (2). The mechanism by which the CPC is recruited to chromosomes in a cell cycle–specific manner remains unknown.

In screening for mitotic histone-binding proteins, we found that the CPC interacts with histones H3/H4 in Xenopus cytostatic factor (CSF)–arrested egg extracts (fig. S1) (5). Given that Ser10 of histone H3 (H3S10) is an excellent substrate of Aurora B, we surmised that the CPC–H3/H4 interaction was mediated by the H3 tail (6, 7). In egg extracts, the CPC did not show appreciable binding to a peptide corresponding to the N-terminal tail of H3 (H31-21) (Fig. 1A). However, all four CPC subunits bound to an H3 peptide phosphorylated on Thr3 (H3T3ph) more strongly than to H31-21, H3S10ph, scrambled H3 sequence peptide (Scr. H3), or a histone H4 N-terminal (H41-18) peptide (Fig. 1A).

Fig. 1

Survivin mediates the interaction between the CPC and H3T3ph. (A) Western blots (WB) of proteins copurified with indicated peptides from metaphase Xenopus egg extracts. Peptides were visualized by use of Coomassie brilliant blue (CBB). (B) Proteins copurified with indicated peptides from ∆CPC extracts supplemented with recombinant Aurora B and the indicated mRNAs. (C) Purified ternary complex consisting of INCENP1-58-His6, Dasra A, and Survivin or the kinase complex consisting of Aurora B60-361, and INCENP790-871 was incubated with the indicated peptide beads. Coomassie staining of input and bead fractions is shown. Asterisk indicates contaminating E. coli protein. (D) Superposition of 1H, 15N-HSQC spectra (red) with and (black) without addition of 0.4 mM H3T3ph peptide (1 to 13) to a 0.2 mM 15N-labeled human Survivin (1 to 120). Some of the NMR signals of the free protein that shift upon titration with peptide are shown. (E) H3T3ph (1 to 13) binding surface of Survivin (1 to 120). Magenta indicates residues whose resonances substantially shift upon addition of the peptide. (F) Unmodified H3 (1 to 20) binding surface of Survivin (1 to 120). Green indicates residues whose resonances substantially shift upon addition of the peptide.

The CPC can be separated into two functional modules (fig. S2A): the kinase module consisting of Aurora B and the C-terminal domain of INCENP (IN box) (8, 9) and the chromosome-localization module consisting of the N-terminal region of INCENP, Survivin, and Dasra, which form a ternary subcomplex through a tight helical bundle (4, 10). By introducing two mutations in INCENP (F21R and L33R) that disrupt this helical bundle (10) (fig. S2B), we found that the chromosome-localization module is important for recruitment of the CPC to H3T3ph (Fig. 1B) (11). A purified ternary complex of Survivin, Dasra A, and INCENP1-58-His6, but not the kinase module of the CPC (Aurora B60-361-INCENP790-871) (9), specifically bound to H3T3ph peptide in vitro (Fig. 1C), demonstrating that the chromosome-localization module of the CPC directly recognizes H3T3ph.

Survivin was able to bind H3T3ph peptides in the absence of other CPC subunits (fig. S2C), implying that it is the receptor for H3T3ph. We further defined this interaction using nuclear magnetic resonance (NMR) spectroscopy, taking advantage of published resonance assignments of human Survivin (12). Analysis of [15N, 1H]–heteronuclear single-quantum coherence spectroscopy (HSQC) spectra of Survivin in the presence of increasing amounts of H3T3ph peptide revealed that several resonances were perturbed (Fig. 1D and fig. S3). The migration pattern of these peaks indicated that the interaction was in slow chemical exchange and came to saturation (fig. S3A), confirming a direct and strong interaction. When mapped onto the structure of Survivin (12), the chemical shift perturbations induced by H3T3ph peptide clustered to form a contiguous binding pocket in its BIR domain (Fig. 1E). Addition of unmodified H3 peptide to Survivin induced only a small subset of these chemical shift perturbations (fig. S3, B and C) demonstrating that in the absence of phosphorylation, H3 interacts with Survivin far less extensively (Fig. 1F). Among the residues within the H3T3ph-binding pocket is D71, which is critical for centromeric localization and function of the CPC in vivo (13, 14).

The binding interface of Survivin and H3T3ph is highly similar to that of the BIR3 domain of XIAP (X-linked inhibitor of apoptosis) and the N terminus of processed SMAC (15, 16) or caspase-9 (17) (fig. S4). In particular, E314 of XIAP (equivalent to Survivin D71) has been shown to coordinate the N-terminal alanine of its ligands and its mutation abrogates binding in vitro (15, 16), suggesting that recognition of the N-terminal alanine of histone H3 by Survivin is carried out in the same manner with D71 (fig. S4D). In contrast, Survivin basic residues H80 and K62, which are not conserved in the BIR3 domain of XIAP, shift upon H3T3ph binding (figs. S3C and S4) and may accommodate the negatively charged phosphate on H3T3.

To further investigate the physiological importance of the CPC-H3T3ph interaction, we depleted the Xenopus homolog of the mitotic H3T3 kinase Haspin (18) from egg extracts (figs. S5 and S6). In control extracts, H3T3ph was found along entire chromosomes, whereas Dasra A and INCENP localized to chromosome arms and were particularly enriched at centromeres (Fig. 2A and fig. S7A). Haspin depletion abolished the majority of H3T3ph and, concomitantly, significantly reduced Dasra A and INCENP signals from chromosomes (Fig. 2A and figs. S7 and S8). The kinase activity of Haspin is important for chromosomal recruitment of the CPC because addition of wild-type Haspin, but not a kinase dead (KD) version (18), to ∆Haspin extracts rescued the ability of Dasra A and INCENP to bind chromosomes (Fig. 2A and figs. S7 and S8).

Fig. 2

The H3T3 kinase Haspin is required for recruitment of the CPC to metaphase chromosomes. (A) Immunofluorescence images of replicated sperm chromosomes in control or ∆Haspin metaphase extracts with nocodazole, supplemented with buffer, wild-type Haspin, or Haspin-KD, and stained with indicated antibodies. Scale bar, 5 μm. (B) Western blots of proteins copurified with DNA beads from control or ∆Haspin extracts supplemented with buffer or 2.5 μM MBP-Haspinc. Proteins were eluted from beads first with 0.6 M NaCl and then with 2M NaCl. Ku70, a DNA-binding protein, serves as a loading control.

We next biochemically tested the effect of Haspin depletion on binding of the CPC to chromatin. In egg extracts, DNA beads assemble functional chromatin, recruiting the CPC and supporting bipolar spindle formation (3, 4). Consistent with Haspin’s role in localizing the CPC to chromosomes, binding of the CPC to DNA-beads was reduced in ∆Haspin extracts, and this defect was rescued by recombinant C-terminal kinase domain of Haspin (MBP-Haspinc) (Fig. 2B and fig. S9). However, Haspin does not appear to serve as a physical bridge to mediate the CPC-chromatin interaction because the CPC bound H3T3ph peptides in ∆Haspin extracts (fig. S10).

Chromatin assembled on DNA stimulated Aurora B autophosphorylation of its activation loop at threonine 248 (T248ph) (9) and hyperphosphorylation of the Aurora B substrate Op18 (4) in metaphase egg extracts (Fig. 3A). Chromatin failed to stimulate Aurora B activity in ∆Haspin extracts, whereas excess MBP-Haspinc accelerated the kinetics of chromatin-dependent Aurora B phosphorylation. Furthermore, MBP-Haspinc rescued the inability of DNA and sperm chromosomes to activate Aurora B in ∆Haspin extracts (fig. S11), establishing the role of Haspin in chromatin-dependent activation of Aurora B. However, it is unlikely that Haspin directly controls intrinsic Aurora B activity because it is not required for activation of Aurora B by stabilized microtubules, which is a chromatin-independent activator (fig. S11) (4, 7).

Fig. 3

Haspin controls chromatin-induced Aurora B activation to promote spindle assembly. (A) Plasmid DNA was incubated for the indicated times at 20°C with control egg extracts, ∆Haspin extracts, or control extracts containing 2.5 μM MBP-Haspinc. Aurora B phosphorylation at T248 and hyperphosphorylation of Op18 (arrowhead) were monitored by means of Western blot. (B) Representative images of bipolar spindles formed on replicated sperm chromosomes in control or ∆Haspin extracts. Blue, DNA; red, rhodamine-tubulin. Scale Bar, 10 μm. (C) Histogram of metaphase spindle length in control and ∆Haspin extracts as measured in table S1. Error bars represent ranges of three experiments.

Chromatin-induced Aurora B activation is required to drive spindle assembly around chromosomes by suppressing microtubule-depolymerizing activities in Xenopus egg extracts (3, 4). In ∆Haspin extracts, this function was compromised, resulting in shorter spindle length (Fig. 3, B and C, and table S1). Although spindles can still form, which may be attributable to residual CPC on chromosomes (Fig. 2 and fig. S8), these results suggest that direct recruitment of the CPC by H3T3ph activates Aurora B on chromosomes to promote spindle assembly.

At the metaphase-to-anaphase transition, the CPC dissociates from chromosomes (2), which is a process critical to subsequent chromosome decondensation and nuclear reformation (19). Because H3T3ph is dephosphorylated upon exit from M phase (18, 20, 21) (fig. S6B), we investigated whether its removal is essential to these processes in egg extracts. Metaphase chromosomes, preassembled in CSF control extracts, were decondensed 45 min after calcium addition, which induces transitioning to interphase (Fig. 4A). At this point, Dasra A and INCENP dissociated from chromosomes, H3T3ph and H3S10ph levels decreased (Fig. 4A), and functional nuclear formation was nearly complete (fig. S12, A and B). However, in extracts containing constitutively active MBP-Haspinc, but not MBP-Haspinc-KD, chromosomes remained condensed with robust staining of Dasra A, INCENP, H3T3ph, and H3S10ph, and nuclear import was not initiated (Fig. 4A and fig. S12, A and B). These phenotypes were not due to a delay in cell cycle transition because H1 kinase activity was not detectable 15 minutes after calcium addition in all samples (Fig. 4B). The chromosome decondensation defect caused by MBP-Haspinc was CPC-dependent, as expected if Haspin worked through CPC activation (Fig. 4, C and D, and fig. S12C).

Fig. 4

Dephosphorylation of H3T3ph is required for chromosome decondensation and nuclear reformation. (A and B) Sperm nuclei incubated with CSF extracts for 45 min were subsequently treated with buffer, 2.5 μM MBP-Haspinc, or MBP-Haspinc-KD, together with calcium to induce exit from metaphase. (A) Indirect immunofluorescence 45 min after calcium addition. (B) Western blot analysis and histone H1 kinase assay (autoradiography). (C and D) Sperm nuclei and calcium were added together to CSF control or ∆CPC extracts containing buffer or MBP-Haspinc. (C) Hoechst 33258 staining at 30 min after calcium addition. (D) Quantification of (C). Mean and SEM of three independent experiments (n > 30 sperm nuclei). Scale bars, 10 μm.

Our study demonstrates that M phase–specific phosphorylation of histone H3 at Thr3 recruits the CPC to chromatin to activate Aurora B and that this interaction is mediated by Survivin. (fig. S13). This interaction controls spindle assembly and nuclear reformation in Xenopus egg extracts and is important for spindle checkpoint signaling at the centromere in human cells (22). These results highlight a mechanism by which the cell cycle control of Haspin activity restricts chromosomal Aurora B localization and activation to M phase through histone modification.

Supporting Online Material

Materials and Methods

Figs. S1 to S13

Table S1


References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. In the mutants, other amino acids were substituted at certain locations; for example, F21R indicates that phenylalanine at position 21 was replaced by arginine. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  3. The GenBank accession number for X. laevis Haspin is HM559585. We thank M. Dasso, J. Higgins, and S. Mochida for reagents; T. Maniar and K. Yap for technical assistance; J. Higgins for sharing results; and the Rockefeller University Bio-Imaging Resource Center. This work was supported by a Charles H. Revson Biomedical Fellowship (A.E.K), Marie-Josée and Henry Kravis and Erwin Schrödinger postdoctoral fellowships (C.Z.), grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (H.K.) and NIH (GM075249, H.F.).
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