Survivin reads phosphorylated histone H3 threonine 3 to activate the mitotic kinase Aurora B

Abstract

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.

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 Coomassie brilliant blue (CBB). (B) Proteins copurified with indicated peptides from ΔCPC extracts supplemented with recombinant Aurora B and the indicated mRNAs. (C) Purified ternary complex consisting of INCENP_1–58-His₆, Dasra A and Survivin or the kinase complex consisting of Aurora B_60–361 and INCENP_790–871 was incubated with the indicated peptide-beads. Coomassie staining of input and bead fractions is shown. Asterisk: contaminating E. coli protein. (D) Superposition of ¹H, ¹⁵N-HSQC spectra with (red) and without (black) addition of 0.4 mM H3T3ph peptide (1–13) to a 0.2 mM ¹⁵N-labeled human Survivin (1–120). Some of the NMR signals of the free protein that shift upon titration with peptide are shown. (E) H3T3ph (1–13) binding surface of Survivin (1–120). Magenta: residues that significantly shift upon addition of the peptide. (F) Unmodified H3 (1–20) binding surface of Survivin (1–120). Green: residues that significantly shift upon addition of the peptide.

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 ΔHaspin metaphase extracts with nocodazole, supplemented with buffer, wild-type Haspin or Haspin-KD, stained with indicated antibodies. Scale bar, 5 µm. (B) Western blots of proteins copurified with DNA-beads from control or ΔHaspin extracts supplemented with buffer or 2.5 µM MBP-Haspin_c. 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.

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, ΔHaspin extracts or control extracts containing 2.5 µM MBP-Haspin_c. Aurora B phosphorylation at T248 and hyperphosphorylation of Op18 (arrowhead) were monitored by Western blot. (B) Representative images of bipolar spindles formed on replicated sperm chromosomes in control or ΔHaspin extracts. Blue, DNA. Red, rhodamine-tubulin. Scale Bar, 10 µm. (C) Histogram of metaphase spindle length in control and ΔHaspin extracts as measured in Table S1. Error bars represent ranges of three experiments.

Fig. 4

Dephosphorylation of H3T3ph is required for chromosome decondensation and nuclear re-formation. (A, B) Sperm nuclei incubated with CSF extracts for 45 min were subsequently treated with buffer, 2.5 µM MBP-Haspin_c or MBP-Haspin_c-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, D) Sperm nuclei and calcium were added together to CSF control or ΔCPC extracts containing buffer or MBP-Haspin_c. (C) Hoechst 33258 staining at 30 min after calcium addition. (D) Quantification of (C). Mean and SEM of three independent experiments (N> 30). Scale bars, 10 µm.