Polo-like kinase-1 triggers histone phosphorylation by Haspin in mitosis

Abstract

Histone modifications coordinate the chromatin localization of key regulatory factors in mitosis. For example, mitotic phosphorylation of Histone H3 threonine-3 (H3T3ph) by Haspin creates a binding site for the chromosomal passenger complex (CPC). However, how these histone modifications are spatiotemporally controlled during the cell cycle is unclear. Here we show that Plk1 binds to Haspin in a Cdk1-phosphorylation-dependent manner. Reducing Plk1 activity decreases the phosphorylation of Haspin and inhibits H3T3ph, particularly in prophase, suggesting that Plk1 is required for initial activation of Haspin in early mitosis. These studies demonstrate that Plk1 can positively regulate CPC recruitment in mitosis.

Figure 1

Plk1 activity is required for normal phosphorylation of Haspin and H3T3 in mitosis.

1. Uninduced HeLa Tet-On/myc-Haspin cells were treated with 0.6 μM nocodazole for 18 h. Mitotic cells collected by “shake-off” were replated in the presence of nocodazole with MG132 and indicated inhibitors for 1 h. Immunoblots of cell lysates are shown.

2. HeLa cells were transfected with control, Plk1, Aurora B or Haspin siRNA and, after 48 h, subjected to immunofluorescence.

3. RPE1 cells were released from double thymidine block and, after 7 h, kinase inhibitors were added for 2 h, followed by immunofluorescence microscopy. Scale bar, 5 μm.

4. Quantification of H3T3ph and H3S10ph staining from the experiment shown in (C). See supplementary Methods.

Figure 2

Phosphorylation by Cyclin B-Cdk1 allows Haspin to bind Plk1-PBD.

1. Kinase-deficient (KD) MBP-Haspin or MBP-CREB was used as a substrate in an in vitro kinase assay for GST-Cyclin B1/GST-Cdk1 (or control GST-Wee1) activity in the presence of γ³²P-ATP.

2. Myc-Haspin was immunoprecipitated from uninduced HeLa Tet-On/myc-Haspin cell extracts, and subjected to immunoblotting or Far-Western analysis.

3. MBP-Haspin KD was phosphorylated in vitro with GST-Cyclin B1/GST-Cdk1 and then subjected to Far-Western analysis.

Figure 3

Plk1 binding to Haspin requires the PBD of Plk1 and T128 of Haspin.

1. Alignment of vertebrate Haspin sequences surrounding the conserved STP motif. See supplementary Methods.

2. Myc-Haspin was immunoprecipitated from extracts of asynchronous or nocodazole-arrested mitotic HeLa cells expressing myc-Haspin (WT or T128A), and subjected to immunoblotting with anti-myc or anti-S-pT-P motif antibodies.

3. Myc-Haspin was immunoprecipitated from extracts of nocodazole-arrested mitotic HeLa cells expressing myc-Haspin (WT, T128A or 11A), and subjected to immunoblotting or Far-Western analysis.

4. Lysates of nocodazole-arrested mitotic HeLa cells expressing myc-Haspin (WT or T128A) were subjected to “pulldowns” using GST-PBD and controls, followed by immunoblotting with anti-myc antibodies.

5. Lysates of nocodazole-arrested mitotic HeLa cells cotransfected with myc-Haspin (WT or T128A) and HA-Plk1 (WT or PBDmut) plasmids were subjected to immunoprecipitation with anti-myc or HA antibodies, followed by immunoblotting. For ease of interpretation, lanes were spliced as shown to correct loading order. Asterisk indicates a non-specific band.

6. HeLa cells were transfected with myc-Haspin WT or T128A plasmids, arrested in mitosis, and treated with inhibitors and MG132 for 1.5 h as described in Fig 1A. Lysates were subjected to immunoblotting.

Figure 4

Phosphorylation of Haspin at T128 and Plk1 target sites is required for full H3T3ph generation and normal Aurora B localization in mitosis.

1. HeLa Tet-On cells were transfected with indicated siRNAs, followed the next day by transfection with doxycycline-inducible myc-Haspin WT or T128A plasmids. At 24 h after DNA transfection, cells were blocked in mitosis by nocodazole treatment for 18 h, in the presence or absence of doxycycline, and lysates of cells collected by shake-off were analyzed by immunoblotting.

2. HeLa Tet-On cells were transfected with inducible myc-Haspin plasmids (WT, 7A or T128A) in the presence of doxycycline and analyzed as in (A).

3. Model for Haspin regulation in mitosis. See text for details.

4. HeLa cells were released from thymidine block and, after 7 h, 0.3 μM nocodazole was added with kinase inhibitors for 3 h before fixation. The ratio of Aurora B at centromeres versus chromosome arms was determined by immunofluorescence microscopy. Means ± s.d. are shown, n = 10, ** P < 0.001 by Bonferroni multiple comparison test. Example images are shown in supplementary Fig S5A.