Phosphorylation of Caspase-9 by CDK1/Cyclin B1 Protects Mitotic Cells against Apoptosis  Lindsey A. Allan, Paul R. Clarke  Molecular Cell  Volume 26, Issue 2, Pages 301-310 (April 2007) DOI: 10.1016/j.molcel.2007.03.019 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Caspase-9 Is Phosphorylated at Thr125 during Mitosis (A) U2.C9-C287A cells were arrested in G1 by a double-thymidine block (time 0) and were released for the times indicated. Cell lysates were immunoblotted for caspase-9 phosphorylated at Thr125 (Casp9 pT125), histone H3 phosphoryated at Ser10 (H3 pS10), phosphorylated and activated ERK1/2 (ppERK), total caspase-9 (Casp9), and total ERK1/2, as indicated (left panels). Right panels show cell cycle status monitored by flow cytometry. Boxed numbers show the percentage of cells with 4N DNA content. (B) U2.C9-C287A cells were treated as in (A), and were then incubated with purvalanol A or PD184352 for 15 min. Cell lysates were immunoblotted as described in (A), except, at 10 hr, only nonadherent, mitotic cells were analyzed. (C) Asynchronous U2.C9-C287A cells were treated with purvalanol A or UO126 for 15 min. Mitotic cells were separated from adherent interphase cells before analysis by immunoblotting. (D) HeLa cells were treated with nocodoazole (nocod) or taxol as indicated. Caspase-9 immunoprecipitates were immunoblotted for phospho-Thr125 caspase-9 and caspase-9. IgG detected by the secondary antibody is indicated by a single asterisk. Cell lysates were also immunoblotted for cyclin B1 (CycB1) and CDK1. A crossreacting band is indicated by a double asterisk. (E) CDK-dependent phosphorylation of caspase-9 at Thr125 in response to nocodazole. U2.C9-C287A cells were treated with nocodazole for 17 hr or 1 μM TPA for 15 min, then with roscovitine, purvalanol A, or PD184352 for 15 min before analysis by immunoblotting. A mouse monoclonal cyclin B1 antibody was used that does not detect the crossreacting band observed in (D). Molecular Cell 2007 26, 301-310DOI: (10.1016/j.molcel.2007.03.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 CDK1/Cyclin B1-Dependent Phosphorylation of Caspase-9 at Thr125 (A) His6-caspase-9 C287A was incubated with extract from asynchronously growing HeLa cells (A) or mitotic cell extract from nocodazole-treated HeLa cells (N) plus 10 μM roscovitine, 1 μM purvalanol A, or 10 μM UO126. Upper panels, phosphorylation of caspase-9 at Thr125 and total caspase-9 protein detected by immunoblotting. Lower panels, CDK1 activity measured by [32P] phosphorylation of histone H1 and autoradiography. (B and C) Mitotic cell extracts were depleted of CDK1/cyclin B1 by using (B) p13suc1 agarose or (C) cyclin B1 antibody, then incubated with His6-caspase-9 and immunoblotted. A crossreacting band is indicated by an asterisk. In (C), depleted extracts were supplemented with recombinant CDK1/cyclin B1. Endogenous cyclin B (single arrow) and recombinant GST-tagged cyclin B1 (double arrow) are indicated. (D) CDK1/cyclin B1 precipitated from asynchronous (A) or nocodazole-treated mitotic cells (N) by using CDK1 antibody was incubated with [γ-32P]ATP His6-caspase-9 WT or T125A (both C287A), then analyzed by SDS-PAGE and autoradiography (upper panels). Lower panels, phosphorylation of histone H1 in duplicate samples. IgG heavy and light chains are indicated by single and double asterisks, respectively. (E) Caspase-9 coprecipitates with CDK1. U2.C9-C287A cells were treated with nocodazole. Mitotic cells (NM) were separated from adherent cells (NA) prior to lysis. Lysates from asynchronous cells (A) were also analyzed by immunoblotting (left panel). A crossreacting band is indicated by a double asterisk. CDK1 immunoprecipitates were blotted for caspase-9 (right panel). IgG detected by the secondary antibody is indicated by a single asterisk. Molecular Cell 2007 26, 301-310DOI: (10.1016/j.molcel.2007.03.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Phosphorylation of Caspase-9 at Thr125 Inhibits Apoptosis in Cells Arrested in Mitosis (A and B) Characterization of stable clones of U2OS cells expressing exogenous caspase-9 WT or T125A after specific ablation of endogenous caspase-9 by siRNA. (A) Sequences of siRNA duplexes used to ablate caspase-9. The nucleotides mutated in caspase-9 cDNA to produce stable clones of cells expressing capase-9 refractory to ablation by using C9-2 duplex are shown (underlined). The sequence of Ctrl duplex correlates to that of C9-2 duplex containing a 4 bp rearrangement (bold). (B) WT-M2.1, T125A-M2.3, and parental U2OS cells were transfected with siRNA duplexes as indicated. Cell lysates were immunoblotted for caspases. (C–E) Inhibition of caspase-9 Thr125 phosphorylation sensitizes cells to nocodazole-induced apoptosis. (C and D) Cells were transfected with siRNA duplexes as shown and were then (C) left untreated or (D) treated with nocodazole for 24 hr prior to analysis of DNA content by flow cytometry. Boxed numbers, percentage of cells with sub-G1 DNA content. (E) Mean ± SEM from three experiments, one of which is shown in (C) and (D). Molecular Cell 2007 26, 301-310DOI: (10.1016/j.molcel.2007.03.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Nocodazole-Induced Apoptosis Is Initiated during Mitosis after Inhibition of Caspase-9 Thr125 Phosphorylation (A) Cells were transfected with the C9-2 siRNA, then treated with nocodazole for 24 hr. Upper panel, percentage of cells with active caspase-3. Lower panel, DNA content of cells containing active caspase-3. Percentage of active caspase-3-positive cells with a 4N DNA content is shown. (B) Cells were transfected with siRNA duplexes as indicated prior to treatment with nocodazole for 16 hr, then analyzed by flow cytometry. The percentage of cells positive for histone H3 phospho-Ser10 (H3 pS10) is shown. (C) Immunofluorescence microscopy of active caspase-3 and H3 pS10. Cells were transfected with C9-2 duplex, treated with nocodazole for 8 hr, then stained for active caspase-3 and H3 pS10. DNA was visualized by using DAPI. The histogram shows the number of cells staining for both active caspase-3 and H3 pS10 as a percentage of the total number of active caspase-3-positive cells after nocodazole treatment for the times indicated. Molecular Cell 2007 26, 301-310DOI: (10.1016/j.molcel.2007.03.019) Copyright © 2007 Elsevier Inc. Terms and Conditions