Mcl-1 is phosphorylated at two sites in mitosis

To characterise the cell cycle-dependent phosphorylation of Mcl-1, the Flag-tagged protein was expressed in HeLa cells, which were then treated with nocodazole for 3 h. Flag-Mcl-1 was immunoprecipitated from asynchronous or mitotic nocodazole-treated cells and analysed by mass spectroscopy of tryptic peptides (Figure 3A). Flag-Mcl-1 was phosphorylated at five sites in asynchronous cells: Ser64, Thr92, Ser121, Ser159 and Thr163. Ser159 was only found in conjunction with Thr163, consistent with a requirement for priming phosphorylation at Thr163 for phosphorylation of Ser159 by GSK-3 (Maurer et al, 2006; Morel et al, 2009). Ser121 has been described as a site phosphorylated together with Thr163 by JNK in response to oxidative stress (Inoshita et al, 2002), whereas Ser64 has been identified as phosphorylated in cells during G2 and/or mitosis (Kobayashi et al, 2007). Thr92 is phosphorylated by ERK in vitro (Ding et al, 2008), but its phosphorylation in cells has not been described earlier. Of these five sites, only Ser64 and Thr92 were detected in cells arrested in mitosis by nocodazole. Phosphorylation of the tryptic peptide-containing Ser64 increased about three-fold in the mitotic sample, whereas the phosphorylated Thr92 peptide increased 50-fold and was five times more abundant than the Ser64 peptide, suggesting that Thr92 is the major site in Mcl-1 that is phosphorylated specifically in mitosis (Figure 3A).

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Figure 3

Mcl-1 is phosphorylated at two sites in mitosis. (A) Flag-Mcl-1 was immunoprecipitated from HeLa cells growing in asynchronous culture (As) or treated with nocodazole for 3 h, followed by collection of the rounded-up, mitotic cells (NM). Tryptic peptides were analysed by mass spectrometry. The relative abundance of the phosphopeptides containing the Thr92 and Ser64 sites are highlighted. (B) Specificity of antibodies generated against Mcl-1 phosphorylation sites. HeLa cells were transiently transfected with either wild-type Mcl-1 (WT) or the phosphorylation site mutants S64A or T92A. After treatment with 250 ng/ml nocodazole for 2 h, samples were analysed by SDS–PAGE and immunoblotting with antibodies raised either against the phosphorylated Ser64 (left) or Thr92 (right) sites. (C, D) Mcl-1 is phosphorylated at Thr92 in mitosis. Endogenous Mcl-1 was immunoprecipitated from asynchronous (As) HeLa cells or after treatment with 100 ng/ml nocodazole for 2 h before separation of mitotic (NM) and adherent cells (NA). Cell lysates (Input) were analysed by conventional SDS–PAGE. Immunoprecipitates were analysed by conventional SDS–PAGE (C) or Phos-tag SDS–PAGE (D) and immunoblotted for Mcl-1 phosphorylated at Thr92 or total Mcl-1. Phosphorylated Mcl-1 is indicated by a bracket. ^*Indicates the heavy chain of the precipitating antibody. (E) Mcl-1 is highly phosphorylated at Thr92 and Ser64 during mitotic arrest. U2OS cells stably transfected with either wild-type Mcl-1 (WT), T92A or S64A mutants were untreated (As) or treated for 2 h with nocodazole before separation of mitotic (NM) and adherent cells (NA). Cell lysates were analysed by conventional SDS–PAGE or Phos-tag SDS–PAGE as indicated and immunoblotted with the specified antibodies. ^*Indicates a band corresponding to cyclin B1 caused by reprobing of the same blot used to identify cyclin B1.

We generated polyclonal antibodies against phosphopeptides containing either the Ser64 or Thr92 sites and validated their specificity (Supplementary Figure 5). The antibodies recognised Flag-Mcl-1 from mitotically arrested HeLa cells when phosphorylated at Ser64 or Thr92, respectively, as recognition of Flag-Mcl-1 was abolished by mutation of the specific residue to alanine. Phosphorylation of Thr92 was not affected by mutation of Ser64 and vice versa, indicating that each site was phosphorylated independently (Figure 3B). We confirmed that Mcl-1 was phosphorylated at Ser64 and Thr92 during mitosis in the absence of microtubule poisons using U2OS cells that were stably transfected with Flag-Mcl-1 and synchronised in the cell cycle (Supplementary Figure 6). Furthermore, we were able to show that immunoprecipitated endogenous Mcl-1 was phosphorylated at Thr92 in both mitotically arrested cells and in untreated mitotic cells, but not in interphase cells (Figure 3C; Supplementary Figure 7). Figure 3D shows that endogenous Mcl-1 immunoprecipitated from mitotically arrested cells migrated as three phosphorylated forms on a Phos-tag gel, which specifically retards the migration of phosphoproteins (Kinoshita et al, 2006). The upper two bands were detected by the pT92 Mcl-1 antibody and accounted for ~50% of the total Mcl-1. This indicates that Mcl-1 is phosphorylated to a high level at Thr92 in mitotically arrested cells. Analysis of stably transfected Mcl-1 proteins on a Phos-tag gel (Figure 3E) showed that mutation of T92 or S64 to alanine produced distinct single bands that migrated in retarded positions, showing that both residues are highly phosphorylated under these conditions.

CDK1–cyclin B1 phosphorylates Mcl-1 at Thr92 directly and is required for phosphorylation at Ser64 in mitosis

To identify the kinase(s) that phosphorylate(s) Mcl-1 at Thr92 and Ser64, we used a cell-free system derived from mitotic HeLa cells (Hutchins et al, 2004). Recombinant Mcl-1-His₆ was phosphorylated at Thr92 and Ser64 (detected by the specific antibodies) in nocodazole-treated mitotic cell extracts. Mitotic Mcl-1 phosphorylation at both sites was inhibited by either of the CDK inhibitors, roscovitine or purvalanol A (Figure 4A), but not by the MEK1/2 inhibitor, UO126, which blocks the ERK1/2 MAPK pathway, nor the p38 inhibitor, SB203580. SP600125 also partially inhibited phosphorylation at these sites; this reagent inhibits JNK, but also other kinases, including CDKs (Bain et al, 2003). As the mitotic extracts contained cyclin B1, but not cyclin A, these results are consistent with a dependency on CDK1–cyclin B1, the major mitotic CDK, for the phosphorylation of Mcl-1 at Thr92 and Ser64.

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Figure 4

Thr92 of Mcl-1 is phosphorylated directly by CDK1–cyclin B1, whereas Ser64 phosphorylation is dependent on CDK1, but may be indirect. (A) The CDK inhibitors roscovitine and purvalanol A abrogate phosphorylation of Thr92 and Ser64. Mcl-1-His₆ was incubated for 1 h with extracts derived from HeLa cells either in asynchronous culture (As) or arrested in mitosis with nocodazole (N). Samples were analysed by SDS–PAGE and immunoblotting with the specified antibodies. (B) Depletion of CDK1–cyclin B1 from mitotic HeLa cell extracts inhibits phosphorylation of Mcl-1-His₆ at Thr92 and Ser64. Mcl-1-His₆ was incubated for 1 h with extracts depleted of CDK1–cyclin B1 by p13^Suc1 beads. Purified recombinant CDK1–cyclin B1 was added back to depleted extracts to restore kinase activity. (C) Purified recombinant CDK1–cyclinB1 directly phosphorylates Mcl-1-His₆ at Thr92, but not at Ser64. Mcl-1-His₆ was incubated for 1 h with CDK1–cyclin B1 or mitotic extract made from nocodazole-treated cells. (D) Active CDK1 immunoprecipitated from HeLa cells arrested in mitosis with nocodazole (Noc) phosphorylates Mcl-1-His₆ on Thr92 (lower panels). Mock immunoprecipitations were performed with an isotype-matched control antibody. Total cell lysates (Input) used for the precipitations are shown in the upper panels.

Depletion of CDK–cyclin complexes from mitotic extracts using p13^suc1 beads resulted in inhibition of phosphorylation at Thr92 and Ser64 (Figure 4B). Similarly, the phosphorylation of Mcl-1 at Thr92 and Ser64 was abrogated in mitotic extracts depleted using anti-cyclin B1 antibodies (data not shown). In p13^suc1-depleted extracts, addition of active recombinant CDK1–cyclin B1 fully restored phosphorylation of Mcl-1 at Thr92, and partially restored phosphorylation at Ser64 (Figure 4B). Incubation of Mcl-1-His₆ with purified recombinant CDK1–cyclin B1 in the absence of mitotic extracts confirmed that Thr92 was phosphorylated directly by this kinase, whereas Ser64 was not (Figure 4C). This was corroborated when CDK1 immunoprecipitated from mitotic HeLa cells was incubated with Mcl-1-His₆, resulting in phosphorylation of Thr92 (Figure 4D). Thus, CDK1–cyclin B1 is directly responsible for phosphorylation of Mcl-1 at Thr92. Ser64 phosphorylation is dependent on CDK1–cyclin B1, but requires an additional component of the mitotic extracts.

Phosphorylation of Thr92 is required for the mitotic degradation of Mcl-1

To investigate the function of the mitotic phosphorylation of Mcl-1, we established cell lines derived from U2OS cells that were stably transfected with Flag-tagged wild-type Mcl-1 (U2-Mcl-1-WT), or alanine mutants of Thr92 (U2-Mcl-1-T92A) or Ser64 (U2-Mcl-1-S64A). These cells were selected because they express similarly low levels of transfected Mcl-1. Cells were also derived that were transfected with an empty vector control (U2-eV). As before, we treated the cells with nocodazole for 2 h and re-plated the mitotic cells for a further 2 or 4 h (Figure 5A). The transfected Flag-Mcl-1 in U2-Mcl-1-WT was degraded in the mitotic block with a similar time course to the endogenous Mcl-1 protein in U2-eV cells. Flag-Mcl-1-S64A was also unstable in mitotically arrested U2-Mcl-1-S64A cells. In contrast, the degradation of Flag-Mcl-1 T92A was blocked in the U2-Mcl-1-T92A cells, showing that the phosphorylation of Thr92 is required specifically for the mitotic degradation of Mcl-1. Identical results were obtained with different clones of each cell type. Mutation of either Thr92 or Ser64 to alanine did not affect the degradation of Flag-Mcl-1 in interphase cells treated with the protein synthesis inhibitor cycloheximide (Figure 5B). In contrast to T92A, mutation of Thr92 to aspartate, which is predicted to mimic the phosphorylated form, did not stabilise Mcl-1 during mitotic arrest (Figure 5C). The T92D protein was also degraded like the wild-type protein in interphase cells treated with cycloheximide (Figure 5D). The destruction of Mcl-1 during mitotic arrest is, therefore, specifically dependent on Thr92 phosphorylation and is mediated by a mechanism different to that operating in interphase.

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Figure 5

Phosphorylation of Thr92 is required for mitotic destruction of Mcl-1. (A) Mcl-1 T92A is stable in cells arrested in mitosis. U2OS cells stably transfected with either wild-type Mcl-1 (WT), T92A or S64A mutants, or empty vector (eV) were treated for 2 h with nocodazole. Mitotic cells were then isolated and re-plated for a further 2 or 4 h so that cells were arrested in mitosis for a total of 2, 4 or 6 h. (B) The T92A mutant is degraded like wild type and S64A Mcl-1 in interphase. Time course after addition of 40 μg/ml cycloheximide (CHX) to asynchronous cell cultures. (C) Mcl-1 T92D phospho-mimetic mutant is degraded like wild-type Mcl-1 during mitotic arrest. U2OS cells stably transfected with either wild-type Mcl-1 (WT) or T92D mutant were treated as in (A). (D) The T92D mutant is degraded like wild-type Mcl-1 in interphase. Time course after addition of 40 μg/ml cycloheximide (CHX) to asynchronous cell cultures.

The degradation of cyclin A was also delayed in U2-Mcl-1-T92A cells during mitotic arrest (Figure 5A). The level of cyclin B1 in mitotically arrested U2-Mcl-1-T92A cells was also elevated and stabilised compared with U2-Mcl-1-WT, U2-Mcl-1-S64A and U2-eV cells. These results indicate that the mechanism of Mcl-1 destruction is closely related to that of the mitotic cyclins, and suggest that Mcl-1 competes for the same degradation machinery. Strong over-expression of Flag-Mcl-1 by transient transfection resulted in stabilisation of even the WT protein during a prolonged mitotic arrest (data not shown), suggesting that a component of the degradation machinery is limiting under these conditions.

Mcl-1 degradation during mitotic arrest requires Cdc20 and APC3

The degradation of mitotic cyclins and other mitotic regulators is mediated by Cdc20 (Fizzy) and the E3 ubiquitin ligase complex APC/C (Yu, 2007). To test the possible function of Cdc20 in the degradation of Mcl-1 during mitotic arrest, we depleted Cdc20 using siRNA in U2OS cells prior to the mitotic arrest (Figure 6A). Cdc20 itself is unstable during mitotic arrest (Nilsson et al, 2008) and the level of Cdc20 was further reduced with 2 h of nocodazole treatment: in control cells, Cdc20 was reduced to a low, basal level by 4 h in nocodazole and was barely detectable in cells treated with the siRNA after this time. The reduction in Cdc20 levels by siRNA completely inhibited the mitotic degradation of endogenous Mcl-1, and the stabilised protein migrated entirely in upshifted forms, whereas the level of Mcl-1 in asynchronous cells was not affected by Cdc20 depletion. Despite the strong reduction of Cdc20 in cells treated with the siRNA, the degradation of cyclin A during mitotic arrest was delayed, but not prevented. This is consistent with the work by Wolthuis et al (2008) showing that a residual level of Cdc20 is sufficient for cyclin A degradation in mitosis. Similarly, depletion of APC3 (CDC27), an APC/C component, which recruits the activators Cdc20 and Cdh1 (Fizzy related) to the complex (Vodermaier et al, 2003), also stabilised Mcl-1 as well as cyclin A and cyclin B1 during mitotic arrest (Figure 6B). In contrast, depletion of Cdh1 (Figure 6C) or Mule (data not shown) failed to stabilise Mcl-1. We found that Cdc20 co-precipitated with Flag-Mcl-1 from both interphase and mitotically arrested cells, indicating that their interaction is independent of mitotic Mcl-1 phosphoryation (Figure 6D). The association of Mcl-1 with Cdc20 is similar to the association between cyclin A and Cdc20, which is also observed in both interphase and mitosis (Wolthuis et al, 2008).

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Figure 6

Destruction of Mcl-1 during mitotic arrest is mediated by Cdc20. (A) Cdc20 is required for Mcl-1 destruction in cells arrested in mitosis. Cdc20 was depleted from U2OS cells using siRNA, then cells were synchronised in a mitotic arrest by treatment with nocodazole for 2 h followed by collection of rounded-up mitotic cells and further treatment with nocodazole for a total of 4 or 6 h. Cells treated with a luciferase siRNA (Luc) were used as a control. (B) APC3 is required for Mcl-1 destruction in cells arrested in mitosis. APC3 was depleted from U2OS cells using siRNA, then cells were synchronised as in (A). Luciferase siRNA (Luc) was used as a control. (C) Depletion of Cdh1 does not prevent Mcl-1 degradation during mitotic arrest. Cdh1 was depleted from U2OS cells using siRNA, then cells were synchronised in mitotic arrest by treatment with nocodazole for 2 h followed by collection of rounded-up mitotic cells and further treatment with nocodazole for a total of 6 h. (D) Cdc20 interacts with Mcl-1 in interphase and mitosis. U2-Mcl-1-WT cells were synchronised by a single thymidine block followed by release for 9 h before addition of nocodazole (100 ng/ml) and MG132 (10 μM) for 3 h. Flag-Mcl-1 was precipitated from mitotic (NM) and adherent (NA) cells using Flag-agarose and immunoblotted for Cdc20 and Mcl-1; HA-agarose was used as a control (Ctrl) for the precipitation (right panels). Cell lysates (Input) used for the precipitations are shown in the left panels.

Mitotic destruction of Mcl-1 requires a putative D-box

Cdc20 recognises proteins through destruction box (D-box) motifs. We scanned the primary sequence of Mcl-1 for a potential D-box and found one region (residues 207–215) with 6 out of 9 residues identical to the D-box of securin (Figure 7A). Within this motif, two residues, an arginine and a leucine in the minimal consensus RxxL, are also conserved in D-boxes from cyclin A and cyclin B, and these residues are essential for mitotic destruction of cyclin B (Glotzer et al, 1991). We also noticed that Mcl-1 carries an isoleucine-arginine motif at its C-terminus, similar to the C-terminal I/M-R motifs found in Cdc20, Cdh1, APC10 and Nek2A (Figure 7B). This motif is required for their interaction with the APC/C through binding to APC3 (Hayes et al, 2006; Kimata et al, 2008). To test the possible function of these motifs in the mitotic destruction of Mcl-1, we generated stable clones of U2OS cells that express a D-box mutant (R207A/L210A) or a mutant lacking the C-terminal isoleucine-arginine motif (ΔIR). We found that the D-box mutant was completely stable during nocodazole-induced mitotic arrest, whereas the ΔIR protein was unstable (Figure 7C). We also confirmed that both the D-box and ΔIR mutants were unstable like the wild type in interphase cells (Figure 7D). The stabilisation of the D-box mutant was not due to a defect in Thr92 phosphorylation, since both the D-box and ΔIR mutants were phosphorylated at this site in mitotically arrested cells (Figure 7E). Interestingly, the mitotic destruction of both cyclin A and cyclin B1 was still slightly delayed in the D-box mutant cells, suggesting that stabilised Mcl-1 interferes with cyclin destruction through a mechanism other than just competition for Cdc20 through its binding to the D-box.

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Figure 7

Mcl-1 contains a D-box motif that is required for its degradation during mitotic arrest. (A) Sequence alignment of putative Mcl-1 D-box motif compared with those from cyclin A, cyclin B1 and securin. The two residues in Mcl-1 mutated to alanine to yield the Mcl-1 D-box mutant (R207A/L210A) are indicated (arrows). (B) Sequence alignment of the C-terminal regions of Cdc20, Cdh1, APC10, Nek2A and Mcl-1. The I/M-R motif is underlined. (C) Mcl-1 D-box mutant is stable in cells arrested in mitosis. U2OS cells stably transfected with either wild-type Mcl-1 (WT), D-box or IR-deletion (ΔIR) mutants were treated for 2 h with nocodazole. Mitotic cells were then isolated and re-plated for a further 2 or 4 h so that cells were arrested in mitosis for a total of 2, 4 or 6 h. (D) Mcl-1 D-box and IR-deletion (ΔIR) mutants are degraded like wild-type Mcl-1 in interphase. Time course after addition of 40 μg/ml cycloheximide (CHX) to asynchronous cell cultures. (E) Mcl-1 D-box and IR-deletion (ΔIR) mutants are phosphorylated at Thr92 like wild-type Mcl-1 during mitotic arrest. U2OS cells stably transfected with either wild-type Mcl-1 (WT), D-box or IR-deletion (ΔIR) mutants were treated for 2 h with nocodazole before collection of mitotic cells (Noc) or left untreated (As). Flag-Mcl-1 was precipitated from cell lysates using Flag-agarose and immunoblotted for Mcl-1 phosphorylated at Thr92 or total Mcl-1. Cell lysates (Input) used for the precipitations are shown in the right panels.

Stabilisation of Mcl-1 during mitotic arrest inhibits apoptosis

To test the possible function of Mcl-1 degradation during mitotic arrest in determining the sensitivity of cells to the induction of apoptosis, we analysed the response of U2-Mcl-1-WT, U2-Mcl-1-T92A, U2-Mcl-1-S64A and U2-eV cells to prolonged exposure to nocodazole. Analysis of apoptosis by measuring sub-G1 (<2N) DNA content using flow cytometry showed that cells expressing the T92A mutant were significantly more resistant to the induction of apoptosis than cells expressing either WT or S64A Mcl-1 (Figure 8A; Supplementary Figure 8). Analysis by immunofluorescence of individual cells showed that the number of apoptotic cells with active caspase-3 was reduced by ~60% in U2-Mcl-1-T92A cells (Figure 8B). We also found that stabilisation of Mcl-1 through mutation of the D-box reduced sensitivity to nocodazole-induced apoptosis, whereas the ΔIR mutant had no inhibitory effect, consistent with its inability to prevent Mcl-1 destruction (Supplementary Figure 9). Cells expressing Mcl-1 T92A or the D-box mutant were protected against apoptosis to the same extent (Supplementary Figure 10). These results show that Thr92 phosphorylation and the subsequent destruction of Mcl-1 determines the sensitivity of cells to apoptosis induced by prolonged mitotic arrest.

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Figure 8

Abolition of Mcl-1 Thr92 phosphorylation inhibits apoptosis induced from a mitotic arrest. (A) U2OS cells stably transfected with Mcl-1 (WT, T92A or S64A) or empty vector cells (eV) were treated with nocodazole (250 ng/ml) for 45 h. Cells were analysed for sub-G1 DNA content by flow cytometry (±s.e.m., n=3). Statistical analyses were performed using a two-tailed unpaired Student's t-test. The percentage of cells with sub-G1 DNA content in WT, T92A or S64A cells was significantly different from the empty vector control (P<0.01) and T92A was significantly reduced compared with WT or S64A (P<0.02). (B) U2OS cells stably transfected with Mcl-1 (WT, T92A or S64A) or empty vector cells (eV) were treated with nocodazole (100 ng/ml) for times indicated. Percentage of cells with active caspase-3 is shown (±s.e.m., n=4). After 24 h, the percentage of caspase-3 positive cells was significantly reduced in T92A cells compared with the empty vector control (P<0.01) and WT (P<0.03).

Cells

HeLa (OHIO) and U2OS (HTB96) were obtained from Cell Services, Cancer Research UK London Research Institute (Lincoln's Inn Fields). To generate stable clones of U2OS cells expressing Mcl-1 [wild-type, T92A, S64A, T92D, D-box (R207A/L210A) or ΔIR (C-terminal 2 amino acids, IR, deleted)], U2OS cells were co-transfected with Mcl-1 subcloned into pCMV-Flag (MRC Protein Phosphorylation Unit, Dundee) and pcDNA3. Single clones were selected in G418 (800 μg/ml, Calbiochem). Cell culture conditions were described earlier (Allan et al, 2003).

Cell treatments

Cells were synchronised at the G1/S boundary by double thymidine block, which involved incubation with 2 mM thymidine for 16 h, followed by an 8 h release into normal medium before the addition of 2 mM thymidine for a further 16 h. To arrest cells in mitosis, cells were treated with the specified concentration of nocodazole or taxol for the times given. To synchronise cells in a mitotic arrest, rounded-up cells were collected after treatment with nocodazole for 2 h and re-plated in the same nocodazole-containing medium. For proteasome inhibition studies, cells were incubated with 10 μM MG132 (Calbiochem) for 2 h before further treatments. To study the stability of proteins in interphase, cells were treated with 40 μg/ml cycloheximide (Calbiochem).

Identification of phosphorylation sites

Flag-Mcl was immunoprecipitated from HeLa cells, either asynchronous or treated with nocodazole for 3 h (mitotic), using Flag-agarose (Sigma). Samples were analysed by electrophoresis through a NuPAGE 4–12% Bis–Tris gel (Invitrogen). Flag-Mcl-1 bands were excised and digested overnight at 30°C with trypsin. Peptides were analysed by liquid chromatography mass spectrometry using a 4000 Q-Trap system (Applied Biosystems) by Dr Nick Morrice of the MRC Protein Phosphorylation Unit, University of Dundee.

Immunoprecipitation

For Mcl-1 precipitations, cells were lysed in IP buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM Na₃VO₄, 50 mM NaF, 5 mM β-glycerophosphate, 1 mM PMSF, 1 μg/ml each pepstatin, leupeptin and aprotinin, 0.07% 2-mercaptoethanol, 1 μM okadaic acid). A total of 1–2 mg of lysate was incubated with 2 μg/ml α-Mcl-1 antibody (mouse monoclonal, BD Pharmingen) at 4°C for 2 h and then with 15 μl protein G sepharose, 4°C, 1 h. Precipitates were washed three times for 5 min before addition of SDS–gel-loading buffer. For Flag-tagged precipitations, cells were lysed in buffer (as IP buffer above, except glycerol, Na₃VO₄, 2-mercaptoethanol and okadaic acid were omitted). A total of 700 μg lysate was incubated with 15 μl Flag-agarose at 4°C for 10 h. Precipitates were washed three times for 5 min before incubation in SDS–gel-loading buffer at 37°C for 3 min; 2-mercaptoethanol was added to the supernatants and the samples were boiled for 3 min.

Analysis of protein phosphorylation by Phos-tag SDS–PAGE

Protein samples were analysed on 10% SDS–PAGE with 30 μM Phos-tag acrylamide (Phos-tag, Hiroshima, Japan) added to the resolving gel. This reagent specifically retards phosphorylated proteins through its Mn²⁺-dependent interaction with phosphomonoester dianions bound to Ser, Thr and Tyr residues (Kinoshita et al, 2006). After electrophoresis, gels were soaked in standard transfer buffer with 1 mM EDTA at room temperature for 10 min, followed by another 10 min incubation in transfer buffer without EDTA.

Mitotic cell extracts and in vitro kinase assays

Human HeLa cell extracts were prepared as described by Hutchins et al (2004). For western analysis of Mcl-1 phosphorylation in extracts, 500 ng Mcl-1-His₆ (expressed in E. coli from a pET21 vector and purified by nickel–agarose affinity chromatography) was incubated with 10 μg extract for 60 min at 30°C in kinase buffer (50 mM Tris–HCl [pH 7.5], 10 mM MgCl₂ and 10 μM DTT) plus 100 μM ATP and kinase inhibitor where indicated. Kinase inhibitors were used at the following concentrations: 10 μM roscovitine, 2 μM purvalanol A, 10 μM UO126, 10 μM SB203580 and 10 μM SP600125. Samples were analysed by SDS–PAGE and immunoblotting. For p13^suc1 depletion of CDK/cyclin complexes, 500 μg HeLa cell extract was depleted by two rounds of incubation for 60 min at 4°C with 20 μl p13^suc1-agarose beads. To restore CDK1–cyclin B1 activity to depleted extracts, 1 μl containing the indicated amount of purified active recombinant CDK1–cyclin B1 (Millipore) was added to give a total volume of 10 μl per incubation.

Immunoprecipitated kinase assays

Asynchronous or mitotic HeLa cells treated with nocodazole were lysed in 50 mM Tris–HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 20 mM EGTA, 1 mM Na₃V0₄, 50 mM NaF, 5 mM β-glycerophosphate, 0.1 mM PMSF, 1 μg/ml each of aprotinin, leupeptin and pepstatin. For CDK1 immunoprecipitations, 1 mg cell lysate was incubated for 60 min at 4°C with 1 μg anti-CDK1 mouse monoclonal antibody, then 25 μl protein A beads were added and the incubation continued for an additional 60 min at 4°C. Precipitated samples were washed three times in lysis buffer, three times in kinase buffer containing 5 mM EGTA, then incubated with 500 ng Mcl-1-His₆ in kinase buffer containing 100 μM ATP for 60 min at 30°C.

RNA interference

siRNA duplexes were directed against Mcl-1 (5′-GGACUUUUAGAUUUAGUGATT-3′; Adams and Cooper, 2007), Cdh1 (5′-AAUGAGAAGUCUCCCAGUCAG-3′; Bashir et al, 2004), Luciferase (5′-CGUACGCGGAAUACUUCGATT-3′) (MWG, Ebersberg, Germany), APC3 (APC3-1, 5′-GGAAAUAGCCGAGAGGUAA-3′; APC3-2, 5′-CAAAAGAGCCUUAGUUUAA-3′; Dharmacon) or Cdc20 (ON-TARGETplus SMARTpool siRNA Cdc20, L-003225; Dharmacon). For Mcl-1 or Cdc20 knockdown, cells were transfected with 10 nM duplex and siLentFect (Bio-Rad) for 5 h according to the manufacturer's instructions, then trypsinised into fresh media and cultured for 43 h before further treatment. To knockdown APC3, cells were transfected with 50 nM each siRNA and LipofectAmine 2000 (Invitrogen) for 24 h followed by a second transfection for 48 h before further treatment. For Cdh1 knockdown, 50 nM duplex was transfected with LipofectAmine 2000 for 48 h before further treatment.

Flow cytometry

For analysis of sub-G1 DNA content, cells were fixed in 70% ethanol and incubated on ice for 2 h. Samples were washed twice in phosphate-citrate buffer (192 mM disodium phosphate, 4 mM citric acid, pH 7.8) and resuspended in PBS containing 100 μg/ml RNase A and 50 μg/ml propidium iodide. DNA content was analysed by flow cytometry using a FACScan machine (Becton Dickinson). At least 10 000 cells were analysed per sample.

We thank Nick Morrice of the MRC Protein Phosphorylation Unit, University of Dundee for phosphopeptide analysis. This work was funded by Cancer Research UK, Association for International Cancer Research, Biotechnology and Biological Sciences Research Council and Tenovus Scotland. MEH was supported by a Medical Research Council Studentship and PRC was a Royal Society-Wolfson Research Merit Awardee.