Fig. 1. Phosphorylation sites on human APC. (A) APC was immunoprecipitated from extracts of HeLa cells arrested with nocodazole (M) or hydroxyurea (S), subjected to SDS–PAGE and silver staining. Positions of subunits phosphorylated in mitosis are indicated (pApc1, pCdc27, pCdc16 and pCdc23). (B) Schematic phosphorylation site map of APC subunits derived by nano-HPLC–ESI-MS/MS. Black numbers indicate amino acid residues specifically phosphorylated in mitosis, numbers in parentheses indicate sites that could not be assigned with certainty, blue numbers depict sites found both on mitotic and S-phase APC, red refers to sites only phosphorylated in S-phase. Sites found on mitotic APC that were not covered in the S-phase analysis are shown in dark green. (Light green segments, TPRs; blue segments, WD40 repeats; orange segment, cullin domain.) (C) Summary of phosphorylation sites found on human APC.

Fig. 2. Characterization of phospho-specific APC antibodies. (A) Cdc27 immunoprecipitates from extracts prepared from nocodazole (M) or hydroxyurea (S) arrested HeLa cells were analysed by immunoblotting using phospho-specific APC antibodies. As a control, non-phospho-specific Cdc27 antibodies were used for immunoblotting. Where indicated, APC was treated with λ-protein phosphatase (PPase). (B) Whole cell extracts prepared from mitotic or S-phase HeLa cells were analysed as in (A). Arrows indicate the corresponding APC subunit. (C) HeLa cells were synchronized by a double-thymidine arrest-and-release protocol and cell extracts were prepared at the indicated timepoints after release. Immunoblot analysis was performed using the indicated antibodies. As controls, samples from non-synchronized cells (log) and from nocodazole-arrested cells (noc) were analysed. pCdc23 signals could only be detected in Cdc27 immunoprecipitates (IP *). (D) HeLa cells were treated as in (C) but released into medium containing 100 ng/ml nocodazole. After 12.5 h, mitotic cells were collected by shake-off and transfered to fresh medium (noc release) and samples were taken as indicated.

Fig. 3. In vitro phosphorylation of APC by Cdk1 and Plk1. (A) Immunopurified S-phase APC was in vitro phosphorylated using recombinant Cdk1 and Plk1 in the presence of [γ-³²P]ATP, as indicated. After stringent washing, the samples were separated by SDS–PAGE, silver stained (upper panel) and analysed by phosphorimaging (lower panel). Lanes 1 and 2, APC incubated in kinase buffer without kinases; lanes 6 and 7, incubation of kinases alone in kinase buffer; lanes 8 and 9, kinases incubated with histone H1 and casein as substrates. (B) APC phosphorylated in vitro as in (A) was analysed by immunoblotting with pApc1-1, pCdc27-1, pCdc27-2, pCdc16 and pCdc23 antibodies. Cdc27 antibodies were used as a control.

Fig. 4. Cdk1 phosphorylation increases APC activity and Cdc20 binding. (A) Xenopus interphase APC was phosphorylated in vitro by Cdk1 and/or Plk1 and incubated with recombinant Cdc20. Binding of Cdc20 to the APC was monitored by immunoblotting. As a control, beads coupled to non-specific antibodies were treated in the same way (lane 5) and one sample was incubated with buffer instead of Cdc20 (lane 6). (B) Interphase APC purified and treated as in (A) was analysed for the ability to ubiquitinate ¹²⁵I-cyclin B. (C) The amount of ¹²⁵I-cyclin B conjugated to ubiquitin is shown as the percentage of the total amount of ¹²⁵I-cyclin B per reaction.

Fig. 5. APC phospho-epitopes appear in prophase when cyclin B1 translocates into the nucleus. (A) HeLa cells were stained with pCdc27-1 antibodies (red), tubulin (green) and DAPI (blue). (B) Staining was performed as in (A) but using non-phospho-specific Cdc27 antibodies (red). (C) HeLa cells treated as in (A) were stained with pCdc27, LAP2β (green) and DAPI (blue). The arrow indicates a nucleus that stains with pCdc27-1 antibodies and is still surrounded by an intact lamina. (D and E) Co-staining of pCdc27-1 with cyclin B1 (D) and Plk1 (E). The arrow in (D) indicates a cell where nuclear uptake of cyclin B1 has just begun and where pCdc27-1 staining can be seen in the nucleus. The arrow in (E) indicates a cell in which Plk1 but no pCdc27-1 staining can be detected in the nucleus. Bar, 10 µm (A and B), 20 µm (C–E).

Fig. 6. Phospho-APC is enriched at centrosomes but not at the mitotic spindle. (A) Logarithmically growing HeLa cells were stained with non-phospho Cdc27 or pApc1 antibodies (red), tubulin (green) and DAPI (blue). (B and C) HeLa cells were extracted with 0.1% Triton X-100 prior to fixation to remove soluble proteins and stained as in (A). Bar, 10 µm.

Fig. 7. Knockdown of Plk1 expression does not prevent APC activation. (A) HeLa cells were synchronized by a double thymidine arrest-release protocol and subjected to Plk1-RNAi treatment. After releasing cells from the second thymidine block for 13 h, cells were collected and analysed by immunoblotting using Plk1, Cdc27 and proteasome antibodies. Note that no Cdc27 mobility shift can be detected in the control cells because they had exited mitosis by this time (see Figure 2C). (B) Cells were treated as in (A), except that extracts were prepared 10 h after the second thymidine release when control cells progress through mitosis (see Figure 2C). The ubiquitination activity of APC immunopurified from these extracts was analysed as described for Figure 4, using myc-tagged cyclin B as a substrate (top panel). The amounts of APC used in these assays were compared by immunoblotting with Cdc26 antibodies (bottom panel). (C–F) Plk1 RNAi cells as in (A) were stained with antibodies to Plk1 (D; green), cyclin B1 (E; green) or cyclin A (F; green) and DNA was visualized with DAPI (blue). Note that cells which show the rosette-like arrangement of chromosomes typical for loss of Plk1 expression are positive for cyclin B but negative for cyclin A (indicated by arrows in E and F). The number of cells with prometaphase chromosome morphology that had lost cyclin A staining was counted in both control and Plk1 RNAi cells (C). Forty-two control cells and 85 RNAi cells were analysed and classified into cyclin A negative (–), weakly cyclin A postive (+/–) or cyclin A positive cells (+). Of all Plk1 RNAi cells with prometaphase morphology, 93% were cyclin A negative, whereas 83% of all control prometaphase cells were cyclin A negative. The increased frequency of cyclin A negative Plk1 RNAi cells may be due to the prolonged period of time these cells spend in prometaphase (see text). Bar, 10 µm. (G) HeLa cells treated as in (A) were microinjected in the nucleus with cyclin A–GFP cDNA (1 mg/ml), and followed by time-lapse fluorescence and DIC microscopy. Images (200-ms exposure) were taken at 3-min intervals. The total cell fluorescence minus background was quantified for each cell in successive images of a time series and plotted over time. A graph of a single cell, representative of 18 cells analysed, is shown. The start of NEBD and anaphase are marked.