Fig 2: ID2-S5 is phosphorylated by CDK1 during mitosis leading to increased stability.a Overexpression of CDH1 does not affect stability of ID2 S5D. b Half-life of ID2 WT and phospho-mutants in HeLa cells treated with CHX. c Quantification of data in (b); data are means ± SEM of two independent experiments. d Regulation of ID2-S5 phosphorylation during cell cycle progression. Western blot of HeLa cells exposed to double thymidine block. e Loss of interaction between phosphorylated ID2-S5 and core APC subunits at entry into mitosis. Western blot of cells expressing FLAG-ID2 WT and phospho-mutants exposed to double thymidine block. f Inhibition of ID2-S5 phosphorylation by the CDK1-specific inhibitor dinaciclib. Western blot of lysates of cells expressing FLAG-ID2 and treated with palbociclib or dinaciclib. g In vitro kinase assay using CDK1-Cyclin B and GST-ID2 proteins. Lower panel, Coomassie blue staining of GST-ID2 proteins used in the assay. Arrowheads indicate full-length proteins. h ID2 phosphorylation by CDK1-Cyclin B prevents the interaction with core APC but not CDH1. GST-ID2 was phosphorylated in vitro by CDK1-Cyclin B before binding with FLAG-APC3 immunopurified from HeLa cells and activated by in vitro translated HA-CDC20 or CDH1. GST-pull-down reactions were analyzed by western blot. i Loss of ubiquitylation by the ID2-S5D. In vitro ubiquitylation of V5-ID2 WT or mutants was performed using the APC complex prepared as in (h). j In vivo ubiquitylation in HeLa cells co-expressing FLAG-ID2 WT or mutants and HA-ubiquitin. Left panel, HA-ubiquitin western blot of FLAG-ID2 immunoprecipitates. Right panel, western blot of whole cellular lysates (WCL). Coomassie staining in g was performed on SDS-gel loaded with the same amounts of GST-fusion proteins used in the binding reactions. APC proteins and CDH1 are from the same blots in each panel; Cyclin A1, Cyclin B1, pS5-ID2, pS14-ID2 and pT27-ID2, FLAG are from independent gels; in i, V5 and HA/FLAG are from two independent gels; in j, HA and FLAG are from independent gels. Loading controls are from the same gel as FLAG. Molecular weight markers are indicated in kDa. Experiments were repeated three times for a–g and two times for h–j with similar results.

Fig 3: The APC/C is a component of the mitotic centrosome. (A) Mitotic RPE1 or HEK293 cells were pre-extracted, fixed in formaldehyde (top) or ice-cold methanol (bottom) and stained with the indicated antibodies. Boxes indicate regions shown in enlarged images. A line scan with a 10-pixel-wide line was performed at the indicated positions, and the normalized values were plotted (right). Data shown are representative of three experiments. (B) HEK293 cells were treated with STLC or nocodazole to induce a prometaphase arrest. Cells were pre-extracted, fixed in formaldehyde and stained with the indicated antibodies (CNT3, centrin-3). Boxes indicate regions shown in enlarged images. The intensity of APC2 at the centrosome was measured inside the dotted circle and normalized against the signal intensity in the presence of STLC (right). N cells=91 (STLC) and 84 (nocodazole). (C) HEK293 cells were treated with centrinone to deplete their centrosomes. Cells were pre-extracted, fixed in formaldehyde and stained with the indicated antibodies. The intensity of APC2 throughout the whole cell was measured and normalized against the signal intensity in DMSO (bottom). N cells=11 (DMSO control) and 14 (centrinone). In A–C, microtubules were labelled using an a-tubulin antibody, and DNA was stained with DAPI. (D) Centrosomes were purified from mitotic HEK293 cells using a sucrose gradient (centr., centrosomal). The eluted fractions were immunoblotted against the indicated proteins. The top shows PCNT, ?Tub, and centrin-1 (CNT1) as centrosomal proteins, the middle shows several APC/C components, and MCC proteins are shown at the bottom. Blots are representative of four experiments. (E) Centrosomes were purified in parallel from interphase HEK293 cells and mitotic HEK293 cells using a sucrose gradient. The first elution fractions were blotted to detect PCNT as a centrosomal marker and APC3. Blots are representative of three experiments. (F) Centrosomes from E were fixed on a coverglass and stained with the indicated antibodies (top). The intensity of APC2 was measured as indicated in B and normalized against the signal intensity on interphase centrosomes (bottom). N centrosomes=142 (interphase) and 123 (mitotic). Box plots show the median (line), 25–75% range (box) and 5–95% range (whiskers). ****P<0.0001 (Mann–Whitney U-test). Scale bars: 5 µm in A–C, 1 µm in F.

Fig 4: Impaired destabilization of the phospho-mimetic ID2 S5D mutant delays mitotic exit.a HeLa cells expressing ID2 WT or S5 phospho-mutants were exposed to double thymidine block and analyzed by flow cytometry. Bar graph shows quantification of cells in G1, S, and G2/M in one representative experiment. Experiments were performed three times with similar results. b HeLa cells treated as in a were immunostained with pHH3 and analyzed by flow cytometry. Bar graph shows quantification of pHH3-positive cells. c Loss of regulation of ID2-S5D during S-G2/M transition. Western blot of HeLa cells expressing FLAG-ID2 WT or S5 phospho-mutants exposed to double thymidine block. d Exit from mitosis is delayed by expression of ID2-S5D mutant. Western blot of HeLa cells expressing FLAG-ID2 WT or ID2-S5D treated with RO3306. e Left panel, schematics of the cell synchronization protocol for the analysis of ubiquitylation of ID2 WT and phospho-mutants at the G2/M and M/G1 transition. Middle panel, western blot of HeLa cells co-expressing FLAG-ID2 WT or phospho-mutants and HA-ubiquitin immunoprecipitated with FLAG antibody. Right panel, western blot of whole cellular lysates (WCL). Molecular weight markers are indicated in kDa. Experiments were repeated two times with similar results. f HeLa cells expressing ID2 wild type or S5 phospho-mutants were transiently transfected with a plasmid expressing the E-box-luciferase cassette. Cells were synchronized by double thymidine block and analyzed for luciferase expression. Bar graph indicates means ± SD of three replicates. p-values are from unpaired t-test, unequal variance. Experiment was repeated three times with similar results. g Loss of binding to chromatin of E47 and HEB in cells overexpressing ID2 S5D. Western blot of chromatin fractions of HeLa cells transfected with ID2 WT or ID2-S5D, synchronized by double thymidine block, and collected at serial times after release. Experiments were repeated two times with similar results. In d, APC3 and Cyclin B1 are from the same blots; pHH3, CDH1 are from independent gels; in e, HA and FLAG are from different gels; in g, E47 and HEB/pHH3 are from different gel. Loading controls are from the same gel as FLAG except in e where a-tubulin is from the same gel as pHH3.

Fig 5: The APC/C is SUMOylated in mitosis(A) APC4 immunoblot of cell lysates (top panel) and tubulin immunoblot of loading control (lower panel). Cells were arrested either in S-phase or in mitosis. The efficiency of the cell cycle arrest was confirmed by immunoblotting against APC3 and phosphorylated histone H3 at Ser10 (Figure S1A). Only mitotically arrested cells show additional SUMOylation bands above APC4.(B) Ubc9 was depleted using 20 nM RNAi. Ubc9 was depleted for 48 h, after which STLC was added to enrich for mitotically arrested cells, and cells were left for an additional 16 h. Immunoblotting against APC4 was performed (top panel). Only the Ubc9-depleted cells (Ubc9a and Ubc9b) showed the disappearance of bands above APC4. RNAi against the luciferase gene (GL2) was used as a control. An anti-Ubc9 immunoblot showed Ubc9 depletion in the Ubc9a- and Ubc9b-treated cells. Actin loading control (middle panel).(C) APC4 SUMOylation is increased in mitotic cells. U2OS HIS10-SUMO2 and U2OS (control) cells, asynchronous (AS) or synchronized in mitosis with STLC, harvested with trypsinization or mitotic shake-off, respectively. Inputs and SUMO2-enriched fractions were analyzed by immunoblotting using antibodies against APC4, SUMO2/3, cyclin A, and securin. Protein loading was verified by Ponceau S staining. The experiment was performed in triplicate and repeats 2 and 3 are shown in Figure S1C. Synchronization was verified by immunoblotting for cyclin A2 and securin and by FACS analysis for DNA content and MPM2 staining and shown in FACS plots (Figure S1D).(D) SUMOylated APC4 from experiments in (C) and its repeats from Figure S1C were quantified and visualized in a graph. Quantification was performed by measuring the band intensity of SUMOylated APC4 in each experiment and comparing it to the intensity of SUMOylated APC4 in AS cells. Data represent the mean with 1 standard deviation (n = 3). Statistical analysis was performed using an unpaired Student’s t test. ****p < 0.001.