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. 2020 Mar 9;11(1):1268.
doi: 10.1038/s41467-020-15059-5.

Antagonistic Activities of CDC14B and CDK1 on USP9X Regulate WT1-dependent Mitotic Transcription and Survival

Affiliations
Free PMC article

Antagonistic Activities of CDC14B and CDK1 on USP9X Regulate WT1-dependent Mitotic Transcription and Survival

Michael Dietachmayr et al. Nat Commun. .
Free PMC article

Abstract

Regulation of mitosis secures cellular integrity and its failure critically contributes to the development, maintenance, and treatment resistance of cancer. In yeast, the dual phosphatase Cdc14 controls mitotic progression by antagonizing Cdk1-mediated protein phosphorylation. By contrast, specific mitotic functions of the mammalian Cdc14 orthologue CDC14B have remained largely elusive. Here, we find that CDC14B antagonizes CDK1-mediated activating mitotic phosphorylation of the deubiquitinase USP9X at serine residue 2563, which we show to be essential for USP9X to mediate mitotic survival. Starting from an unbiased proteome-wide screening approach, we specify Wilms' tumor protein 1 (WT1) as the relevant substrate that becomes deubiquitylated and stabilized by serine 2563-phosphorylated USP9X in mitosis. We further demonstrate that WT1 functions as a mitotic transcription factor and specify CXCL8/IL-8 as a target gene of WT1 that conveys mitotic survival. Together, we describe a ubiquitin-dependent signaling pathway that directs a mitosis-specific transcription program to regulate mitotic survival.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. CDC14B and CDK1 regulate phosphorylation of USP9X at serine 2563 in mitosis.
a Co-immunoprecipitation of FLAG-tagged CDC14B with endogenous USP9X from HEK 293T cells that were either left untreated or arrested in mitosis using nocodazole (EV = expression vector). Immunocomplexes and respective WCE were probed with antibodies to the indicated proteins. b Immunoblot analysis of U2OS cells that were either synchronized in G1/S phase using a double thymidine block (left panel), or in mitosis using sequential thymidine and nocodazole treatment (right panel). Cells were then released into the cell cycle and collected at the indicated time points. c Immunoblot analysis of U2OS cells that were treated with siRNA directed against CDC14B and synchronized in mitosis as described above. Mitotic shake-off was performed and samples were analyzed by western blot with the indicated antibodies. d Quantification of n = 3 biologically independent experiments conducted as described in c. Ratio paired t-test was applied with **p = 0.0045. e Immunoblot analysis of mitotic U2OS cells after synchronization with thymidine and nocodazole. Mitotic cells were shaken off and kept in nocodazole-containing medium during treatment with either the CDK1 inhibitor RO-3306 or DMSO for 0.5 h. f In vitro kinase assay with C-terminal truncates of USP9X (aa 2165–2570), either the USP9X wild-type form (USP9XWT) or a USP9X form with a serine to alanine mutation on position 2563 (USP9XS2563A), that were purified from Escherichia coli and exposed to recombinant active CDK1-Cyclin B in the presence of radioactive 32P-ATP (CBB, Coomassie Brilliant Blue), *Cyclin B. g Quantification of two independent experiments conducted as described in f. 32P signals are normalized to the respective Coomassie signal. Mean is displayed from n = 2 biologically independent experiments. h Enzyme kinetics of USP9XWT and USP9XS2563A proteins purified from mitotic HEK 293T cells were measured at different Ubiquitin-AMC concentrations. The resulting values for KM of USP9XWT or USP9XS2563A were 0.9090 or 0.7835 µM, respectively, and for Vmax 36.69 or 31.06 µM/min, respectively. The values indicated are from n = 1 experiment. Throughout this figure means and standard deviations as error bars are displayed.
Fig. 2
Fig. 2. WT1 is a substrate of pUSP9X (serine 2563) in mitosis.
a Mass spectrometric analysis of the USP9X-dependent ubiquitome in mitotic HEK 293T cells. USP9X knockdown cells were cultured in heavy (“H”), control knockdown cells in medium (“M”) SILAC media. b Co-immunoprecipitation of FLAG-tagged WT1 with endogenous USP9X from HEK 293T cells (EV, expression vector). Cells were exposed to nocodazole, collected, lysed (WCE, whole cell extracts), and subjected to anti-FLAG immunoprecipitation before analysis by western blot. c Co-immunoprecipitation of USP9XWT rather than USP9XS2563A with WT1. Expression vector (EV), FLAG-tagged USP9XWT or USP9XS2563A was overexpressed in asynchronous (AS) or mitotically arrested (Mit, 15 h nocodazole) HEK 293T cells, and purified by immunoprecipitation before western blot analysis. d Quantification of WT1 co-immunoprecipitated with either USP9XWT or USP9XS2563A from n = 4 biologically independent experiments as described in c. Ratio paired t-test was applied with *p = 0.0248. e Immunofluorescence imaging of mitotic U2OS cells showing colocalization of WT1 (red) and USP9X (green) in two representative mitotic cells. Cells were transfected with FLAG-tagged WT1 and arrested in mitosis using nocodazole (15 h) before fixing and staining. Values for Pearson’s and Manders’ coefficients (tM1 and tM2) are the mean and SD calculated from n = 17 biologically independent cells. Scale bar, 10 µm. f In vivo ubiquitylation assay showing pUSP9X-dependent ubiquitylation of WT1. HEK 293T cells transfected with FLAG-tagged USP9XWT or USP9XS2563A, 2xStrep-tagged WT1, and HA-tagged Ubiquitin were synchronized in mitosis using nocodazole. Fourteen hours before collection nocodazole, bortezomib, and the caspase inhibitor Z-VAD-FMK were added. Cells were lysed under denaturing conditions and 2xStrep affinity purification was performed. g Cycloheximide time course showing USP9X-dependent WT1 stability. U2OS cells were treated with control or USP9X siRNA and arrested in mitosis using sequential thymidine and nocodazole (11 h). Mitotic cells were collected by shake-off and cycloheximide, bortezomib (where indicated), and Z-VAD-FMK added before collection and western blot analysis. h Quantification of WT1 protein levels from n = 3 biologically independent experiments as described in g. Western blot bands of WT1 were quantified using ImageJ software and normalized to loading control and time point 0. One sample t-test was applied with *p = 0.0219; *p = 0.0389. Throughout this figure mean and standard deviations as error bars are displayed.
Fig. 3
Fig. 3. WT1 regulates IL-8 transcription and secretion in mitosis.
a Heatmap of RNA-Seq analysis depicting differentially regulated genes in mitotic control cells versus WT1 knockdown U2OS cells. Each column displays an independent biological replicate, negatively regulated genes on the top, positively regulated genes on the bottom. b RNA-Seq analysis showing differentially regulated genes in mitotic USP9XWT versus USP9XMut U2OS cells. c Quantitative RT-PCR showing accumulation of IL-8 mRNA in mitotically synchronized versus asynchronous U2OS cells. Mean is shown from n = 4 biologically independent experiments. One sample t-test was applied with *p = 0.0198. d Extracellular IL-8 measured by ELISA is increased in the supernatant of mitotic versus asynchronous U2OS cells. Mean is shown from n = 3 biologically independent experiments. Ratio paired t-test was applied with ***p = 0.0002. e Quantitative RT-PCR from mitotic U2OS cells showing reduced IL-8 mRNA levels in response to USP9X, WT1, or double compared to control knockdown. Mean is shown from n = 3 biologically independent experiments. One-way ANOVA was applied with ****p<0.0001 followed by Dunnett’s test with ****p < 0.0001 (p(USP9X versus WT1) = 0.211; p(USP9X versus USP9X+WT1) > 0.9999; p(WT1 versus USP9X+WT1) = 0.2061). f Secreted IL-8 measured by ELISA in the supernatant of mitotic U2OS cells following USP9X, WT1, or double versus control knockdown. Mean is shown from n = 3 biologically independent experiments. One-way ANOVA was applied with ***p = 0.0001 followed by Dunnett’s test with ***p(Ctrl versus USP9X) = 0.0004, **p(Ctrl versus WT1) = 0.0055, ***p(Ctrl versus USP9X+WT1) = 0.0002; p(WT1 versus USP9X) = 0.3081, p(WT1 versus USP9X+WT1) = 0.0791, p(USP9X versus USP9X+WT1) = 0.6772. g Quantitative RT-PCR showing reduced IL-8 mRNA in USP9XMut versus USP9XWT U2OS cells that were mitotically synchronized using nocodazole. Mean is shown from n = 3 biologically independent experiments. One sample t-test was applied with **p = 0.002. h ELISA showing reduction of secreted IL-8 in the supernatant of mitotically synchronized USP9XMut compared to USP9XWT U2OS cells. Mean is shown from n = 3 biologically independent experiments. Ratio paired t-test was applied with *p = 0.0277. i Chromatin immunoprecipitation (ChIP) showing WT1 occupancy on the CXCL8 promoter in nocodazole-synchronized U2OS cells. ChIP signal was quantified using RT-PCR. WT1 occupancy was normalized to total input DNA. Mean from n = 3 biologically independent experiments is shown. Ratio paired t-test was applied with **p = 0.00277. j CXCL8 reporter assay performed in nocodazole-treated U2OS cells that were transfected with luciferase reporter construct harboring the human CXCL8 promoter or an empty reporter construct and a WT1 overexpressing vector as indicated. Luminescence was normalized to the background luminescence of the empty reporter construct. Mean is from n = 4 biologically independent experiments. Ratio paired t-test was applied with ***p = 0.0007. Throughout this figure standard deviations are displayed as error bars.
Fig. 4
Fig. 4. CDK1/CDC14B-dependent phosphorylation of USP9X at serine 2563 promotes mitotic survival via WT1 and IL-8.
a Mitotic apoptosis in response to CXCL8 versus control knockdown detected by immunoblot in U2OS cells that were treated with the respective siRNA and arrested in mitosis using nocodazole for 8 h. Samples were collected, lysed, and analyzed by western blot with the indicated antibodies. b Quantification of relative amount of cleaved caspase 3 in n = 3 biologically independent experiments conducted as described in a. Ratio paired t-test was applied with **p = 0.0055. c Immunoblot analysis showing increased mitotic apoptosis in U2OS cells in response to treatment with nocodazole (8 h) and the CXCR1/2 inhibitor reparixin (RPX) or DMSO for 48 h. d Quantification of relative amount of cleaved caspase 3 in n = 4 biologically independent experiments conducted as described in c. Ratio paired t-test was applied with **p = 0.0031. e Immunoblot analysis confirming reversal of mitotic apoptosis following exogenous reconstitution of IL-8 in CXCL8-depleted cells. Experiment was performed as in a, with addition of exogenous IL-8 for the last 48 h. Cells were treated with nocodazole for 8 h. f Immunoblot analysis detecting induction of mitotic apoptosis in response to WT1, CXCL8, or WT1 and CXCL8 knockdown compared to control knockdown in U2OS cells that were arrested in mitosis using nocodazole (8 h). g Immunoblot analysis showing increased mitotic apoptosis by WT1 knockdown only in USP9XWT but not in USP9XMut U2OS cells. Analyzed cells were treated with control or WT1 siRNA and then arrested in mitosis using nocodazole. h Induction of mitotic apoptosis in USP9XMut U2OS cells that were kept asynchronous or treated with nocodazole for 32 h, stained with PI and measured by flow cytometry. PI-positive cells were quantified in each sample using FlowJo software. Mean is shown from n = 3 biologically independent experiments. Paired t-test was applied with **p(Mitosis) = 0.00278, p(AS) = 0.0745. i Immunoblot analysis revealing decreased mitotic apoptosis after CDC14B knockdown in USP9XWT but not in USP9XMut U2OS cells. Before lysis cells were transfected with control or CDC14B-directed siRNA, arrested in mitosis, and collected for analysis by western blot. Throughout this figure, mean and standard deviations as error bars are displayed.

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