The TRIM69-MST2 signaling axis regulates centrosome dynamics and chromosome segregation

Abstract

Stringent control of centrosome duplication and separation is important for preventing chromosome instability. Structural and numerical alterations in centrosomes are hallmarks of neoplastic cells and contribute to tumorigenesis. We show that a Centrosome Amplification 20 (CA20) gene signature is associated with high expression of the Tripartite Motif (TRIM) family member E3 ubiquitin ligase, TRIM69. TRIM69-ablation in cancer cells leads to centrosome scattering and chromosome segregation defects. We identify Serine/threonine-protein kinase 3 (MST2) as a new direct binding partner of TRIM69. TRIM69 redistributes MST2 to the perinuclear cytoskeleton, promotes its association with Polo-like kinase 1 (PLK1) and stimulates MST2 phosphorylation at S15 (a known PLK1 phosphorylation site that is critical for centrosome disjunction). TRIM69 also promotes microtubule bundling and centrosome segregation that requires PRC1 and DYNEIN. Taken together, we identify TRIM69 as a new proximal regulator of distinct signaling pathways that regulate centrosome dynamics and promote bipolar mitosis.

Graphical Abstract

Figure 1.

TRIM69A expression is associated with Centrosome Amplification 20 (CA20) gene expression signature, Homologous Recombination-Deficiency (HRD) and Aneuploidy in basal breast cancers. (A) Heatmap showing relative expression of CA20 signature genes in relation to TRIM69A mRNA expression and various other classifiers. Annotations for PAM50 subtype, BRCA1 germline mutation status, TP53 RNA subtype (43), self-reported race, and DNA Repair group are provided along with continuous CA20, DDR, and HRD signature scores. Heatmap clustering was performed by both samples (columns) and genes (rows) using centroid linkage. (B) Violin plot depicting distribution of CA20 scores by TRIM69A expression (low = bottom three quartiles, high = top quartile). P-value represents comparison of CA20 between TRIM69A high and low groups by Wilcoxon rank-sign test. (C) Violin plot of aneuploidy scores separated by TRIM69A expression group. P-value represents comparison of aneuploidy scores (44) in TRIM69A high versus low samples by Wilcoxon rank-sign test t. (D) Violin plot of HRD scores according to TRIM69A expression group. P-value represents comparison of HRD scores (44) in TRIM69A high versus low samples by Wilcoxon rank-sign test. This is a violin plot of samples separated as TRIM69A High or Low. HRD score is plotted on the y-axis. Wilcoxon P-value was performed.

Figure 2.

TRIM69A interacts with MST2 and MST1 mainly in a detergent-insoluble subcellular compartment. (A) Immunoblots showing relative distribution of TRIM69A and TRIM69B between detergent-soluble and detergent-insoluble cell fractions in H1299 and MDA-MB-231 cells. (B) Representative confocal microscopy images of HA-TRIM69A/B-expressing cells showing subcellular distribution of HA-TRIM69 (green) in relation to pericentrin (red) for each cell cycle stage (identified based on nuclear morphology). Scale = 5 μm. (C) H1299 cells transduced with viruses encoding HA-TRIM69A, HA-TRIM69B, or an ‘empty’ adenovirus vector for control. Cells were biochemically-fractionated to generate detergent-soluble and detergent-insoluble extracts. The detergent-insoluble extracts were dissociated using DNAse and sonication. HA-TRIM69A and HA-TRIM69B complexes were immunopurified from the extracts and analyzed by mass-spectrometry. The table lists the most abundant proteins that specifically co-purified from with HA-tagged TRIM69 variants. Max Ratios were selected with a stringent filter to avoid a ratio <2 for any sample. The bar chart shows fold-enrichment of various TRIM69-associated proteins in anti-HA-immunoprecipitates from HA-TRIM69A/B-expressing cells when compared with control (empty vector) cultures. (D) Immunoblot showing co-immunoprecipitation of MYC-TRIM69A with HA-MST2. (E) Confocal microscopy images of a representative H1299 cell showing co-localization of MYC-TRIM69A (red) with HA-MST2 (green). (F) Confocal microscopy images of representative H1299 cells at different cell cycle stages showing subcellular distribution of HA-MST2 (green) in relation to pericentrin (red). Scale = 5 μm. (G) Yeast 2-hybrid assays showing extent to which TRIM69A or TRIM69B interact with MST2 and MST1.

Figure 3.

TRIM69A regulates MST2 ubiquitination and subcellular distribution. (A) TRIM69 (WT), but not catalytically-inactive TRIM69A (E3 mut) promotes conjugation of Ub to MST2. (B) TRIM69 WT but not catalytically-inactive TRIM69A (E3 mut) undergoes autoubiquitination. (C) MYC-TRIM69A does not affect MST2 stability in cycloheximide (CHX, 10 μg/ml)-treated 239T cells. The half-life of MST2 was determined based on results from two different independent experiments. Relative levels of MST2 as detected by immunoblotting were quantified using ImageJ software and normalized to GAPDH levels. (D) Wild-type MYC-TRIM69A (but not MYC-TRIM69A E3 mut or MYC-TRIM69B) redistributes MST2 to the detergent insoluble compartment in H1299 cells. (E) Confocal microscopy images of representative H1299 cells showing TRIM69A-induced subcellular redistribution of HA-MST2. Scale bar represents 5 μm. (F) Immunoblot showing that multiple independent TRIM69-directed siRNAs reduce the amount of endogenous MST2 associated with the detergent-insoluble compartment in H1299 and MDA-MB-231 cells.

Figure 4.

TRIM69A promotes MST2-PLK1 interaction in the detergent-insoluble compartment. (A) Effect of TRIM69 on the protein-interaction network of MST2 in detergent-soluble and detergent-insoluble compartments. 1% FDR was used to filter all peptides/proteins; only proteins with >1 peptides are reported. (B) TRIM69A promotes co-localization of MST2 (red) and PLK1 (green) in H1299 cells. Scale bar represents 10 μm. (C) TRIM69A promotes interaction between MST2 and PLK1 in the detergent-insoluble compartment. (D) Phosphoproteome profiling analysis showing TRIM69-inducible MST2 phosphorylation sites in the detergent-insoluble compartment. 1% FDR was used to filter all peptides/proteins; only proteins with >1 peptides are reported. (E) Immunoblots showing phosphorylation of MST2 by TRIM69A in CSK insoluble fraction using SDS-PAGE and Phos-tag phosphate-affinity gel electrophoresis. (F) Immunoblots showing TRIM69A promoted phosphorylation of NEK2A by MST2 in CSK insoluble fraction using SDS-PAGE and Phos-tag phosphate-affinity gel electrophoresis. (G) Effect of MST2 phosphorylation site mutations on co-localization of MST2 and PLK1. Co-localization was quantified using the ImageJ plug-in JAcoP. Each column represents the mean ± standard error of the mean (SEM) from two independent experiments, n = 20 cells for each condition, ∗∗P < 0.01. Scale bar represents 5 μm. (H) Effect of MST2 phosphorylation site mutations on MST2 interactions with TRIM69A and PLK1.

Figure 5.

TRIM69 and MST2 promote centrosome separation. (A–D) U2OS cells were transfected with the indicated plasmids for 24 h before a single thymidine block/release and treatment with 5 mM Eg5 inhibitor (STLC) to trap cells in prometaphase. (A-B) Representative confocal microscopy images showing simultaneous immunostaining for pericentrin (green) and C-Nap1 (red) in U2OS cells harboring ectopically-expressed NEK2A, MST2, or TRIM69A. Scale = 5 μm. (C) Effect of ectopically-expressed NEK2A, MST2, or TRIM69A on inter-centrosome distance. Results are from two independent experiments. n > 20 cells were analyzed for each condition. Data are mean ± standard error of the mean (SEM) (∗∗P < 0.0001). (D) Effect of ectopically-expressed NEK2A, MST2 or TRIM69A on intensity of C-NAP1 signals at centrosomes. Results are from two independent experiments. n > 20 cells were analyzed for each condition. Data are mean ± standard error of the mean (SEM) (∗∗P < 0.0001). (E, F) Effect of siTRIM69 and siMST2/1 on inter-centrosome distance. Results are from two independent experiments; n > 20 cells were analyzed for each condition. Data are mean ± standard error of the mean (SEM) (∗∗P < 0.0001). (G) U2OS cells were transfected with indicated siRNAs for 48 h before a single thymidine block/release and treatment with 5 uM Eg5 inhibitor (STLC) to trap cells in prometaphase. The bar chart shows the effect of siTRIM69 and siMST2/1 on intensity of C-NAP1 signals at centrosomes. Results are from two independent experiments. n > 20 cells were analyzed for each condition. Data are mean ± standard error of the mean (SEM). ∗∗P < 0.0001.

Figure 6.

TRIM69A promotes triple-negative breast cancer (TNBC) cell proliferation and allows breast cancer cells to tolerate mitotic stresses due to centrosome amplification. (A) Clonogenic survival assays using parental MDA-MB-231 (TRIM69^+/+) cells or a TRIM69^−/− derivative cell line. Quantitative analysis of colony formation is presented in a bar chart (lower panel). Clonogenic survival of TRIM69^−/− cells was normalized to colony survival of WT cells. Data points represent the mean of triplicate determinations ± SEM. **P ≤ 0.001. (B) Immunofluorescence images of pericentrin (red) and β-tubulin (green) staining in taxol-treated MDA-MB-231 cells showing representative normal and clustered centrosomes. Scale = 5 μm. The bar chart shows quantification of normal vs. scattered centrosomes in TRIM69^+/+ and TRIM69^−/− derivative cells. Each column represents the mean ± range from two independent experiments, n = 100 cells for each condition. (C) Immunofluorescence images of pericentrin (red) and β-tubulin (green) staining in HAUS1 siRNA-treated MDA-MB-231 cells showing representative normal and clustered centrosomes. Scale = 5 μm. The bar chart shows quantification of cells with normal or scattered centrosomes. Bars represent the mean ± range from two independent experiments, n = 100 cells for each condition. (D) Immunoblots showing doxycycline-inducible expression of TRIM69A WT or TRIM69A E3 mut. (E) The bar chart shows quantification of normal versus scattered centrosomes in TRIM69^+/+ and TRIM69^−/− cells after reconstituting TRIM69A WT or TRIM69A E3 mut. Each column represents the mean ± range from two independent experiments, n = 50 cells for each condition. ∗P < 0.05. (F) The bar chart shows quantification of colony formation in TRIM69^+/+ and TRIM69^−/− cells after inducing TRIM69A WT or TRIM69A E3 mut expression with or without taxol treatment. Data points represent the mean of triplicate determinations ± SEM. *P < 0.05.

Figure 7.

TRIM69A prevents mitotic defects in PLK4-overexpressing cells. (A) Effect of TRIM69A siRNA on clonogenic survival of U2OS cells harboring doxycycline (Dox)-inducible GFP-PLK4. The left panel shows representative plates containing stained colonies. On the bar chart, each column represents the mean survival ± SEM of an independent biological replicate. (B) Doxycycline-inducible expression of GFP-PLK4 results in increased pericentrin (purple)-staining in a representative U2OS cell stably expressing RFP-H2B. Scale bar represents 5 μm. (C) Effect of siRNAs targeting TRIM69A, MST2 and MST1 on mitotic timing of mRFP-H2B-expressing U2OS cells in the presence and absence of excessive PLK4, as determined by time-lapse live cell fluorescence microscopy. The results presented are compiled from three independent experiments. +Dox: siCon, n = 69 cells; siTRIM69A1, n = 87 cells; siTRIM69A2, n = 93 cells; siMST2 + MST1, n = 100 cells; –Dox: siCon, n = 98; siTRIM69A1, n = 101 cells; siTRIM69A2, n = 109 cells; siMST2 + MST1, n = 116 cells. Data are mean ± SEM. *P ≤ 0.05. Quantification of time-lapse imaging experiments, performed exactly as described in the legend for Figure 7. The results show the effect of siTRIM69A and siMST2/4 treatments on the following measurements: time from NEB to anaphase (D); number of daughter cells with micro-/multi-nucleation (E); mitotic cells with multipolar spindles (F); and mitotic cells with lagging chromosomes and/or anaphase bridges (G). Data are mean ± SEM. *P ≤ 0.05, **P ≤ 0.001. (H) Immunoblots showing effective depletion of MST2 and MST1 by siRNA treatments in U2OS cells.

Figure 8.

TRIM69A promotes reorganization of microtubules, their associated motors and nucleoporins. (A) Representative images showing effect of TRIM69A and TRIM69A E3 mut expression on immunostaining pattern of β-tubulin (green) in H1299 cells. Scale bar represents 5 μm. (B) Effect of TRIM69A expression on immunostaining pattern of acetylated α-Tubulin (red) in H1299 cells (left panel). Scale bar represents 5 μm. Immunoblots showing TRIM69A promoted acetylation of α-Tubulin in CSK insoluble fraction (right panel). (C) The bar charts illustrate the quantification of tubulin spindle intensity after TRIM69A knockdown, number under bars are number of cells quantified. Data are mean ± standard error of the mean (SEM). ∗∗P < 0.0001; n.s., not significant difference. (D) Confocal microscopy images of a representative H1299 cell showing co-localization of MYC-TRIM69A (green) with mCherry-PRC1 (red) (Scale bar represents 5 μm). (E) Effect of TRIM69A expression on immunostaining of β-tubulin (green) and distribution of endogenous PRC1 (red) in H1299 cells. Scale bar represents 5 μm. (F) Confocal microscopy images showing the effect of mCherry-PRC1 (red) overexpression on MT bundling (green) in H1299 cells. (G) Effect of TRIM69 expression on immunostaining pattern of DYNLL1 (green) and β-tubulin (red) in H1299 cells. Scale bar represents 5 μm. (H) The bar charts illustrate that TRIM69-induced centrosome clustering in paclitaxel-treated cells is inhibited by siRNAs against PRC1 and Dynein. Bars represent mean ± range from two independent experiments. (I) Representative images showing effect of PRC1 and DYNEIN knockdown on interphase MT-bundles after TRIM69A overexpression. Immunostaining pattern of β-tubulin (green) is shown. All cells were imaged with the same imaging parameters. Scale bar represents 5 μm. (J) The bar charts illustrate the quantification of interphase MT-bundle width at the base of the peak represented in Figure I. n is the number of bundles-five bundles per cell. Data are mean ± standard error of the mean (SEM), ∗∗P < 0.0001; ∗0.001 > P > 0.0001; n.s., not significant. (K) The bar charts illustrate the quantification of interphase MT-bundle cross section intensity represented in figure (I). n is the number of bundles-five bundles per cell. Data are mean ± standard error of the mean (SEM), ∗∗p < 0.0001; ∗0.001 > p > 0.0001; n.s., not significant. (L) Representative images showing effect of PRC1, DYNEIN and MST2/MST1 knockdown on intensity of individual spindles after TRIM69A overexpression in metaphase H1299 cells. Immunostaining pattern of β-tubulin (green) and Pericentrin (red) is shown. All cells were imaged with the same imaging parameters. Scale bar represents 5 μm. (M) The bar charts illustrate the quantification of tubulin spindle intensity represented in (L), numbers under the bars indicate the number of cells quantified. Data are mean ± standard error of the mean (SEM). ∗∗P < 0.0001; n.s., not significant differences. (N)The immunoblots validate effective downregulation of PRC1 and DYNEIN protein in siRNA experiments.