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. 2017 Jan 3;3:16048.
doi: 10.1038/celldisc.2016.48. eCollection 2017.

Arginine Methylation of USP9X Promotes Its Interaction With TDRD3 and Its Anti-Apoptotic Activities in Breast Cancer Cells

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Free PMC article

Arginine Methylation of USP9X Promotes Its Interaction With TDRD3 and Its Anti-Apoptotic Activities in Breast Cancer Cells

Nithya Narayanan et al. Cell Discov. .
Free PMC article

Abstract

The Tudor domain-containing proteins are characterized by their specific interactions with methylated protein motifs, including methyl-arginines and methyl-lysines. The Tudor domain-containing protein 3 (TDRD3) is one of the major methyl-arginine effector molecules that recognizes methylated arginine residues on histones and the C-terminal domain of RNA polymerase II, and activates transcription. However, majority of the cellular TDRD3 localizes to the cytoplasm and its functions there are still elusive. Here, we have identified ubiquitin-specific protease 9 X-linked (USP9X) as a TDRD3-interacting protein by GST (glutathione S-transferase) pull-down and co-immunoprecipitation. Detailed characterization suggests that the interaction between TDRD3 and USP9X is mediated through the Tudor domain of TDRD3 and the arginine methylation of USP9X. This interaction plays a critical role in TDRD3 protein stability, as knockdown of USP9X expression leads to increased TDRD3 ubiquitination. We also found that USP9X co-localizes with TDRD3 in cytoplasmic stress granules and this localization is diminished in Tdrd3-null mouse embryonic fibroblast cells, suggesting that TDRD3 is essential for USP9X stress granule localization. Furthermore, we found that one of the USP9X de-ubiquitination targets, myeloid cell leukemia protein 1, is regulated by TDRD3, indicating that TDRD3 potentially regulates USP9X de-ubiquitinase activity. Finally, we show that knockdown of TDRD3 expression sensitizes breast cancer cells to chemotherapeutic drug-induced apoptosis, likely due to its regulation of USP9X. This study provides a novel candidate strategy for targeting apoptosis pathways in cancer therapy.

Keywords: Tudor domain; USP9X; apoptosis; arginine methylation; stress granule.

Figures

Figure 1
Figure 1
TDRD3 interacts with USP9X. (a) GST pull-down assays were performed using recombinant GST, GST-Tudor and GST-Tudor (E691K) proteins with the HeLa cell total cell lysates. Both the input samples and pull-down samples were detected with an anti-USP9X antibody (left panel). The GST-tagged recombinant proteins in the pull-down samples were visualized by Ponceau S staining (right panel). (b) TDRD3 and USP9X co-IP. HeLa cells were IPed with rabbit control IgG and two different rabbit polyclonal anti-TDRD3 antibodies. Both the input and the eluted protein samples were detected with anti-TDRD3 and anti-USP9X antibodies. Two different sources of TDRD3 antibody were used to confirm the results—anti-TDRD3 serum [13] and TDRD3 antibody from Cell Signaling Technology (Danvers, MA, USA) (TDRD3 CST). (c) TOP3B does not interact with USP9X. Both the HeLa cells and HEK293 cells were IPed with rabbit control IgG, anti-TDRD3 and anti-TOP3B antibodies and detected with anti-TDRD3 and anti-USP9X antibodies. (d) HeLa cells transiently transfected with GFP empty vector, GFP-TDRD3 and GFP-TOP3B were IPed with an anti-GFP antibody. The input and IPed protein complexes were detected with anti-GFP and anti-USP9X antibodies.
Figure 2
Figure 2
Mapping the interaction regions of TDRD3 with USP9X. (a) A series of GFP-fusion deletions of TDRD3 were generated. The locations of the OB fold (OB-fold), the ubiquitin-binding domain (UBA) and the Tudor domain (Tudor) are indicated. A summary of the interactions observed in b is shown. (b) A co-IP assay was performed in HeLa cells transfected with the different TDRD3 GFP-fusion vectors. The cell lysates were IPed with anti-GFP, and the eluted samples were blotted with anti-USP9X and anti-TOP3B. The input samples were blotted with anti-USP9X, anti-TOP3B and anti-GFP. (c) A diagram indicates the GST-fusion deletions of USP9X generated for the pull-down assays described in d. (d) GST pull-down assays were performed using recombinant GST, GST-USP9X (N1), GST-USP9X (N2), GST-USP9X (C1) and GST-USP9X (C2) with the HeLa cell total cell lysates. Both the input samples and pull-down samples were detected with anti-TDRD3 (upper panel). The GST-tagged recombinant proteins in the pull-down samples were visualized by Ponceau S staining (bottom panel). (e) A graphical summary of the protein regions that mediate the TDRD3–USP9X interaction.
Figure 3
Figure 3
The interaction of TDRD3 with USP9X is regulated by the arginine methylation of USP9X. (a) USP9X is arginine-methylated in cells. HeLa cells were first treated with the methylation inhibitor AdOx for 4 days and the total cell lysates were IPed with rabbit control IgG, anti-ADMA (pan-antibody that detects asymmetrically dimethylated proteins) and anti-SDMA (pan-antibody that detects symmetrically dimethylated proteins). The IPed protein complexes were detected with anti-USP9X. The input samples were detected with anti-ADMA and anti-SDMA to monitor the efficiency of the methylation inhibition. Protein expression was detected using anti-USP9X and anti-ACTIN. (b) HeLa cells were treated with AdOx for 4 days and the total cell lysates were IPed with anti-USP9X. The eluted protein samples were detected with an anti-ADMA and anti-USP9X. The arrow indicates the USP9X asymmetrical dimethylation. (c) The TDRD3 interaction with USP9X is reduced when methylation is inhibited. HeLa cells were treated with AdOx for 4 days to inhibit methylation. GST pull-down assays were performed using recombinant GST-Tudor with HeLa cell lysates. Both the input and the pull-down samples were detected with anti-USP9X. The GST-Tudor proteins in the pull-down samples were visualized by Ponceau S staining. (d) ADMA modification regulates USP9X interaction with TDRD3. HeLa cells were treated with PRMT inhibitor MS023 (10μm) for 48 h to inhibit ADMA modification. Co-IP assays were performed to detect USP9X interaction with TDRD3. Both the input and the IPed samples were detected with anti-USP9X, anti-TDRD3 antibody. The effect of MS023 treatment on cellular ADMA modification and USP9X methylation was detected using an anti-ADMA antibody. (e) PRMT1 regulates the TDRD3 interaction with USP9X. HeLa cells were transfected with either control or PRMT1-specific siRNA for 3 days and the total cell lysates were IPed with anti-USP9X. The eluted protein samples were detected with anti-TDRD3, anti-ADMA and anti-USP9X antibodies. The expression levels of individual proteins in the input samples were detected with anti-USP9X, anti-TDRD3, anti-PRMT1 and anti-ACTIN.
Figure 4
Figure 4
USP9X protects TDRD3 from ubiquitination. (a) TDRD3 is ubiquitinated in cells. HeLa cells were transiently transfected with HA-ubiquitin and GFP-TDRD3 (as indicated). After 24 h of transfection, the cells were either treated with DMSO or 10 μm of MG132 for an additional 16 h. TDRD3 in vivo ubiquitination was detected by IP with anti-TDRD3. The eluted protein samples were detected with anti-HA and anti-TDRD3. The input samples were detected with anti-p53 and anti-ACTIN to monitor the efficiency of proteasome inhibition by MG132. (b) HeLa cells were treated with either DMSO or MG132 for 16 h and the total cell lysates were IPed with anti-TDRD3. The eluted protein samples were detected with anti-ubiquitin (P4D1) and anti-TDRD3. The input samples were detected with anti-TDRD3 and anti-ACTIN. (c) Loss of USP9X promotes TDRD3 ubiquitination in the cells. HeLa cells were transfected with either control or USP9X-specific siRNA and either treated with DMSO or MG132 for 16 h. The total cell lysates were IPed with anti-TDRD3 and the eluted protein samples were detected with anti-ubiquitin (P4D1) and anti-TDRD3. The input samples were detected with anti-USP9X, anti-TDRD3 and anti-ACTIN. (d) Inhibition of USP9X de-ubiquitinase (DUB) activity destabilizes TDRD3. HeLa cells were treated with 5 μm of WP1130 for 24 h. The expression levels of TDRD3 and USP9X were detected with anti-TDRD3 and anti-USP9X. Anti-ACTIN was used as a loading control.
Figure 5
Figure 5
TDRD3 regulates USP9X localization to SGs in response to arsenite treatment. (a) HeLa cells cultured on glass coverslips were left untreated (Control) or treated with 0.5 mm sodium arsenite (Arsenite) for 30 min. The cells were fixed and immunostained with anti-USP9X and anti-TDRD3 to detect the localization of endogenous proteins. DAPI was used to stain the nuclear DNA. The arrows indicate USP9X and TDRD3 co-localization in SGs. (b) HeLa cells were treated as in a and immunostained with anti-USP9X and anti-G3BP. (c) Wild-type and TDRD3-knockout MEF cells were both treated with 0.5 mm sodium arsenite for 30 min. The cells were fixed and immunostained with anti-USP9X and anti-G3BP antibodies.
Figure 6
Figure 6
TDRD3 regulates the stability of a USP9X target protein—MCL-1. (a) Reduction of TDRD3 levels destabilizes MCL-1. MDA-MB-231 cells were stably transfected with an inducible shRNA vector targeting TDRD3 mRNA. The cells were either left untreated or treated with doxycycline (Dox) (1 μg ml−1) for 6 days. The protein expression levels were detected by western blotting using anti-MCL-1, anti-USP9X and anti-TDRD3. Anti-ACTIN served as a loading control. (b) The re-expression of TDRD3 stabilizes the MCL-1 protein. LCD cells were stably transfected with GFP or GFP-TDRD3 and the total cell lysates were immunoblotted with anti-MCL-1, anti-USP9X and anti-TDRD3. Anti-ACTIN served as a loading control. (c) The mRNA levels of MCL-1 in MDA-MB-231 cells treated using the conditions described in a were detected by RT-qPCR (quantitative reverse transcription PCR). Error bars represent the s.d. calculated from triplicate qPCR reactions. (d) Reduction of TDRD3 levels promotes MCL-1 ubiquitination. Both control and Dox-inducible TDRD3 shRNA-expressing MDA-MB-231 cells were treated with DMSO or MG132 (10 μm) for 16 h. The cells were lysed in RIPA buffer and IPed with an anti-MCL-1 antibody. The eluted protein samples were detected with anti-ubiquitin (P4D1) and anti-MCL-1 (left panel). The input samples were detected with anti-MCL-1, anti-TDRD3 and anti-USP9X. Anti-ACTIN served as a loading control. (e) Reduction of PRMT1 levels destabilizes MCL-1. MDA-MB-231 cells were transfected with control or PRMT1-specific siRNA. The total cell lysates were detected with anti-MCL-1, anti-USP9X, anti-TDRD3 and anti-PRMT1. Anti-ACTIN served as a loading control.
Figure 7
Figure 7
TDRD3 knockdown sensitizes MDA-MB 231 cells to apoptosis. (a) Knockdown of TDRD3 sensitizes cells to DUB inhibition-induced apoptosis. Both control and Dox-inducible TDRD3 shRNA-expressing MDA-MB-231 cells were treated with increasing concentration of WP1130 (0, 5, or 10 μm) for 16 h. Apoptosis was detected by FACS analysis. A representative figure is shown. (b) Summary of the results from a for increasing concentrations of WP1130. (c) Western blotting was performed to examine the TDRD3 and MCL-1 protein level in cells treated with or without WP1130 (5 μm, 16 h) and knockdown of TDRD3. (d, e) Knockdown of TDRD3 sensitizes cells to BCL-2 inhibitor ABT-199 and ABT-263 induced apoptosis. The experiments were performed similar to a, except that the cells were treated with increasing concentrations of ABT-199 (0, 2, 5, 10 μm) and ABT-263 (0, 1 μm) for 48 h. Error bars represent s.d. calculated from triplicate assays of one representative experiment. (f) Graphic summary of the results. PRMT-mediated arginine methylation of USP9X promotes its interaction with methyl-arginine effector molecule TDRD3, which is essential for its stress granule localization and regulation of its DUB activity, that is, de-ubiquitination of MCL-1. Both processes could contribute to USP9X anti-apoptotic activity.

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References

    1. Bedford MT, Clarke SG. Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009; 33: 1–13. - PMC - PubMed
    1. Yang Y, Bedford MT. Protein arginine methyltransferases and cancer. Nat Rev Cancer 2013; 13: 37–50. - PubMed
    1. Lasko P. Tudor domain. Curr Biol 2010; 20: R666–R667. - PubMed
    1. Selenko P, Sprangers R, Stier G, Buhler D, Fischer U, Sattler M. SMN tudor domain structure and its interaction with the Sm proteins. Nat Struct Biol 2001; 8: 27–31. - PubMed
    1. Ponting CP. Tudor domains in proteins that interact with RNA. Trends Biochem Sci 1997; 22: 51–52. - PubMed
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