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. 2017 Mar 1;37(6):e00419-16.
doi: 10.1128/MCB.00419-16. Print 2017 Mar 15.

Interactome Analysis Reveals a Novel Role for RAD6 in the Regulation of Proteasome Activity and Localization in Response to DNA Damage

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Interactome Analysis Reveals a Novel Role for RAD6 in the Regulation of Proteasome Activity and Localization in Response to DNA Damage

Hongli An et al. Mol Cell Biol. .
Free PMC article

Abstract

RAD6, an E2 ubiquitin-conjugating enzyme, is a key node for determining different DNA damage repair pathways, controlling both the error-prone and the error-free DNA damage repair pathways through differential regulation of the ubiquitination of the proliferating cell nuclear antigen (PCNA) protein. However, whether other pathways are involved in the RAD6-mediated regulation of DNA damage repair is still unclear. To deeply understand the molecular mechanisms of RAD6 in DNA damage repair, we performed a proteomic analysis and identified the changes of the protein-protein interaction (PPI) networks of RAD6 before and after X-ray irradiation. Furthermore, our study indicated that a proteasome-related event is likely involved in the DNA damage repair process. Moreover, we found that RAD6 promotes proteasome activity and nuclear translocation by enhancing the degradation of PSMF1 and the lamin B receptor (LBR). Therefore, we provide a novel pathway that is employed by RAD6 in response to DNA damage.

Keywords: DNA damage; LBR; PSMD3; PSMF1; RAD6; interactome analysis; proteasome.

Figures

FIG 1
FIG 1
Proteomic analysis of RAD6 interaction networks in the DNA damage response. (A) Schematic of the experimental procedures used to identify RAD6-interacting partners before and after DNA damage. HEK293T cells transfected with the GFP control or GFP-tagged RAD6A were treated with X-ray irradiation (X-ray 2.5 h) or not treated with X-ray irradiation [X-ray (−)], as indicated, at a dosage of 80 kV for 5 min, and the cells were recovered after 2.5 h. Cell extracts were prepared and subjected to co-IP assays with anti-GFP antibodies. The precipitated proteins were then subjected to mass spectrometry analysis. (B) After depletion of the nonspecific binding background by comparison with the GFP control, a number of RAD6A-interacting proteins were identified in cells without or with X-ray irradiation. The data are summarized in the Venn diagram. (C) The well-established RAD6-interacting proteins that were identified in our proteomics analysis are listed. The score refers to the obtained value analyzed by Mascot software on the basis of the original mass spectrum data. The following references mentioned in the figure appear at the indicated reference numbers in the References section: Wood et al., 2003, reference ; Kim et al., 2009a, reference ; Kim et al., 2009b, reference ; Kim and Roeder, 2011, reference ; Lyakhovich and Shekhar, 2003, reference ; Chen et al., 2012, reference ; and Read et al., 2014, reference . (D) All RAD6A-interacting proteins that appeared specifically under the control or X-ray irradiation conditions are listed. (E) The results of cellular component analysis of the RAD6A-interacting proteins that appeared under both control and X-ray irradiation conditions are shown. (F) The results of GO and pathway analyses of the RAD6A-interacting partners that appeared under both control and X-ray irradiation conditions are shown. (G) Some of the interacting proteins were selected and subjected to experimental validation. HEK293T cells were transfected with a Myc-tagged RAD6A construct combined with different randomly selected HA-tagged RAD6-interacting partners identified in our immunoprecipitation-mass spectrometry analysis for 48 h. Cells were then lysed and subjected to co-IP assays with anti-Myc antibodies. The normal mouse IgG (NIgG) antibodies were used as a negative control. The precipitated proteins were then subjected to Western blot analysis with the indicated anti-HA antibodies. CoA, coenzyme A; IP, immunoprecipitation; IB, immunoblotting.
FIG 2
FIG 2
Proteasome-related events potentially participate in the RAD6-mediated DNA damage response. (A) Network analysis of the RAD6-interacting proteins before X-ray irradiation. The numbers near the connecting lines between proteins refer to the number of times that the two specific proteins appeared in one pathway, which was analyzed by use of the indicated database. (B) KEGG pathway analysis of the RAD6-interacting proteins before X-ray irradiation. Drug metabolism-cytochrome P4 …, drug metabolism-cytochrome P450; Metabolism of xenobiotics by cy …, metabolism of xenobiotics by cytochrome P450; Leukocyte transendothelial migr, leukocyte transendothelial migration; Complement and coagulation casc, complement and coagulation cascade; Nicotinate and nicotinamide met, nicotinate and nicotinamide metabolism. (C) Network analysis of the RAD6-interacting proteins after X-ray irradiation. The numbers near the connecting lines between proteins refer to the number of times that the two specific proteins appeared in one pathway, which was analyzed by use of the indicated database. (D) KEGG pathway analysis of the RAD6-interacting proteins after X-ray irradiation. Phosphatidylinositol signaling …, phosphatidylinositol signaling system; Valine, leucine and isoleucine …, valine, leucine, and isoleucine degradation; Benzoate degradation via CoA li …, benzoate degradation via coenzyme A ligase; Synthesis and degradation of ke …, synthesis and degradation of ketone bodies; Amyotrophic lateral sclerosis …, amyotrophic lateral sclerosis (ALS). (E) STRING protein network analysis of the RAD6-interacting proteins before X-ray irradiation. (F) STRING protein network analysis of the RAD6-interacting proteins after X-ray irradiation.
FIG 2
FIG 2
Proteasome-related events potentially participate in the RAD6-mediated DNA damage response. (A) Network analysis of the RAD6-interacting proteins before X-ray irradiation. The numbers near the connecting lines between proteins refer to the number of times that the two specific proteins appeared in one pathway, which was analyzed by use of the indicated database. (B) KEGG pathway analysis of the RAD6-interacting proteins before X-ray irradiation. Drug metabolism-cytochrome P4 …, drug metabolism-cytochrome P450; Metabolism of xenobiotics by cy …, metabolism of xenobiotics by cytochrome P450; Leukocyte transendothelial migr, leukocyte transendothelial migration; Complement and coagulation casc, complement and coagulation cascade; Nicotinate and nicotinamide met, nicotinate and nicotinamide metabolism. (C) Network analysis of the RAD6-interacting proteins after X-ray irradiation. The numbers near the connecting lines between proteins refer to the number of times that the two specific proteins appeared in one pathway, which was analyzed by use of the indicated database. (D) KEGG pathway analysis of the RAD6-interacting proteins after X-ray irradiation. Phosphatidylinositol signaling …, phosphatidylinositol signaling system; Valine, leucine and isoleucine …, valine, leucine, and isoleucine degradation; Benzoate degradation via CoA li …, benzoate degradation via coenzyme A ligase; Synthesis and degradation of ke …, synthesis and degradation of ketone bodies; Amyotrophic lateral sclerosis …, amyotrophic lateral sclerosis (ALS). (E) STRING protein network analysis of the RAD6-interacting proteins before X-ray irradiation. (F) STRING protein network analysis of the RAD6-interacting proteins after X-ray irradiation.
FIG 3
FIG 3
Proteasome activities are essential for RAD6-mediated HR and NHEJ repair. (A) Diagrams of the HR and NHEJ reporter systems. Detailed descriptions are provided in Materials and Methods. Pcmv, cytomegalovirus promoter; SV40, simian virus 40. G and FP indicate the separated parts of the GFP gene. (B) Elevated levels of HR and NHEJ repair regulated by RAD6 depend on the presence of proteasome activities. Cells carrying the HR or NHEJ reporters were transfected with or without Myc-tagged RAD6A, and cells were treated with 25 μM MG132 for another 8 h or not treated, as indicated. The HR and NHEJ repair efficiencies were calculated using the specific reporters as previously described (12). The error bars indicate the standard deviations from three biological replicates. (C) RAD6 overexpression promotes DNA damage repair, while this accelerating effect was abolished by the inhibition of proteasome activity. HEK293T cells transfected with an empty vector expressing GFP as a control or GFP-tagged RAD6A were treated with 25 μM MG132 for 8 h or not treated, as indicated. Cells were then subjected to X-ray irradiation at a dosage of 80 kV for 5 min and recovered at the indicated times. Lastly, cells were harvested and lysed for Western blot analyses with the indicated antibodies. (D) RAD6 promotes the upregulation of proteasome activities during DNA damage repair, while knockdown of RAD6 expression abolishes the proteasome activity increase during DNA damage repair. Control HEK293T cells and cells transfected with Myc-tagged RAD6A or RAD6A/B-specific siRNAs were subjected to X-ray irradiation as described above. Cells were harvested at specific recovery times, and total cell extracts were prepared. The proteasome activities were examined using the Amplit fluorimetric proteasome 20S activity assay kit (AAT Bioquest, CA; catalog number 13456) according to the manufacturer’s protocol. The error bars indicate the standard deviations from three biological replicates. **, P < 0.05. (E) X-ray irradiation induces an increase in nuclear proteasome activities, depending on the presence of RAD6. HEK293T cells were treated with X-ray irradiation as described above. Nuclear (Nu) and cytoplasmic (Cyto) fractions were prepared using a nuclear and cytoplasmic protein extraction kit (product number P0028; Beyotime, Jiangsu Province, People's Republic of China) according to the manufacturer's instruction. Then, the proteasome activities of each fraction were examined. The error bars indicate the standard deviations from three biological replicates. Cont, control.
FIG 4
FIG 4
X-ray irradiation promotes the degradation of PSMF1 through the ubiquitin-proteasome pathway regulated by RAD6. (A) PSMF1 and PSMD3 are potential RAD6-interacting partners according to the data from our mass spectrometry analysis in Fig. 1. Score refers to the obtained value analyzed by Mascot software on the basis of the original mass spectrum data. (B) DNA damage stimulates the interactions of RAD6A with PSMF1 or PSMD3 and results in a decrease in PSMF1 protein levels. (Top) HEK293T cells were transfected with an empty vector expressing GFP, Myc-tagged RAD6A, and HA-tagged PSMF1 and PSMD3, as indicated, and cells were subjected to X-ray irradiation (X-ray 2.5 h) or not irradiated (as a control), as indicated. Cell extracts were prepared and subjected to co-IP assays with anti-Myc antibodies, followed by Western blot analyses with the indicated antibodies. The empty vector expressing GFP was used as an external control to indicate that the observed changes were not due to the different transfection efficiencies. (Bottom) For endogenous co-IP assays, HEK293T cells were harvested and subjected to co-IP assays with anti-RAD6 antibodies, followed by Western blot analyses with the indicated antibodies. (C) The X-ray-induced decrease in PSMF1 depends on proteasome activity, suggesting that the observed PSMF1 downregulation occurs through the proteasome-mediated protein degradation pathway. HEK293T cells treated with 25 μM MG132 for 8 h or not treated were or were not subjected to X-ray irradiation, as indicated. Cell extracts were prepared and subjected to Western blot assays with the indicated antibodies (top), and the bands were semiquantified (bottom). (D) X-ray irradiation promotes the ubiquitination of PSMF1. HEK293T cells transfected with HA-tagged PSMF1 were subjected to X-ray irradiation or not irradiated, as indicated. HA-PSMF1 was precipitated with anti-HA antibodies under denaturing conditions, and the precipitates were subjected to Western blot analyses with the indicated antibodies. (E) X-ray irradiation-induced PSMF1 degradation depends on the presence of RAD6. HEK293T cells transfected with a control siRNA or RAD6A/B-specific siRNAs were treated with X-ray irradiation or not irradiated as previously described. Cell extracts were prepared and subjected to Western blot analyses with the indicated antibodies (top), and the bands were semiquantified (bottom). (F) RAD6 regulates PSMF1 ubiquitination under X-ray irradiation conditions. HEK293T cells expressing HA-tagged PSMF1 were transfected with a control siRNA (−) or RAD6A/B-specific siRNAs (+) for 48 h, and cells were treated with MG132 for 8 h (+) or not treated (−), as indicated. Cells were then subjected to X-ray irradiation for 5 min and recovered after another 2.5 h. Immunoprecipitation assays were performed under denaturing conditions with anti-HA antibodies followed by Western blotting analyses with the indicated antibodies (left). HEK293T cells were transfected with HA-tagged PSMF1 with or without a Myc-tagged RAD6A plasmid for 48 h, and cells were treated with MG132 for 8 h or not treated, as indicated. Cells were then subjected to X-ray irradiation for 5 min and recovered after another 2.5 h. Immunoprecipitation assays were performed under denaturing conditions with anti-HA antibodies, followed by Western blot analyses with the indicated antibodies (right). (ub)n, ubiquitination.
FIG 5
FIG 5
LBR regulates the nuclear localization of proteasomes, and the degradation of LBR is affected by RAD6. (A) LBR is a potential RAD6-interacting protein, as indicated in our mass spectrometry analysis in Fig. 1. The score refers to the obtained value analyzed by Mascot software on the basis of the original mass spectrum data. (B) Knockdown of LBR results in the nuclear translocation of proteasomes, as indicated by PSMD3 immunostaining. HEK293T cells were transfected with a GFP-tagged PSMD3 plasmid and a DsRed2-tagged LBR together with a control siRNA (siCont) or an LBR-specific siRNA (siLBR), as indicated, for 48 h. Cells were irradiated with X rays for 2.5 h or not irradiated. Cells were then subjected to immunofluorescence assays by confocal laser microscopy. DAPI (4′,6-diamidino-2-phenylindole) staining was used to indicate the cell nucleus. Bar, 10 μm. (C) RAD6 overexpression promotes the nuclear translocation of proteasomes. HEK293T cells stably expressing a Myc-tagged RAD6A plasmid were transfected with GFP-tagged PSMD3 to indicate the proteasomes and DsRed2-tagged LBR for 48 h. Cells were irradiated with X rays for 2.5 h or not irradiated. (Top) The cell nucleus is indicated by DAPI staining. Cells were then subjected to immunofluorescence assays by confocal laser microscopy. Bar, 10 μm. (Bottom) The results of validation of Myc-RAD6A overexpression by Western blotting (WB). (D) RAD6 interacts with LBR, and X-ray irradiation downregulates LBR protein levels. Myc-tagged RAD6A and HA-tagged LBR were cotransfected into HEK293T cells for 48 h. Cells were then subjected to X-ray irradiation (X-ray 2.5 h) or not irradiated (control) as indicated. Cell extracts were prepared and subjected to co-IP analyses with anti-Myc antibodies, and Western blot assays were performed with the indicated antibodies. Reverse transcription-PCR (RT-PCR) assays were also performed using cells treated similarly. The detected genes are indicated (left). gapdh, glyceraldehyde-3-phosphate dehydrogenase gene. For endogenous co-IP assays, HEK293T cells were harvested and subjected to co-IP assays with anti-RAD6 antibodies, followed by Western blot analyses with the indicated antibodies (right). (E) RAD6 controls LBR protein levels in a proteasome-dependent manner. HEK293T cells transfected with or without (control) a Myc-tagged RAD6A plasmid were treated with MG132 for 8 h or not treated, as indicated. (Top) Cell extracts were prepared and subjected to Western blot analyses with the indicated antibodies. (Bottom) Cells transfected with a control siRNA or RAD6A/B-specific siRNAs (siRAD6A/B) were lysed and subjected to Western blot assays with the indicated antibodies. (F) Immunostaining assays support the conclusion that RAD6 regulates LBR protein levels. HEK293T cells were transfected with an equal amount of DsRed2-tagged LBR together with or without (control) a GFP-tagged RAD6A plasmid for 48 h. Cells were then irradiated with X rays for 2.5 h or not. Cells were subjected to immunofluorescence assays by confocal laser microscopy. DAPI staining was used to indicate the cell nucleus. Bar, 10 μm. (G) RAD6 regulates the degradation of LBR. (Top) Cells transfected with an empty vector expressing Myc or Myc-tagged RAD6A were treated with 50 μg/ml cycloheximide (CHX) for the indicated times. (Left) Cells were then harvested and subjected to Western blot analyses with the indicated antibodies. (Right) The bands were quantified, and the bars indicate the standard deviations from three biological replicates. (Bottom) Cells transfected with a control siRNA or RAD6A/B-specific siRNAs were treated with 50 μg/ml CHX for the indicated times. (Left) Cells were then harvested and subjected to Western blot analyses with the indicated antibodies. (Right) The bands were quantified, and the bars indicate the standard deviations from three biological replicates. (H) RAD6 regulates the ubiquitination of LBR. (Left) HEK293T cells expressing HA-tagged LBR were transfected with a control siRNA (−) or RAD6A/B-specific siRNAs (+) for 48 h, and cells were treated with MG132 for 8 h or not treated, as indicated. Immunoprecipitation assays were performed under denaturing conditions with anti-HA antibodies, followed by Western blot analyses with the indicated antibodies. (Right) HEK293T cells were transfected with the HA-tagged LBR plasmid with or without a Myc-tagged RAD6A plasmid for 48 h, and cells were treated with MG132 for 8 h or not treated, as indicated. Immunoprecipitation assays were performed under denaturing conditions with anti-HA antibodies, followed by Western blot analyses with the indicated antibodies.
FIG 6
FIG 6
DNA damage-induced downregulation of LBR protein levels occurs through the proteasome-mediated pathway and is regulated by RAD6 and PSMD3. (A) The X-ray-induced decrease in LBR depends on proteasome activity, suggesting that the observed LBR downregulation occurs through the proteasome-mediated protein degradation pathway. HEK293T cells treated with 25 μM MG132 for 8 h or not treated were subjected to X-ray irradiation or not irradiated, as indicated, and cells were recovered after another 2.5 h. Cell extracts were prepared and subjected to Western blot assays with the indicated antibodies. (B) X-ray irradiation does not alter the ubiquitination of LBR. HEK293T cells expressing HA-tagged LBR were treated with (+) MG132 for 8 h or not treated (−). Cells were subjected to X-ray irradiation or not irradiated as described above and recovered after another 2.5 h. Cells were harvested and subjected to immunoprecipitation assays under denaturing conditions with anti-HA antibodies. The precipitates were then used for Western blot analyses with the indicated antibodies. (C) The X-ray-induced degradation of LBR depends on the presence of RAD6 and PSMD3. HEK293T cells transfected with a control siRNA, RAD6A/B-specific siRNAs, or a PSMD3-specific siRNA (siPSMD3) were treated with X-ray irradiation or not irradiated, and cells were recovered after 2.5 h. Cell extracts were prepared and subjected to Western blot analyses with the indicated antibodies. (D) PSMD3 interacts with LBR, and their interaction is enhanced by X-ray irradiation. (Top two panels) HEK293T cells were cotransfected with a DsRed2-tagged PSMD3 plasmid and a GFP-tagged LBR plasmid for 48 h. Cell extracts were then prepared and subjected to co-IP assays with anti-GFP antibodies. Western blot analyses were performed with the indicated antibodies. (Bottom) HEK293T cells expressing GFP-LBR and DsRed2-PSMD3 were treated with MG132 for 8 h, and cells were then subjected to X-ray irradiation or not irradiated (control). The cells were recovered after another 2.5 h. Co-IP assays were employed with anti-GFP antibodies, and Western blot analyses were performed with the indicated antibodies. (E) RAD6 is essential for the interaction of PSMD3 and LBR under X-ray irradiation conditions. HEK293T cells expressing GFP-LBR and DsRed2-PSMD3 were transfected with a control siRNA (−) or RAD6A/B-specific siRNAs (+) for 48 h. Cells were then treated with MG132 for 8 h and were subjected to X-ray irradiation (+) or not irradiated (−). Cells were recovered after another 2.5 h. Cell extracts were prepared and subjected to co-IP assays with anti-GFP antibodies, followed by Western blot analyses with the indicated antibodies. IgGH, IgG heavy chain.
FIG 7
FIG 7
Working model. Under normal conditions, proteasomes are primarily localized in the cytoplasm and proteasome activities are inhibited by the inhibitory protein PSMF1. However, when cells encounter DNA damage stress, such as X-ray irradiation, the proteasome activity is enhanced and more proteasomes enter the nucleus. The proteasome activity is regulated by RAD6 through controlling the ubiquitination and degradation of PSMF1, while the nuclear translocation of proteasomes is also regulated by RAD6 via the control of LBR ubiquitination and degradation. When cells are exposed to X-ray irradiation, the degradation of PSMF1 and LBR by RAD6 is enhanced, further increasing proteasome activity and nuclear localization.

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