Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage
The Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of Ku70 and document...
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MDPI AG
2023-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/8/7041 |
| _version_ | 1797605160061501440 |
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| author | Sanna Abbasi Laila Bayat Caroline Schild-Poulter |
| author_facet | Sanna Abbasi Laila Bayat Caroline Schild-Poulter |
| author_sort | Sanna Abbasi |
| collection | DOAJ |
| description | The Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of Ku70 and documented an altered DNA damage response in cells expressing a Ku70 S155D phosphomimetic mutant. Here, we conducted proximity-dependent biotin identification (BioID2) screening using wild-type Ku70, Ku70 S155D mutant, and Ku70 with a phosphoablative substitution (S155A) to identify Ku70 S155D-specific candidate proteins that may rely on this phosphorylation event. Using the BioID2 screen with multiple filtering approaches, we compared the protein interactor candidate lists for Ku70 S155D and S155A. TRIP12 was exclusive to the Ku70 S155D list, considered a high confidence interactor based on SAINTexpress analysis, and appeared in all three biological replicates of the Ku70 S155D-BioID2 mass spectrometry results. Using proximity ligation assays (PLA), we demonstrated a significantly increased association between Ku70 S155D-HA and TRIP12 compared to wild-type Ku70-HA cells. In addition, we were able to demonstrate a robust PLA signal between endogenous Ku70 and TRIP12 in the presence of double-stranded DNA breaks. Finally, co-immunoprecipitation analyses showed an enhanced interaction between TRIP12 and Ku70 upon treatment with ionizing radiation, suggesting a direct or indirect association in response to DNA damage. Altogether, these results suggest an association between Ku70 phospho-S155 and TRIP12. |
| first_indexed | 2024-03-11T04:57:11Z |
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| id | doaj.art-7334146d3fdc43e4a2fb1a6802a24cc4 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T04:57:11Z |
| publishDate | 2023-04-01 |
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| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-7334146d3fdc43e4a2fb1a6802a24cc42023-11-17T19:34:18ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-04-01248704110.3390/ijms24087041Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA DamageSanna Abbasi0Laila Bayat1Caroline Schild-Poulter2Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 3K7, CanadaDepartment of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 3K7, CanadaDepartment of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON N6A 3K7, CanadaThe Ku heterodimer, composed of subunits Ku70 and Ku80, is known for its essential role in repairing double-stranded DNA breaks via non-homologous end joining (NHEJ). We previously identified Ku70 S155 as a novel phosphorylation site within the von Willebrand A-like (vWA) domain of Ku70 and documented an altered DNA damage response in cells expressing a Ku70 S155D phosphomimetic mutant. Here, we conducted proximity-dependent biotin identification (BioID2) screening using wild-type Ku70, Ku70 S155D mutant, and Ku70 with a phosphoablative substitution (S155A) to identify Ku70 S155D-specific candidate proteins that may rely on this phosphorylation event. Using the BioID2 screen with multiple filtering approaches, we compared the protein interactor candidate lists for Ku70 S155D and S155A. TRIP12 was exclusive to the Ku70 S155D list, considered a high confidence interactor based on SAINTexpress analysis, and appeared in all three biological replicates of the Ku70 S155D-BioID2 mass spectrometry results. Using proximity ligation assays (PLA), we demonstrated a significantly increased association between Ku70 S155D-HA and TRIP12 compared to wild-type Ku70-HA cells. In addition, we were able to demonstrate a robust PLA signal between endogenous Ku70 and TRIP12 in the presence of double-stranded DNA breaks. Finally, co-immunoprecipitation analyses showed an enhanced interaction between TRIP12 and Ku70 upon treatment with ionizing radiation, suggesting a direct or indirect association in response to DNA damage. Altogether, these results suggest an association between Ku70 phospho-S155 and TRIP12.https://www.mdpi.com/1422-0067/24/8/7041Ku70TRIP12proximity-dependent biotin identification (BioID2)phosphorylationdouble-stranded breaksproximity ligation assay |
| spellingShingle | Sanna Abbasi Laila Bayat Caroline Schild-Poulter Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage International Journal of Molecular Sciences Ku70 TRIP12 proximity-dependent biotin identification (BioID2) phosphorylation double-stranded breaks proximity ligation assay |
| title | Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage |
| title_full | Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage |
| title_fullStr | Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage |
| title_full_unstemmed | Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage |
| title_short | Analysis of Ku70 S155 Phospho-Specific BioID2 Interactome Identifies Ku Association with TRIP12 in Response to DNA Damage |
| title_sort | analysis of ku70 s155 phospho specific bioid2 interactome identifies ku association with trip12 in response to dna damage |
| topic | Ku70 TRIP12 proximity-dependent biotin identification (BioID2) phosphorylation double-stranded breaks proximity ligation assay |
| url | https://www.mdpi.com/1422-0067/24/8/7041 |
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