Abstract A03: The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair
Notice bibliographique
Résumé
Abstract Introduction: EMSY, a putative DNA damage repair gene, is amplified in over 10% of high-grade serous ovarian carcinoma (HGSOC) cases. EMSY overexpression has been hypothesized to antagonize BRCA2 via direct interaction and compromise the homology-directed repair (HDR) of DNA double strand breaks. Its role as a transcription factor has been described in a protein kinase AKT1 phosphorylation-dependent manner. The purpose of this study was to decipher EMSY's role in HDR and to assess the importance of its phosphorylation in this context. Experimental procedures: We measured HDR activity in several cell lines (U2OS osteosarcoma, H1299 non-small cell lung carcinoma and OVCAR8 HGSOC) using the DR-GFP reporter assay and RAD51 foci assessment. Endogenous immunoprecipitations (IPs) were performed with low stringency lysis buffer and protein A/G-plus agarose. V5-tagged EMSY constructs were made using the Invitrogen's Gateway TOPO cloning system. These constructs were further used to create EMSY phospho-mutants. Cells were transfected by either electroporation or FuGene reagent. For the in vitro kinase assays, EMSY constructs were sub-cloned and expressed in BL21 STAR bacteria and purified using Invitrogen's Champion pET102 Expression kit. Recombinant protein kinases were obtained from Active Motif and CellSignaling. Forskolin and H-89 were obtained from Santa Cruz Biotechnology. Summary of the data: EMSY overexpression resulted in decreased HDR activity in all three DR-GFP cell lines, thus supporting the hypothesis that EMSY overexpression impairs HDR. V5-tagged EMSY overexpressing and endogenous immunoprecipitation experiments demonstrated no interaction between EMSY and BRCA2, suggesting EMSY's role in HDR to be BRCA2-independent. We confirmed that EMSY is phosphorylated by AKT1 at serine 209 phospho-site and identified a previously unknown phospho-site at threonine 207. We identified protein kinase A (PKA) as a kinase targeting EMSY T207. Furthermore, by performing both DR-GFP assay and RAD51 foci assessment in OVCAR8 cells that overexpress WT EMSY or either phospho-mutant, we demonstrated that mutant EMSY-S209A affects HDR activity similar to the WT EMSY while EMSY-T207A does not. This suggests the importance of PKA and the T207 phospho site for the EMSY-driven HDR suppression. Conclusions: EMSY-overexpressing cells show decreased HDR activity, demonstrating EMSY's relevance to the HDR pathway. Our data support the notion that EMSY-driven HDR impairment is BRCA2-interaction-independent and challenges the currently held impression that EMSY overexpression mimics the BRCA2-depleted phenotype via direct interaction. We found a new phospho-site at EMSY T207 and identified PKA as a targeting kinase. Phosphorylation of EMSY at T207, but not S209 phospho-site is necessary for EMSY-driven suppression of HDR. We suggest that an increase in EMSY's T207 phosphorylation in patients bearing EMSY¬-amplified tumors could enhance BRCAness and render these patients more sensitive to drugs effective in HDR-impaired setting, such as PARP inhibitors. Citation Format: Petar Jelinic, Laura Eccles, Jill Tseng, Paulina Cybulska, Simon N. Powell, Douglas Levine. The EMSY threonine 207 phospho-site is required for EMSY-driven suppression of DNA damage repair [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A03.
Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.
Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,001 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,001 | 0,001 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».