@article{LAFONT2020129705,

title = {DNA-PKcs Ser2056 auto-phosphorylation is affected by an O-GlcNAcylation/phosphorylation interplay},

author = {Florian Lafont and Fabrice Fleury and Houda Benhelli-Mokrani},

url = {http://www.sciencedirect.com/science/article/pii/S0304416520302178},

doi = {https://doi.org/10.1016/j.bbagen.2020.129705},

issn = {0304-4165},

year  = {2020},

date = {2020-01-01},

journal = {Biochimica et Biophysica Acta (BBA) - General Subjects},

volume = {1864},

number = {12},

pages = {129705},

abstract = {Background DNA dependent Protein Kinase (DNA-PK) is an heterotrimeric complex regulating the Non Homologous End Joining (NHEJ) double strand break (DSB) repair pathway. The activity of its catalytic subunit (DNA-PKcs) is regulated by multiple phosphorylations, like the Ser2056 one that impacts DSB end processing and telomeres integrity. O-GlcNAcylation is a post translational modification (PTM) closely related to phosphorylation and its implication in the modulation of DNA-PKcs activity during the DNA Damage Response (DDR) is unknown. Methods Using IP techniques, and HeLa cell line, we evaluated the effect of pharmacological or siOGT mediated O-GlcNAc level modulation on DNA-PKcs O-GlcNAcylation. We used the RPA32 phosphorylation as a DNA-PKcs activity reporter substrate to evaluate the effect of O-GlcNAc modulators. Results We show here that human DNA-PKcs is an O-GlcNAc modified protein and that this new PTM is responsive to the cell O-GlcNAcylation level modulation. Our findings reveal that DNA-PKcs hypo O-GlcNAcylation affects its kinase activity and that the bleomycin-induced Ser2056 phosphorylation, is modulated by DNA-PKcs O-GlcNAcylation. Conclusions DNA-PKcs Ser2056 phosphorylation is antagonistically linked to DNA-PKcs O-GlcNAcylation level modulation. General significance Given the essential role of DNA-PKcs Ser2056 phosphorylation in the DDR, this study brings data about the role of cell O-GlcNAc level on genome integrity maintenance.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lafont2018,

title = {Assessment of DNA-PKcs kinase activity by quantum dot–based microarray},

author = {Florian Lafont and Nizar Ayadi and Cathy Charlier and Pierre Weigel and Igor Nabiev and Houda Benhelli-Mokrani and Fabrice Fleury},

doi = {10.1038/s41598-018-29256-2},

issn = {20452322},

year  = {2018},

date = {2018-01-01},

journal = {Scientific Reports},

volume = {8},

number = {1},

pages = {1--12},

abstract = {Therapeutic efficacy against cancer is often based on a variety of DNA lesions, including DNA double-strand breaks (DSBs) which are repaired by homologous recombination and non-homologous end joining (NHEJ) pathways. In the past decade, the functions of the DNA repair proteins have been described as a potential mechanism of resistance in tumor cells. Therefore, the DNA repair proteins have become targets to improve the efficacy of anticancer therapy. Given the central role of DNA-PKcs in NHEJ, the therapeutic efficacy of targeting DNA-PKcs is frequently described as a strategy to prevent repair of treatment-induced DNA damage in cancer cells. The screening of a new inhibitor acting as a sensitizer requires the development of a high-throughput tool in order to identify and assess the most effective molecule. Here, we describe the elaboration of an antibody microarray dedicated to the NHEJ pathway that we used to evaluate the DNA-PKcs kinase activity in response to DNA damage. By combining a protein microarray with Quantum-Dot detection, we show that it is possible to follow the modification of phosphoproteomic cellular profiles induced by inhibitors during the response to DNA damage. Finally, we discuss the promising tool for screening kinase inhibitors and targeting DSB repair to improve cancer treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Silva2016,

title = {Rapid diminution in the level and activity of DNA-dependent protein kinase in cancer cells by a reactive nitro-benzoxadiazole compound},

author = {Viviane A O Silva and Florian Lafont and Houda Benhelli-Mokrani and Magali {Le Breton} and Philippe Hulin and Thomas Chabot and Fran{ç}ois Paris and Vehary Sakanyan and Fabrice Fleury},

doi = {10.3390/ijms17050703},

issn = {14220067},

year  = {2016},

date = {2016-05-01},

journal = {International Journal of Molecular Sciences},

volume = {17},

number = {5},

publisher = {MDPI AG},

abstract = {The expression and activity of DNA-dependent protein kinase (DNA-PK) is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR) is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-yl)sulfanyl]-2,1,3-benzoxadiazole (NSC), bearing a nitro-benzoxadiazole (NBD) scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK) rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}