DNA-PKcs Ser2056 auto-phosphorylation is affected by an O-GlcNAcylation/phosphorylation interplay

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 12; p. 129705
• Main Authors: Lafont, Florian, Fleury, Fabrice, Benhelli-Mokrani, Houda
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2020; Elsevier
• Subjects: Acetylglucosamine - metabolism; Acylation; DNA; DNA damage; DNA repair; DNA-Activated Protein Kinase - metabolism; DNA-PKcs; genome; HeLa Cells; human cell lines; Humans; Life Sciences; NHEJ; O-GlcNAcylation; Phosphorylation; post-translational modification; protein kinases; Protein Processing, Post-Translational; protein subunits; telomeres; DNA-PKcs; O-GlcNAcylation; Phosphorylation; HR; NHEJ; DNA repair
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129705

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. 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. 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. DNA-PKcs Ser2056 phosphorylation is antagonistically linked to DNA-PKcs O-GlcNAcylation level modulation. 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. [Display omitted] •Human DNA-PKcs is O-GlcNAc modified.•Lower DNA-PKcs O-GlcNAcylation decreases its kinase activity.•Higher DNA-PKcs O-GlcNAcylation has no effect on its kinase activity.•Pharmacological modulation of DNA-PKcs O-GlcNAcylation affects its Ser2056 Phosphorylation in an antagonistic manner.•DNA-PKcs PSer2056 regulation model: Antagonistic interplay between DNA-PKcs Ser2056 Phosphorylation and the O-GlcNAcylation of a proximal in silico predicted site.