DNA-PKcs Ser2056 auto-phosphorylation is affected by an O-GlcNAcylation/phosphorylation interplay
Graphical abstract
Introduction
DNA-PK is a heterotrimeric enzyme composed of the KU70/80 heterodimer and DNA-PKcs, a catalytic subunit of 460 kDa, member of the Phosphatidyl Inositol 3-Kinase related Kinase (PIKK) serine/threonine kinase family [1]. DNA-PKcs activity is enhanced by the recognition of Double Strand Breaks (DSB) and plays an essential role in their repair [2]. DNA-PKcs is a key player of the NHEJ repair pathway, and its absence or miss function causes IR sensitivity and inefficient V(D)J recombination associated with Severe Combined Immune Deficiency (SCID) [3]. The DNA-PKcs kinase has multiple phosphorylation sites organized in clusters, some of which are auto-phosphorylated after DNA damage [4,5]. The auto-phosphorylation of the 2609 residue, located in the ABCDE cluster, is essential for Artemis nuclease activity during the Non Homologous End Joining (NHEJ) repair pathway [6] and is also implicated in the dissociation of DNA-PKcs from the Ku heterodimer and from DNA [4,7,8]. The phosphorylation of Ser2056 (PSer2056), located in the PQR cluster, regulates the conformation and activity of DNA-PKcs, and has an essential role in the DNA damage response orientation [9,10]. This phosphorylation limits DSB end processing by Artemis and thus limits the NHEJ repair pathway [4]. The PSer2056 is also implicated in telomeres capping and plays an essential role in genome maintenance by preventing telomeres fusions [11]. DNA-PKcs phosphorylations are therefore fine tuners of its activity essential to the maintaining of genome integrity.
Ten years ago, Zachara's work, consisting in MS-coupled SILAC screening of the effect of heat stress on cellular protein O-GlcNAcylation, showed that DNA-PKcs is an O-GlcNAc modified protein in green monkey cells [12].
O-GlcNAcylation is a post translational modification (PTM) consisting in the O-linkage of the sugar group (β-N-acetylglucosamine) of the UDP-GlcNAc donor on serine and threonine residues [13]. Discovered in 1984 by Gerald Hart [14], O-GlcNAcylation is found in prokaryotic (bacteria, viruses) and eukaryotic cells (animal, plants) and is found on many proteins implicated in various cellular pathways [15,16]. The dynamic of this PTM is due to the O-GlcNAc transferase (OGT) and the O-GlcNAc-ase (OGA) ubiquitous enzymes which catalyse respectively the addition and removal of O-GlcNAc from cytosolic and nuclear proteins [17]. Protein O-GlcNAcylation affects their cellular localisation, interactions, half-life [18] and activity [19,20]. There is a crosstalk between O-GlcNAcylation and phosphorylation [21]. These two PTM can be antagonistic by competing for binding to the same residue, or by steric hindrance when two proximal residues are modified, as is the case with p53 [22]. In some cases, O-GlcNAcylation and phosphorylation can act synergistically depending on the residues and the proteins concerned [21,23]. Cellular level of O-GlcNAcylation is directly linked to the glucose intake and is involved in many stress signaling pathways, including heat stress, oxidative stress and DNA Damage Response (DDR) [12,24,25]. Whereas the implication of phosphorylation in the DDR signalisation is well studied, the implication of O-GlcNAcylation remains poorly understood. However, some recent studies reveal the implication of O-GlcNAcylation in the DDR. For example, it has been shown that the ATM kinase (a PIKK family member), which participates in DNA damage signalisation, is O-GlcNAc modified in mouse primary cultured neurons [24]. This work showed that an elevation of cellular O-GlcNAcylation level led to an increase of radiation-induced ATM phosphorylation on its Ser1981 residue. This means that ATM activity is positively regulated by an increase of O-GlcNAcylation. In contrast, this study showed that a decrease of O-GlcNAcylation has no effect on ATM activity [24].
Taking into account the close relationship between O-GlcNAcylation and phosphorylation, the aim of our study is to explore the effect of DNA-PKcs O-GlcNAcylation on its phosphorylation by focusing on the PSer2056 residue. We examined the effects of O-GlcNAcylation level modulation on DNA-PKcs Ser2056 phosphorylation after inducing DNA damage with the radio mimetic bleomycin treatment.
Section snippets
Materials
The bleomycin sulfate (Bleo) (ref:S1214). was purchased from Selleckchem The inhibitor of O-GlcNAcase (OGA), O-(2-AcetamidO-2-deoxy-D-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc) (ref:A7229), the inhibitor of glutamine fructose-6-aminotransferase, 6-diazO-5oxO-L-norleucine (DON) (ref:D2141) and the topoisomerase I inhibitor, (S)-(+)-Camptothecin (CPT) (ref:C9911) were purchased from Sigma-Aldrich. Antibodies used in this study were anti-O-GlcNAc (CTD110.6, Santa Cruz Biotechnology,
Human DNA-PKcs is an O-GlcNAc modified protein
To look for the presence of O-GlcNAc on human DNA-PKcs we made an immunoprecipitation from HeLa nuclear extracts. We show, in Fig. 1, a western blot analysis where DNA-PKcs O-GlcNAcylation is revealed by the CTD110.6 antibody that recognizes this PTM. This antibody was validated by the disappearance of the signal in the presence of 0,5 M competing free Glc-NAc (see supplementary fig. S1). Our results show that in control condition, DNA-PKcs is O-GlcNAc modified in human cells. Knowing the
Discussion
The DNA-PKcs kinase has a key regulatory role in double strand break DDR and its Ser2056 auto-phosphorylation regulates DNA end processing which influences the choice between an accurate (HR) or an inaccurate (NHEJ) repair pathways. We show here that human DNA-PKcs is also an O-GlcNAc modified protein, as shown by Zachara's work in 2011 in green monkey cells [12]. O-GlcNAcylation and phosphorylation are two closely linked PTM that can be antagonistic or synergic depending on the affected
Conclusion
With ATM and ATR, DNA-PKcs is one of the three major kinases implicated in the early DDR. We show here that the modulation of DNA-PKcs O-GlcNAcylation level affects its bleomycin-induced Ser2056 phosphorylation. Indeed, we observed an antagonistic relationship between these two PTM of DNA-PKcs during the bleomycin induced DDR. Considering the essential role of the DNA-PKcs Ser2056 phosphorylation in DSB repair pathways choice and its implication in telomeres capping, our results provide some
Funding
This work was partially supported by the Ligue contre le cancer 44.
Declaration of Competing Interest
None.
Acknowledgments
We thank Professor Charles Tellier for his impulsion to discover the O-GlcNAcylation world and for the helpful discussions. A special thank you to Professor Tony Lefebvre for his valuables advice. We thank the IMPACT platform for making available all the necessary equipment. We thank Dr. Milena Popova for her advice and for English proof reading of the manuscript.
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