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

Highlights

• 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.

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.

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.