Biochemical and Biophysical Research Communications
Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance
Introduction
DNA double-stranded breaks (DSBs) are deleterious DNA lesions that are primarily repaired by two pathways; non-homologous end-joining (NHEJ) and homologous recombination (HR) [1]. NHEJ is the prominent pathway responsible for repairing DSBs in human cells [2]. A central player in NHEJ is the DNA-dependent protein kinase (DNA-PK) (see reviews for more details) [3], [4]. DNA-PK consists of a DNA binding subunit (Ku70/80, Ku) and a catalytic subunit (DNA-PKcs). DNA-PKcs is composed of HEAT (Huntington-elongation-A-subunit-TOR) repeats in its N-terminus, which produce a pincer-shaped structure that forms a central channel and a C-terminal region that contains the PI3 kinase domain, which is flanked by the FAT (FRAP, ATM, TRRAP) domain at its N-terminal side and by the FATC domain at its C-terminal side [5], [6]. Following DSB induction, the Ku heterodimer quickly binds to the DSB ends and recruits DNA-PKcs to the break site, which is mediated by the N-terminal region of DNA-PKcs [7], [8], [9]. Upon interacting with the DSB-Ku complex, DNA-PKcs is activated. It is believed that activation of DNA-PKcs is dependent on a conformational change in both the FAT and FATC domains [4]. Once activated, DNA-PKcs phosphorylates a number of substrates with the best characterized being itself.
Following DSB formation, DNA-PKcs is heavily phosphorylated and these phosphorylations are critical for its role in DSB repair [4], [10]. DNA-PKcs autophosphorylates itself and is also phosphorylated by the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) protein kinases [11], [12]. A large number of the phosphorylation sites are clustered in different regions of DNA-PKcs [13], [14], [15], [16]. Two prominent clusters phosphorylated in response to DSB induction are the T2609 [13], [17] and S2056 [11], [18], [19] phosphorylation clusters. S2056 is an autophosphorylation site [18], whereas phosphorylation of the T2609 cluster can be mediated by DNA-PKcs, ATM, or ATR. Blocking phosphorylation of DNA-PKcs at the T2609 phosphorylation cluster by mutating the serines/threonines to alanine results in blocking the release of DNA-PKcs from DNA ends, reduced DSB repair capacity, and increased radiosensitivity [17], [20], [21]. Blocking phosphorylation of the S2056 cluster causes increased radiosensitivity and results in increased DNA end processing, suggesting that it is required for NHEJ [19], [22]. Although, regulation of DNA-PKcs through its phosphorylation has been extensively investigated, other posttranslational modifications of DNA-PKcs are not well studied.
Acetylation of lysine residues is a reversible posttranslational modification, which neutralizes the positive charge of this amino acid and changes the functionality of the protein in diverse ways [23]. There are many individual reports of protein acetylation modulating diverse biological processes, suggesting that lysine acetylation has broad regulatory functions [24]. For example, it plays a key role in the regulation of gene expression through the modification of core histone tails by histone acetyltransferases (HATs) or histone deacetylases (HDACs) [25]. Lysine acetylation has also been implicated to play a role in modulating the activity of DSB repair proteins, including Ku70 [26], ATM [27], and CtIP [28]. A recent proteomic study revealed that there are at least 16 lysine residues acetylated in DNA-PKcs [29], but the biological function of DNA-PKcs acetylation is not characterized. Here, we show that DNA-PKcs is acetylated, and we identified two lysine residues (K3241 and K3260) that are potentially acetylated on DNA-PKcs. Mutating these lysine residues to block acetylation results in increased radiosensitivity and chromosomal aberrations, suggesting that these sites are important for DNA-PKcs-dependent DSB repair. Collectively, the data provide initial evidence that acetylation may modulate DNA-PKcs.
Section snippets
Cell culture and transfections
Chinese hamster ovary (CHO) DNA-PKcs-deficient (V3) cell line [30] and V3 cells stably expressing YFP-tagged DNA-PKcs were cultured in Hyclone MEM media containing 10% Fetal Bovine Serum and Newborn Calf Serum (1:1 mixture), 100 U/mL penicillin and 100 U/mL streptomycin. The cells were incubated at 37 °C in a humidified incubator with 5% CO2. For generation of stable cell lines, cells were transfected with the linearized expression plasmid using Lipofectamine® 2000 transfection reagent
Mutating 8 potential acetylation sites of DNA-PKcs results in increased radiosensitivity
A proteomic study identified 16 potential lysine residues acetylated in DNA-PKcs; therefore, we postulated that acetylation modulates DNA-PKcs activity [29]. To test this hypothesis, we first examined whether DNA-PKcs is acetylated in vivo. Immunoprecipitated DNA-PKcs from HeLa nuclear extract was probed with anti-pan-acetyl-lysine antibodies, and we found that DNA-PKcs is acetylated in normal cycling cells (Fig. 1A). Next, we aimed to identify acetylation sites that may modulate the activity
Discussion
For the past decade, extensive work has been performed to identify the regulatory mechanisms governing the activity of DNA-PKcs. A number of groups have revealed that phosphorylation of DNA-PKcs is a key post-translational modification that modulates DNA-PKcs [4], [10]. In this study, we aimed to provide evidence that acetylation regulates DNA-PKcs. We showed that DNA-PKcs is acetylated in vivo in normal cycling cells. Furthermore, blocking potential acetylation at two sites previously
Acknowledgements
The work was supported by the National Institute of Health [CA50519 and CA13499 to DJC] and the Cancer Prevention Research Institute of Texas [RP110465 to DJC].
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