DNA-PKcs post-translational modifications and associated diseases
Review Article
Zongpei Guo, Ping-Kun Zhou & Teng Ma
Genome Instability & Disease 3, 136–143 (2022)
Abstract
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) forms DNA-PK holoenzyme with Ku70/Ku80 heterodimer, which initiates the (non-homologous end-joining) NHEJ repair upon double-strand breaks (DSBs). Besides function in NHEJ, DNA-PKcs also plays multiple roles in other biological processes including transcription, telomere maintenance, autophagy, cell cycle checkpoint, and lymphocyte development. Dysregulation of DNA-PKcs associates with various diseases such as genome instability and cancer, immunological deficiency, and neurological disorders. DNA-PKcs function is strictly controlled by post-translational modifications, especially phosphorylation. DNA-PKcs phosphorylation affects either the end-processing or the end-joining in DSB repair or Variable (V) Diversity (D) and Joining (J)/Class switch recombination in lymphocytes development. With increasing evidence of proteomic advances in DNA-PKcs study, other PTMs have been added including ubiquitination, neddylation, acetylation, and PARylation. Different DNA-PKcs PTMs may be involved different pathophysiological activities. Moreover, complexed crosstalk between DNA-PKcs phosphorylation and other PTMs will further aid the understanding of DNA-PKcs biology.
Introduction
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a colossal polypeptide with 4128 amino acids encoded by PRKDC gene (Hartley et al., 1995). It was first discovered and characterized as part of SP1 transcription complexes about three decades ago (Jackson et al., 1990). As a member of the phosphoinositide 3-kinase-related protein kinases (PIKKs) family, DNA-PKcs shares similar domain organization and structural features with Ataxia telangiectasia mutated (ATM) and Ataxia telangiectasia and Rad3 related (ATR). All the three kinases contain a HEAT (Huntingtin, elongation, factor 3, a subunit of protein phosphatase 2A, TOR1) repeats domain at their N-terminal and a kinase domain located in their C-terminal surrounded by upstream FAT (FRAP-ATM-TRRAP) and downstream FATC (FAT C-terminal) (Blackford & Jackson, 2017; Bosotti et al., 2000).
A number of studies have illuminated the important roles of DNA-PKcs in diverse biological processes, including transcription, non-homologous end-joining (NHEJ) repair, VDJ recombination, and class switch recombination in B-cell development, metabolism, ribosomal RNA processing, and haematopoiesis (Bertocci et al., 2006; Goodwin & Knudsen, 2014; Goodwin et al., 2015; Park et al., 2017; Shao et al., 2020; Song et al., 2019).
In NHEJ repair, Ku70/Ku80 heterodimers rapidly recognize the broken DNA ends and recruit DNA-PKcs, forming the active Ser/Thr kinase DNA-PK holoenzyme. Structural studies of DNA-PKcs have shown that its N-terminal portion creates a pincer structure that forms a central channel for possible binding to dsDNA, and the C-terminal structural domain forms a coronal structure located at the top of the pincer structure. The binding of DNA-PKcs to the DNA-Ku complex leads to the inward displacement of the Ku heterodimer on the dsDNA strand and ultimately to the activation of DNA-PKcs kinase activity. The NHEJ complex undergoes structural transition either from inactive state to active state or from long-range synapsis to short-range synapsis to fulfill the end-processing and ligation. Trans-autophosphorylation on the ABCDE cluster (aa 2609-2647) is essential for the transition. Finally, the XRCC4-XLF-Ligase IV complex seals the DNA end (Chaplin et al., 2021a, 2021b; Chen, Lee, et al., 2021; Chen, Xu, et al., 2021; Liang et al., 2022).
With increasing advances in proteomic technology, more post-translational modifications of DNA-PKcs have been identified. Here, we will summarize the progresses in DNA-PKcs post-translational modifications (PTMs) studies and how these DNA-PKcs PTMs associate with diseases.
Phosphorylation of DNA-PKcs
Among all the DNA-PKcs PTMs, phosphorylation is most critical for DNA-PK activity and being extensively studies, especially in DSBs repair and VDJ recombination, and class switch recombination.
DNA-PKcs phosphorylation in DSBs’ repair
DSBs are the most lethal DNA lesions, which are canonically repaired through homologous recombination (HR) or NHEJ in eukaryotic cells (Ceccaldi et al., 2016). Different from HR, NHEJ can function throughout the cell cycle, because it does not need the homologous DNA as template. Therefore, in eukaryotic cells, most DSBs are repaired via the NHEJ pathway (Guo et al., 2020). Upon DSBs, the heterodimer of Ku70/80 senses and binds broken DNA ends rapidly to facilitate the recruitment and activation of DNA-PKcs. The DNA-dependent protein kinase (DNA-PK) complex can maintain the conformation of broken DNA ends and protect the DNA damage sites from resection by nucleases and subsequently promote broken DNA joining (Shao et al., 2012). DNA-PKcs, ATM, and ATR are activated by DSBs and initiate the DNA damage response signaling (Zhao et al., 2020).
DNA-PKcs phosphorylation is essential for NHEJ repair. Once activated, DNA-PKcs phosphorylates many substrates including Ku70/80 (Chan et al., 1999) and downstream NHEJ factors such as Artemis (Ma et al., 2005), the X-ray cross-complementing protein 4(XRCC4) (Leber et al., 1998), the XRCC4-like factor (XLF) (Yu et al., 2008), DNA ligase IV (Wang et al., 2004), and itself (Jiang et al., 2015a). However, except for DNA-PKcs itself, other six substrates’ phosphorylation is not required for efficient NHEJ repair (Neal & Meek, 2011).
DNA-PKcs can be auto- or trans-phosphorylated on more than 40 sites distributed in different phosphorylation clusters such as JK, PQR, and ABCDE after induction of DSBs (Davis et al., 2014; Dobbs et al., 2010). But which one is most important? Among them, the best-characterized clusters are PQR and ABCDE, which are surrounded by Ser2056 and flanked by Thr-2609, respectively (Fig. 1). Both Ser2056 and Thr-2609 can be auto-phosphorylated and are required for efficient DSBs repair (Cui et al., 2005). However, further studies demonstrated that Thr-2609 was preferentially trans-phosphorylated by ATM or ATR under DSBs or replication stress (Chan, 2002) (Chen et al., 2007) (Yajima et al., 2006). Mice expressing DNA-PKcs3A, in which Thr2605, Thr2634, and Thr2643 (human Thr-2609, Thr2638, and Thr2647) are replaced by three alanine residues, show congenital bone marrow failure and early postnatal lethality. In addition, cells derived from DNA-PKcs3A mice show hypersensitivity to DNA cross-linking agents (Zhang et al., 2011). DNA-PKcs phosphorylation at Thr-2609 is required for Artemis-mediated endonuclease activity and regulates Artemis access to DNA ends to promote subsequent homologous recombination pathway (Goodarzi et al., 2006; Jiang et al., 2015a). Phosphorylated Thr-2609 co-localizes with PKL1 through the mitotic phase. And the deficiency of DNA-PKcs can result in abnormal chromosome segregation and failed cytokinesis in response of DNA damage (Huang et al., 2014). Recently, Shao et al. identified that DNA-PKcs can bind U3 small nucleolar RNA in a Ku-dependent manner and then be activated at Thr-2609 cluster but not Ser2056 cluster to promote 18S rRNA processing and the global protein synthesis in haematopoietic cells. Additionally, blocking of Thr-2609 phosphorylation causes bone marrow failure in mice (Shao et al., 2020).
Fig. 1
A differential phosphorylation mode of DNA-PKcs in DSBs repair. Upper, the autophosphorylation of DNA-PKcs Ser2056 is required for end-ligation by the LIG4-XRCC4-XLF complex. Lower; ATM-mediated DNA-PKcs Thr2069 phosphorylation is required for Artemis recruitment for end processing
Full size imageAnother well-characterized phosphorylation cluster is Ser2056, which is known as a strict autophosphorylation site in response to DSBs and is essential for NHEJ repair (Davis et al., 2014). The mutation protein DNA-PKcs Ser2056A is released slower from DSB sites compared with the wild type and Thr2609A, but neither mutant show a behavior like 7A mutant (Uematsu et al., 2007). This suggests that DNA-PKcs phosphorylation at Ser2056 is also responsible for its disassociation from DSB ends. Mutagenesis studies revealed the opposing roles of phosphorylation at Ser2056 and Thr-2609. Phosphorylation at Ser2056 limits broken DNA ends resection to promote the NHEJ repair, whereas Thr-2609 facilitates the access of DNA ends to nucleases such as Artemis to promote HR when NHEJ is failed [25, 36, (Meek et al., 2007).
In addition, numbers of other phosphorylation sites have been identified in the N- and C-terminal regions of DNA-PKcs. Phosphorylation of DNA-PKcs at Ser56, Ser72 in N-terminus and Thr3950 in its kinase domain can result in abrogation of DNA-PKcs kinase activity but not complex assembly (Leber et al., 1998). Phosphorylation at Ser1004 and Thr946 does not impair the enzymatic activity of DNA-PKcs but it inhibits NHEJ repair efficiency (Neal et al., 2011). Another amino acid, Ser3205, can be phosphorylated in response to DNA damage in an ATM-dependent manner, but its phosphorylation does not affect NHEJ and only has a minimal effect on HR proficiency. Neither S3205A nor S3205D affects DNA-PKcs kinase activity and cell radioresistance (Neal et al., 2011). Although, how the phosphorylation status of DNA-PKcs regulates its functionality has much been reported, it does not well characterized how these phosphorylations affect the NHEJ and other DSB responses, and this deserves to be researched with great interest.
DNA-PKcs phosphorylation in VDJ recombination and class switch recombination (CSR)
The canonical NHEJ pathway also plays key roles in VDJ recombination and class switch recombination, which is required for immunoglobulin diversity and B-cell maturation (Chi et al., 2020). Briefly, during the VDJ recombination process, DNA-PK mediated NHEJ rejoins the hairpins and processes the RSS (single recombination signal sequence) ends derived from RAGs (Recombination-Activation Genes)-RSS-HMGB1 (High-Mobility Group1) complex. For CSR, DNA-PK mediated NHEJ repairs the S region DSBs generated by AID (Activation-induced cytidine deaminase). PRKDC gene deficiency or expression of kinase-dead DNA-PKcs protein causes severe combined immunodeficiency (SCID) in mice (Blunt et al., 1995; Miller et al., 1995).
In mice, the counterpart phosphorylation site of DNA-PKcs Ser 2056 is Ser 2053. DNA-PKcs Ser 2053 phosphorylation can be induced by ionizing radiation. Knockin mouse models with alanine- (DNA-PKcs PQR) or phospho-mimetic aspartate (DNA-PKcs SD) substitutions at the S2053 cluster showed normal kinase activity, and underwent efficient V(D)J recombination and class switch recombination, suggesting phosphorylation of DNA-PKcs at the S2053 (S2056) cluster likely dispensable for end-ligation and hairpin-opening function during lymphocyte development (Jiang et al., 2019).
Similarly, DNA-PKcs Thr-2609 is not required for hairpin opening and V(D)J recombination (Jiang et al., 2015b; Lee et al., 2013; Shao et al., 2020; Zhang et al., 2011). Crowe et al. used the DNA-PKcs 5A mouse model carrying alanine substitutions at the T2609 cluster and found that loss of DNA-PKcs T2609 does not affect the CSR efficiency, but increased alternative end-joining was used by CSR. Therefore, DNA-PKcs T2609 plays a role in the repair pathway choice of CSR (Crowe et al., 2020).
Ubiquitination of DNA-PKcs
Ubiquitin (Ub) is a highly evolutionarily conserved protein with 76 amino acid residues which was first identified to mediate protein degradation in reticulocyte extracts (Wilkinson et al., 1980). In human cells, UBA52, UBA80, UBC, and UBB four genes are responsible for the ubiquitin production. Among them, UBA52 and UBA80 are first transcribed fused to ribosomal proteins, whereas UBC and UBB express linear poly-ubiquitin chains, which require to be processed to ubiquitin monomers (Baker & Board, 1987). Ub precursor needs to be cleaved to expose its carboxyl-terminal di-glycine motif which is required for the covalent conjugation via its carboxyl-terminal glycine to the lysine of target protein. Ubiquitination involves three-step processes, which needs Ub-activating (E1) enzyme, Ub-conjugating (E2), and Ub-ligating (E3), respectively. E3s are responsible for specific substrate recognition, so they are crucial for drugs design which can only target specific substrates of the E3 ligase (Hoeller & Dikic, 2009).
DNA-PKcs was reported to be degraded under different conditions. About 20 years ago, Jane Parkinson and collages found that Herpes simplex virus (HSV-1) infection could cause DNA-PKcs degradation and inactivation, which was dependent on the expression of the HSV-1 immediate–early protein Vmw110. Vmw110 itself could directly cause degradation of DNA-PKcs, but reduced the kinase activity indirectly. Ultimately, the degradation and inactivity of DNA-PKcs seem to be beneficial to HSV-1 infection and facilitate the virus replication (Parkinson et al., 1999).
Valosine-containing protein (VCP), a chaperon protein that regulates target protein for ubiquitin–proteasome-dependent degradation, was reported to promote DNA-PKcs degradation in glioblastoma cells and increase the sensitivity of glioblastoma cells to radiation (Jiang et al., 2013).
However, which E3 ubiquitin ligase is responsible for proteasome degradation of DNA-PKcs is unknown until 2014. Shiuh-Rong Ho et al. identified the ring between ring (RBR) finger-domain containing protein RNF144A as the first E3 ubiquitin ligase for DNA-PKcs degradation. Adriamycin treatment can induce a significant expression of RNF144A mRNA in human colon cancer HCT116 P53 wild-type cells but not isogenic HCT116 P53 deficient cells. RNF144A can bind and ubiquitinate cytoplasmic DNA-PKcs for proteasome-dependent degradation, which reduces DNA-PKcs both in cytoplasm and nucleus and sensitizes cancer cells to DNA damage agents (such as neocarzinostatin and adriamycin) and then promotes apoptosis (Ho et al., 2014) (Fig. 2).
Fig. 2
Up-to-date identified DNA-PKcs PTMs. Generally, DNA-PKcs ubiquitination leads to the degradation of DNA-PKcs protein in cells. Acetylation, neddylation, and PARylation are all connected to DNA damage response
Full size imageHigh throughput proteomics analysis identified many ubiquitinated lysine on DNA-PKcs, but not anyone was furthermore studied to illuminate the function (Kim et al., 2011). Many conservative lysines on DNA-PKcs such as K71, K99, K108, K117, and so on deserve to be studied.
Acetylation of DNA-PKcs
Acetylation of lysine is also a reversible process which is regulated by acetyltransferases and deacetylase. Protein acetylation modulates many biological processes. Acetylation of histone at the N-terminal lysine residues can remove the positive charge and then decrease the interaction of N-termini of histones with the negatively charged phosphate groups of DNA. Therefore, acetylation of histones plays an important role in gene expression regulation (Shahbazian & Grunstein, 2007). In addition, lysine acetylation was reported to modulate some DSB repair protein activity such as Ku70, ATM, CtIP, and DNA-PKcs (Mori et al., 2016). David J Chen group identified two conservative lysine residues that can be acetylated, K3241 and K3260. Mutating the two lysines did not affect cell profiling but impaired DSB repair and resulted in radiosensitivity (Parkinson et al., 1999). Proteomic study has revealed at least 16 potential acetylated lysine residues on DNA-PKcs, but their functions need to be explored (Choudhary et al., 2009) (Fig. 2).
Neddylation of DNA-PKcs
Since the discovery of ubiquitin, many ubiquitin-like (UBL) proteins have been identified including NEDD8 (neural precursor cell expressed developmentally down-regulated protein 8), SUMO1-3 (small ubiquitin-related modifier-1, -2, and -3), UFM1 (ubiquitin-fold modifier-1), ATG8 (autophagy-related protein 8), ATG12 (autophagy-related protein 12), and so on (Brown & Jackson, 2015). Among these UBLs, NEDD8 is about 60% identical and 80% homologous to ub, the highest one of UBLs (Guo et al., 2020; Ma et al., 2013). Like ubiquitination, NEDD8 binds its targets covalently through neddylation system enzymes, E1 (the dimer of UBA3 and APPBP1), E2 (UBE2M or UBE2F), and E3 (Enchev et al., 2015; Ma et al., 2013). NEDD8 could be recruited to DNA damage sites induced by micro-radiation and play an important role during DNA damage signaling or even regulate DSBs repair efficiency and change the DSBs repair pathway (Brown et al., 2015; Jimeno et al., 2015; Ma et al., 2013).
Zhenqiang Pan et al. first mentioned DNA-PKcs neddylation through mass spectrometry analysis in NEDD8 overexpressed HEK293 cells (Jones et al., 2008). However, the detection of endogenous neddylation is indispensable to identifying a genuine substrate, because ubiquitination system can be involved when NEDD8 is overexpressed (Enchev et al., 2015). We identified that both exogenous and endogenous NEDD8 can modify DNA-PKcs at its kinase domain and this modification could be enhanced by ionizing radiation-induced DSBs. Then, we mutated eight conserved lysine residues within the kinase domain and identified the K4007 as the mainly neddylated site. Then, through mass spectrometry, we found that the K4007 was indeed modified with GG and identified HUWE1 was responsible for DNA-PKcs neddylation. When inhibiting DNA-PKcs neddylation via knockdown UBA3, UBE2M or HUWE1, DNA-PKcs autophosphorylation at Ser2056 but not Thr-2609 was dramatically decreased as well as the NHEJ repair efficiency (Guo et al., 2020) (Fig. 2).
PARylation of DNA-PKcs
Protein ADP-ribosylation is a general process in human cells that utilizes ADP-ribosyltransferases (ARTs) to transfer an ADP-ribose moiety from NAD+ to the amino acid side-chains of target proteins or to an already protein-bound ADP-ribose, to form mono-ADP-ribosylation (MAR) or poly-ADP-ribosylation (PAR) termed MARylation and PARylation, respectively (Ma et al., 2013; Martello et al., 2016). ADP-ribosylation is a reversible process which is regulated by writer enzymes, poly (ADP-ribose) polymerases (PARPs), and eraser enzyme, PAR glycohydrolase (PARG). Protein ADP-ribosylation is involved into diverse biological processes including replication (Helleday, 2011), transcription (Oei et al., 1998), metabolism (Alemasova & Lavrik, 2019), apoptosis (Zhao et al., 2019), and DNA damage response (Ray Chaudhuri & Nussenzweig, 2017).
The PARP family contains 16 members; among them, PARP1 and PARP2 are closely associated with DSBs’ repair, especially PARP1, which affects many DSB repair proteins such as BRCA1, BRCA2, and EXO1 (Luo & Kraus, 2012). Additionally, PARP1 ADP-ribosylates DNA-PKcs and stimulates its kinase activity (Ruscetti et al., 1998). In turn, DNA-PKcs phosphorylates PARP1 in a DNA-dependent manner and suppresses PARP1 activity to alter the pattern of ADP-ribosylation (Ariumi et al., 1999). However, the suppression of PAPR1 is not caused by its phosphorylation, because it happens without ATP existence[07].
Additionally, PARP1 and DNA-PKcs interact with each other and DNA-PKcs PARylation regulates its autophosphorylation at Ser2056 during DSBs response (Zou et al., 2015) (Han et al., 2019). When simultaneous inhibition of DNA-PKcs kinase activity and PARylation with NU7441 and olaparib, cell survival was more significantly reduced than treatment with olaparib or NU7441 alone (Han et al., 2019). Besides, Trp-tRNA synthetase (TrpRS) bridges DNA-PKcs and PAPR1 to form a nuclear complex and then facilities DNA-PKcs PARylation and subsequently PARP1 phosphorylation (Sajish et al., 2012).
Although DNA-PKcs has been confirmed PARylated and its PARylation indeed promotes DSBs repair and cell survival, which sites are mainly modified remains to be further identified. And then, the function of DNA-PKcs PARylation could be described more thoroughly (Fig. 2).
Conclusions
DNA-PKcs phosphorylation has been demonstrated as keys to DNA-PKcs roles in DNA end-processing, DNA end-joining, or the DNA damage signaling. It can affect the conformation of DNA-PKcs binding to dsDNA ends and promote the eviction of DNA-PKcs. However, more structural biological studies are needed to address this point. Interestingly, other DNA-PKcs PTMs have shown cross-talks with DNA-PKcs autophosphorylation, such as neddylation and PARylation. How these PTMs orchestrate with each other in DSBs repair remains fascinating. For DNA-PKcs ubiquitination, it targets DNA-PKcs for proteasome degradation and plays important role in determining DNA-PKcs homeostasis in cells. Identification of the different ubiquitin chains on DNA-PKcs may be an interesting future direction. How DNA-PKcs acetylation impacts the DSBs repair or chromatin needs further investigation. With more works addressing if these newly identified DNA-PKcs PTMs are involved in diseases, our understanding of DNA-PKcs will be significantly substantiated.
Data availability
The authors confirm that the data supporting the findings of this study are available within the article.
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Funding
This study was supported by the Lung Cancer Project from Beijing Municipal Health Commission 2020–2022.
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Department of Radiation Toxicology and Oncology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing, 100850, People’s Republic of China
Zongpei Guo & Ping-Kun Zhou
Chinese Institute for Brain Research, Beijing, 102206, People’s Republic of China
Zongpei Guo
Institute for Environmental Medicine and Radiation Hygiene, School of Public Health, University of South China, Hengyang, Hunan, 421001, People’s Republic of China
Ping-Kun Zhou
Cancer Research Center, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, 101149, People’s Republic of China
Teng Ma
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Correspondence to Ping-Kun Zhou or Teng Ma.
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Guo, Z., Zhou, PK. & Ma, T. DNA-PKcs post-translational modifications and associated diseases. GENOME INSTAB. DIS. 3, 136–143 (2022). https://doi.org/10.1007/s42764-022-00073-2
Received04 January 2022
Revised02 April 2022
Accepted06 April 2022
Published28 April 2022
Issue DateJune 2022
DOIhttps://doi.org/10.1007/s42764-022-00073-2
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DNA-PKcs
NHEJ
Auto-phosphorylation
Ubiquitination
Neddylation
Acetylation
PARylation
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