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  • The kinase activity of DNA PKcs is essential for NHEJ

    2019-11-07

    The kinase activity of DNA-PKcs is essential for NHEJ, but the exact role of the enzymatic activity of DNA-PKcs plays in NHEJ is not fully understood [57]. In vitro, DNA-PKcs can phosphorylate each of the canonical NHEJ factors including Ku70/80 [58], [59], XRCC4 [60], [61], rgd peptide IV [62], and XLF [63], but surprisingly none of these phosphorylations are required for NHEJ. DNA-PKcs has also been shown to phosphorylate a number of other factors implicated in NHEJ and the DNA damage response, including Artemis, polynucleotide kinase/phosphatase (PNKP), the histone H2AX and p53 [9], [64], [65]. But, considering these factors are also phosphorylated by ATM, the role of DNA-PKcs-mediated phosphorylation of these proteins is not exactly clear. However, it was recently found that phosphorylation of the implicated NHEJ factor Werner (WRN) by DNA-PKcs is required for efficient DSB repair, possibly identifying a DNA-PKcs mediated phosphorylation of a substrate that is important for NHEJ [66]. Furthermore, a number of new in vivo substrates of DNA-PKcs have emerged [65]. Proteins that are phosphorylated by DNA-PKcs following DNA damage include Akt/PKB [67], [68], the nuclear receptor 4A [69], heat shock protein HSP90α [70], [71], and the scaffold attachment factor A [72], but it is not known if phosphorylation of any of these proteins is important for NHEJ or the cellular response to DSBs. In respect to the NHEJ pathway, the best characterized DNA-PKcs substrate is DNA-PKcs itself as it autophosphorylates itself on a number of residues in different regions of the polypeptide [73], [74], [75], [76]. Autophosphorylation of DNA-PKcs results in kinase inactivation and dissociation from the DNA-Ku70/80 complex in vitro, but even the specific role that DNA-PKcs autophosphorylation plays in NHEJ is not completely understood [73], [77].
    Phosphorylation of DNA-PKcs Following induction of a DSB, DNA-PKcs is phosphorylated on more than 40 sites including at a number of phosphorylation clusters (discussed sites outlined in Fig. 2) [78]. The best characterized DNA-PKcs phosphorylation cluster is the threonine 2609 (Thr2609) cluster. The Thr2609 cluster was original identified as an autophosphorylation site but further analyses revealed that the Thr2609 cluster is primarily targeted by ATM and ATR in response to DSBs and replication stress, respectively [79], [80], [81], [82]. Phosphorylation of the Thr2609 cluster is important for NHEJ as ablating the phosphorylation of these sites via alanine substitutions results in severe radiosensitivity and diminished DNA end-joining ability in vitro [79], [81], [83], [84]. Furthermore, DNA-PKcs3A knock-in mutant mice lacking a functional Thr2605 cluster (human Thr2609) confer early lethality and congenital bone marrow failure [85]. Cells derived from DNA-PKcs3A mice are highly sensitive toward replication stress agents and have impairment of multiple DNA repair pathways including HR and FA. These findings demonstrated the critical function and requirement of Thr2609 cluster phosphorylation in vivo under physiological conditions. Protein biochemistry and structure studies suggest that DNA-PKcs phosphorylation at the Thr2609 cluster causes a large conformational change of DNA-PKcs, which promotes its dissociation from the Ku70/80 heterodimer and release from DSB ends [12], [47]. Additionally, Thr2609 cluster phosphorylation and/or conformational change of DNA-PKcs facilitate its association with other DNA repair molecules. For example, the coordination between ATM and Artemis in DSB repair requires DNA-PKcs phosphorylation at the Thr2609 cluster as phosphorylated DNA-PKcs recruits Artemis to DNA ends and facilitates its endonuclease activity [86]. This notion is further supported by in vivo evidence that a functional Thr2609 cluster in conjunction with ATM kinase is able to promote the coding end joining during V(D)J recombination, a process requiring Artemis endonucleases activity [87]. The other well characterized phosphorylation cluster of DNA-PKcs is the serine 2056 (Ser2056) cluster [88], [89]. Serine 2056 is a bona fide autophosphorylation site in response to DSBs in vivo and phosphorylation of the Ser2056 cluster is important for NHEJ as ablating phosphorylation of this cluster causes increased radiosensitivity and less efficient DSB repair. The Ser2056 and Thr2609 phosphorylation clusters may play opposing roles in regard to protecting the ends of the DSB. Phosphorylation at Ser2056 limits DNA end processing whereas the Thr2609 promotes it [88], [90]. A number of phosphorylation sites have been found in the N- and C-terminal regions of DNA-PKcs. Phosphorylation at threonine 946 and serine 1004 inhibits NHEJ but does not affect the enzymatic activity of DNA-PKcs [91]. Three phosphorylation sites have been shown to directly affect the kinase activity of DNA-PKcs. A phosphorylation site has also been identified in the kinase domain of DNA-PKcs at threonine 3950 [74]. Ablation of this site by alanine substitution does not affect the kinase activity of DNA-PKcs but the phospho-mimic aspartic acid substitution results in ablation of DNA-PKcs kinase activity, implicating that autophosphorylation at this site may be a method to directly turn off DNA-PKcs kinase activity. Phosphorylation of DNA-PKcs in its N-terminus at serines 56 and 72 also results in abrogation of DNA-PKcs enzymatic activity, but how phosphorylation of these two sites regulates the kinase activity of DNA-PKcs is unknown [91]. Finally, serine 3205 is phosphorylated in response to IR and this phosphorylation is mediated by ATM, similar to the Thr2609 cluster [91]. Although much has been learned about how the phosphorylation status of DNA-PKcs regulates the functionality of the protein, the exact role of these phosphorylations in NHEJ and other DSB responses is still unclear and will continue to serves as a research topic of great interest.