Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase

Abstract

DNA ligase IV (Lig4), x-ray cross-complementation group 4 (XRCC4), and DNA-dependent protein kinase (DNA-PK) are essential mammalian nonhomologous end joining proteins used for V(D)J recombination and DNA repair. Previously a Lig4 peptide was reported to be an in vitro substrate for DNA-PK, but the phosphorylation state of Lig4 protein in vivo is not known. In this study, we report that a full-length Lig4 construct was expressed as a phosphoprotein in the cell. Also the fulllength Lig4 protein, in complex with XRCC4, was an in vitro substrate for DNA-PK. Using tandem mass spectrometry, we identified a DNA-PK phosphorylation site at Thr-650 in human Lig4 and a potential second phosphorylation site at Ser-668 or Ser-672. Phosphorylation of Lig4 per se was not required for Lig4 DNA end joining activity. Substitution of these amino acids with alanine, individually or in combination, led to changes in Lig4 protein stability of mouse Lig4. The phosphomimetic mutation S650D returned Lig4 stability to that of the wild-type protein. Furthermore DNA-PK was found to negatively regulate Lig4 protein stability. Our results suggest that Lig4 stability is regulated by multiple factors, including interaction with XRCC4, phosphorylation status, and possibly Lig4 conformation.Nonhomologous end joining (NHEJ) 1 is an efficient mechanism used by mammalian cells to repair DNA double strand breaks (1, 2) and is also required for the process of V(D)J recombination, the rearrangement of immunoglobulin and Tcell receptor genes essential for the generation of a diverse immune response (3). XRCC4 and DNA ligase IV (Lig4), which form a ligation complex in the cell (4, 5), are two critical proteins involved in these two processes (6 -8).The five most well studied proteins that are required for both NHEJ and V(D)J recombination in mammalian cells are Ku70, Ku80, DNA-dependent protein kinase catalytic subunit (DNAPKcs), XRCC4, and Lig4. The heterodimeric Ku protein, comprising subunits Ku70 and Ku80, binds strongly to doublestranded DNA ends (9). When bound to DNA, Ku recruits DNA-PKcs and activates its kinase activity (10, 11). The XRCC4⅐Lig4 protein complex is recruited to the DNA ends to complete ligation (4,5,12). An additional protein, Artemis, which has nuclease activity and can open DNA hairpin intermediates with DNA-PKcs during V(D)J recombination (13), has also been shown to be involved in both NHEJ and V(D)J recombination (14, 15).Mice lacking NHEJ components have abnormal lymphocyte development due to defective V(D)J recombination (16). Mice deficient in Ku70, Ku80, XRCC4, or Lig4 also exhibit growth defects, premature senescence, and hypersensitivity to ionizing irradiation. Deficiency of XRCC4 or Lig4 is most severe, resulting in significant neuronal apoptosis and embryonic lethality (7,8,17). Embryonic fibroblast cells that are deficient for either DNA-PKcs or Artemis also exhibit cell type-specific ionizing irradiation hypersensitivity (16). Defects in the NHEJ factors have also been implicated in the generation of chromosome i...

Ataxia telangiectasia-mutated protein and DNA-dependent protein kinase have complementary V(D)J recombination functions

“…Given the more substantial impairment of extrachromosomal SE joining in the absence of ATM and DNA-PKcs, relevant common substrates might be a core NHEJ factor or even RAG. Although previous mutagenesis analyses failed to identify a definitive role for ATM and DNA-PKcs phosphorylation sites of any single NHEJ factor during V(D)J recombination (36)(37)(38), it is possible that phosphorylation of two or more NHEJ factors might lead to overlapping functions that could mask effects of mutagenesis of a single factor. Alternatively, RAG, which generates and associates with SEs, also has potential phosphorylation sites for DNA-PKcs and ATM.…”

“…Regarding the apparently overlapping ATM and DNA-PKcs substrates involved in SE joining, these two kinases phosphorylate a large number of shared substrates in response to DSBs, including both chromatin-associated repair factors (e.g., H2AX, SMC1, and Kap1) and members of the NHEJ pathway (e.g., KU, XRCC4, Ligase 4, Artemis, and DNA-PKcs itself) (36)(37)(38). Given the more substantial impairment of extrachromosomal SE joining in the absence of ATM and DNA-PKcs, relevant common substrates might be a core NHEJ factor or even RAG.…”

Eukaryotic DNA Ligases: Structural and Functional Insights

“…The stability of DNA ligase IV is also regulated by phosphorylation at Ser650. Notably, DNA ligase IV is phosphorylated by the DNA-dependent protein kinase, DNA-PK, a key NHEJ factor that negatively regulates DNA ligase IV stability (89). As expected, targeted inactivation of LIG4 in mouse and human cells dramatically reduced V(D)J recombination and resulted in hypersensitivity to ionizing radiation because of the defect in the repair of doublestrand breaks by NHEJ (90,91).…”

DNA Double-Strand Break Repair Through Non-Homologous End-Joining: Recruitment and Assembly of the Players

“…It has been awaited to find the biological consequence of XRCC4 phosphorylation by DNA-PK through the identification and elimination of the phosphorylation site(s). Several groups, employing mass spectrometry, identified Ser260 and Ser318 as the major phosphorylation sites in XRCC4 by DNA-PK in vitro (Lee et al, 2002;Yu et al, 2003;Lee et al, 2003;Wang et al, 2004). However, it is presently unclear whether these sites are phosphorylated in living cells, especially, in response to DNA damage.…”