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PMID:15194694
| Citation |
Wang, YG, Nnakwe, C, Lane, WS, Modesti, M and Frank, KM (2004) Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase. J. Biol. Chem. 279:37282-90 |
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| 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 full-length 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. |
| Links |
PubMed Online version:10.1074/jbc.M401217200 |
| Keywords |
Amino Acid Sequence; Base Sequence; Cell Line; DNA Ligases/metabolism; DNA Primers; DNA-Activated Protein Kinase; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Enzyme Stability; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Proteins; Phosphorylation; Protein-Serine-Threonine Kinases/chemistry; Protein-Serine-Threonine Kinases/genetics; Protein-Serine-Threonine Kinases/metabolism; Sequence Homology, Amino Acid |
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Significance
Annotations
| Gene product | Qualifier | GO ID | GO term name | Evidence Code | with/from | Aspect | Notes | Status |
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