Phosphorylation at S2053 in Murine (S2056 in Human) DNA-PKcs Is Dispensable for Lymphocyte Development and Class Switch Recombination

J Immunol. 2019 Jul 1;203(1):178-187. doi: 10.4049/jimmunol.1801657. Epub 2019 May 17.


The classical nonhomologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large DNA-PK catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs-/- ) abrogates end processing (e.g., hairpin opening), but not end-ligation, whereas expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD ) abrogates end-ligation, suggesting a kinase-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs-/- and DNA-PKcsKD/KD mice because of the requirement for both hairpin opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056 cluster is the best-characterized autophosphorylation site in human DNA-PKcs. In this study, we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053. We also generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster. Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retained normal kinase activity and underwent efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053 cluster of murine DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Differentiation / genetics
  • Cell Line
  • DNA-Activated Protein Kinase / genetics
  • DNA-Activated Protein Kinase / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Fibroblasts / physiology*
  • Fibroblasts / radiation effects
  • Gene Knock-In Techniques
  • Humans
  • Immunoglobulin Class Switching / genetics
  • Lymphocyte Activation
  • Lymphocytes / physiology*
  • Lymphocytes / radiation effects
  • Mice
  • Mice, Knockout
  • Mutation / genetics
  • Radiation Tolerance
  • Serine / genetics


  • DNA-Binding Proteins
  • Serine
  • DNA-Activated Protein Kinase
  • Prkdc protein, mouse