Acetylation of the nuclear localization signal in Ku70 diminishes the interaction with importin-α.

Proteins are functionally regulated by various types of posttranslational modifications (PTMs). Ku, a heterodimer complex of Ku70 and Ku80 subunits, participates in DNA repair processes. Ku is distributed not only in the nucleus but also in the cytoplasm, suggesting that the function of Ku is regulated by its subcellular localization. Although Ku70 undergoes PTMs including phosphorylation or acetylation, it remains unknown whether the PTMs of Ku70 affect the subcellular localization of Ku. Using a cell-free pull-down assay technique, we show that Nε-acetylation of lysine residues in the synthetic peptide matched to Ku70's nuclear localization signal (NLS) reduces the peptide's interaction with the nuclear transport factor importin-α. The reduced interaction by acetylation was supported by molecular simulation analysis. In addition, when expressed in the endogenous Ku80-defective Chinese hamster ovary xrs-6 cells, some full-size human Ku70 mutants with substitutions of glutamine, a possible structural mimetic of Nε-acetyl-lysine, for lysine at the specific NLS positions exhibited no nuclear distribution. These findings imply that acetylation of particular lysine residues in the Ku70 NLS regulates nuclear localization of Ku.