The nuclear localization signal of the human Ku70 is a variant bipartite type recognized by the two components of nuclear pore-targeting complex.

Ku protein is a complex of two subunits, Ku70 and Ku80. Ku is suspected to participate in both DNA double-strand break repair and transcription. Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins. In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70. Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells. Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells. The NLS of Ku70 was located at the region composed of 18 amino acid residues (positions 539 to 556). This region overlapped with the Ku80-independent DNA-binding domain reported previously. The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region. All the subregions were necessary for complete NLS activity. The amino acids in the nonbasic intervening region of Ku70 might be important for full NLS activity not only to provide sufficient length between the two separated clusters of basic amino acids but also to have an adequate amino acid sequence. All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70. The structure of the Ku70 NLS resembled the consensus of a bipartite-type NLS. The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97.

Koike M ，Ikuta T ，Miyasaka T ，Shiomi T ... -  《EXPERIMENTAL CELL RESEARCH》

Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal.

Koike M ，Ikuta T ，Miyasaka T ，Shiomi T ... -  《ONCOGENE》

Ku70 can translocate to the nucleus independent of Ku80 translocation and DNA-PK autophosphorylation.

Ku plays an important role in multiple nuclear processes, e.g., DNA repair, chromosome maintenance, and transcriptional regulation. Although some evidence suggests that the nuclear translocation of Ku plays a key role in regulating the function of Ku, the mechanism is poorly understood. Using the site-directed mutagenesis technique, we demonstrate here that Ku70 can translocate to the nucleus without heterodimerization with Ku80. The nuclear accumulation of Ku70 mutants of the nuclear localization signal, which retained their binding ability with Ku80, was diminished. On the other hand, Ku70 mutants which lacked the ability to bind with Ku80 could translocate to the nuclei. Human Ku70, when transfected, accumulated within the nuclei of hamster xrs-6 cells which had undetectable DNA-PK activity and Ku80. Ku70 and Ku80 mutants of DNA-PK phosphorylation sites showed normal heterodimerization and nuclear translocation. These findings also support the idea that Ku70 can translocate to the nucleus independent of DNA-PK autophosphorylation.

Koike M ，Shiomi T ，Koike A 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》

KARP-1 works as a heterodimer with Ku70, but the function of KARP-1 cannot perfectly replace that of Ku80 in DSB repair.

Ku, the heterodimer of Ku70 and Ku80, plays an essential role in the DNA double-strand break (DSB) repair pathway, i.e., non-homologous end-joining (NHEJ). Two isoforms of Ku80 encoded by the same genes, namely, Ku80 and KARP-1 are expressed and function in primate cells, but not in rodent cells. Ku80 works as a heterodimer with Ku70. However, it is not yet clear whether KARP-1 forms a heterodimer with Ku70 and works as a heterodimer. Although KARP-1 appears to work in NHEJ, its physiological role remains unclear. In this study, we established and characterized EGFP-KARP-1-expressing xrs-6 cell lines, EGFP-KARP-1/xrs-6. We found that nuclear localization signal (NLS) of KARP-1 is localized in the C-terminal region. Our data showed that KARP-1 localizes within the nucleus in NLS-dependent and NLS-independent manner and forms a heterodimer with Ku70, and stabilizes Ku70. On the other hand, EGFP-KARP-1 could not perfectly complement the radiosensitivity and DSB repair activity of Ku80-deficient xrs-6 cells. Furthermore, KARP-1 could not accumulate at DSBs faster than Ku80, although EGFP-KARP-1 accumulates at DSBs. Our data demonstrate that the function of KARP-1 could not perfectly replace that of Ku80 in DSB repair, although KARP-1 has some biochemical properties, which resemble those of Ku80, and works as a heterodimer with Ku70. On the other hand, the number of EGFP-KARP-1-expressing xrs-6 cells showing pan-nuclear γ-H2AX staining significantly increases following X-irradiation, suggesting that KARP-1 may have a novel role in DSB response.

Koike M ，Yutoku Y ，Koike A 《-》

Nuclear localization of Ku antigen is promoted independently by basic motifs in the Ku70 and Ku80 subunits.

Bertinato J ，Schild-Poulter C ，Haché RJ 《JOURNAL OF CELL SCIENCE》