35. The Subcellular Localization and the Subcellular Translocation Mechanism of Ku70 and Ku80

Manabu Koike, Aki Koike, and Tadahiro Shiomi

Keywords: Ku7O, Ku80, DNA-PKcs, nuclear localization signal, cell cycle

The Ku protein is a complex of two subunits, Ku7O and Ku80. Ku plays an important role in DNA-PKcsdependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expressuon and the subcellular localization of Ku and DNA PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku7O and Ku8O in the nuclei in interphase cells. In mitotic cells (1) most of the Ku protein was found diffused in the cytoplasm, (2) a fraction was .deLetted at the periphery of condensed chromosomes, and (3) no Ku protein was present in the chromesome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel func tion of Ku in the G2/M phase, which does not require DNA-PKcs.

Ku is generally believed to always form and function as heterodimers on the basis of in vitro observa tions. Here, we demonstrate that the localization of Ku8O does not completely coincide with that of Ku7O. Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/ early Gl phase of the cell cycle. Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to unvestugate the molecular mechanisms underlying the nuclear translocation of Ku. The nuclear translocation of Ku8O started during the late telophase/ early Gl phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku7O Furthermore, we found that the Ku8O protein was transported to the nucleus without heterodimerization with Ku7O. To understand in detail the mechanism of transport of Ku8O, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined a region spanning nine amino acid residues (positions 561-569). The Ku8O NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97. All these findings support the idea that Ku80 can translocate to the nucleus using its own NUS independent of the translocation of Ku7O.

Publicattons:
1)Koike, M., Ikuta, T., Miyasaka, T. and Shromt, T.: Oncogene, 18, 7495-7505, 1999.
2)Koike, M., Awaji, T., Kataoka, M., Tsujimoto, G K3rtasova, T., Koike, A. and Shiomi, T.: J. Cell. S.,. 112, 4031-4039, 1999.
3)Koike, M., Ikuta, T., Miyasaka, T. and Shiomi, T., Exp.Cell Res., 250, 401-413, 1999.
4)Suzuki, T., Kurosaki, T., Shimada, K., Kansaku, N. Kuhnlein, U., Zadworny, D., Agata, K. Hashimoto, A., Koide, M., Koike, M., Takata, M. Kuroiwa, A., ]VIinai, S., Namikawa, T. and Matsuda Y.: Cytogenet Cell Genet., 87, 32-40 1999.