Annual Report

30. Dimerization and Nuclear Localization of Ku70 and Ku80 Proteins

Manabu Koike, Tadahiro Shiomi, and Aki Koike

Keywords: Ku70, Ku80, dimerization, nuclear localization signal, GFP


Ku, a heterodimer of Ku70 and Ku80, plays a key role in multiple nuclear processes, e.g. DNA repair, chromosome maintenance, and transcription regulation. Heterodimerization is essential for Ku-dependent DNA repair in vivo, although its role is poorly understood. Some lines of evidence suggest that heterodimerization is required for the stabilization of Ku70 and Ku80. Here we show that the heterodimerization of these Ku subunits is important for their nuclear entry. When transfected into Ku-deficient xrs-6 cells, exogenous Ku70 and Ku80 tagged with green fluorescent protein (GFP) accumulated into the nucleus, whereas each nuclear localization signal (NLS)-dysfunctional mutant was undetectable in the nucleus, supporting the idea that each Ku can translocate to the nucleus through its own NLS. On the other hand, the nuclear accumulation of each NLS-dysfunctional mutant was markedly enhanced by the presence of an exogenous wild-type counterpart. In Ku-expressing HeLa cells, each NLS-dysfunctional mutant, as well as wild-type Ku70 and Ku80, was still detectable in the nucleus, whereas the double mutant of each Ku subunit, with decreased functions of both nuclear targeting and dimerization, was undetectable there. Our results indicate that each Ku subunit can translocate to the nucleus not only through its own NLS, but also through heterodimerization with each other.

In conclusion, we have shown a novel role for the heterodimerization of Ku70 and Ku80. Ku70 and Ku80 appear to have multiple functions as a monomeric form and a heterodimeric form. There are at least two nuclear translocation pathways of each Ku subunit. We speculate that the Ku subunits may use the NLS-dependent nuclear translocation pathway to perform some function(s) independent of each other and Ku subunits may use the nuclear translocation pathway through heterodimerization to perform the same functions dependent on each other. The control mechanism for nuclear localization of Ku70 and Ku80 plays, at least in part, a key role in regulating the physiological function of Ku in vivo. Further studies to elucidate the molecular mechanisms of nuclear transport of the Ku subunits will lead to a better understanding of the regulation mechanism of nuclear proteins.

Publications:
1) Koike, M., Shiomi, T., and Koike, A.: J. Biol. Chem. 276, 11167-11173, 2001.
2) Koike, M., Kuroiwa, A., Koike, A., Shiomi, T., and Matsuda, Y. : Cytogenetics and Cell Genetics. in press.
3) Koike, M., Shiomi, T. and Koike, A. Biochem. Biophys. Res. Commun. 276, 1105-1111, 2000.


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