Dimerization and Nuclear Localization of Ku Proteins
2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês
10.1074/jbc.m010902200
ISSN1083-351X
AutoresManabu Koike, Tadahiro Shiomi, Aki Koike,
Tópico(s)RNA Research and Splicing
ResumoKu, 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 repairin 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 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 in the nucleus. 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. 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 repairin 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 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 in the nucleus. 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. DNA-dependent protein kinase DNA double-strand break nuclear localization signal green fluorescent protein propidium iodide yellow variant GFP cyan variant GFP enhanced GFP glutathioneS-transferase Ku is a complex composed of two protein subunits of 70 and 80 kDa, hereafter designated as Ku70 and Ku80, respectively (1Mimori T. Hardin J.A. Steitz J.A. J. Biol. Chem. 1986; 261: 2274-2278Abstract Full Text PDF PubMed Google Scholar). It was shown that Ku is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK)1 that phosphorylates several nuclear proteins in vitro,e.g. p53, RNA polymerase II, or Ku itself and is involved in DNA double-strand break (DSB) repair and V(D)J recombination (2Anderson C.W. Trends Biochem. Sci. 1993; 18: 433-437Abstract Full Text PDF PubMed Scopus (235) Google Scholar, 3Weaver D.T. Adv. Immunol. 1995; 58: 29-85Crossref PubMed Scopus (59) Google Scholar). Besides this main function, the Ku protein has other functions, some of which may be independent of DNA-PK activity. Both Ku70- and Ku80-knockout mice exhibited not only deficiencies in DNA DSB repair but also growth retardation (4Nussenzweig A. Chen C. da Costa Soares V. Sanchez M. Sokol K. Nussenzweig M.C. Li G.C. Nature. 1996; 382: 551-555Crossref PubMed Scopus (579) Google Scholar, 5Gu Y. Seidl K.J. Rathbun G.A. Zhu C. Manis J.P. van der Stoep N. Davidson L. Cheng H.L. Sekiguchi J.M. Frank K. Stanhope-Baker P. Schlissel M.S. Roth D.B. Alt F.W. Immunity. 1997; 7: 653-665Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). In addition, Ku70- and Ku80-mutant embryo fibroblasts in primary cultures have prolonged doubling times compared with normal embryo fibroblasts due to the rapid loss of proliferating cells and have shown signs of senescence (4Nussenzweig A. Chen C. da Costa Soares V. Sanchez M. Sokol K. Nussenzweig M.C. Li G.C. Nature. 1996; 382: 551-555Crossref PubMed Scopus (579) Google Scholar, 5Gu Y. Seidl K.J. Rathbun G.A. Zhu C. Manis J.P. van der Stoep N. Davidson L. Cheng H.L. Sekiguchi J.M. Frank K. Stanhope-Baker P. Schlissel M.S. Roth D.B. Alt F.W. Immunity. 1997; 7: 653-665Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). However, this appears not to be the case for cs DNA-PK-knockout mice (6Gao Y. Chaudhuri J. Zhu C. Davidson L. Weaver D.T. Alt F.W. Immunity. 1998; 9: 367-376Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). These findings suggest that Ku plays some role in growth regulation and/or senescence independent of the function of DNA-PK. Ku has been generally believed to always exist and function as a heterodimer. The heterodimerization is essential for DNA DSB repairin vivo and is also important in activating DNA-PK, which is one of the main functions of Ku (7Jin S. Weaver D.T. EMBO J. 1997; 16: 6874-6885Crossref PubMed Scopus (126) Google Scholar). The interacting regions of Ku70 and Ku80 have been identified by many research groups (8Wu X. Lieber M.R. Mol. Cell. Biol. 1996; 16: 5186-5193Crossref PubMed Scopus (110) Google Scholar, 9Osipovich O. Durum S.K. Muegge K. J. Biol. Chem. 1997; 272: 27259-27265Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 10Cary R.B. Chen F. Shen Z. Chen D.J. Nucleic Acids Res. 1998; 26: 974-979Crossref PubMed Scopus (41) Google Scholar, 11Koike M. Miyasaka T. Mimori T. Shiomi T. Biochem. Biophys. Res. Commun. 1998; 252: 679-685Crossref PubMed Scopus (30) Google Scholar, 12Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), but the role of this interaction in the Ku functions remains unknown. Loss of one of the Ku subunits results in a significant decrease in the steady-state level of the other (5Gu Y. Seidl K.J. Rathbun G.A. Zhu C. Manis J.P. van der Stoep N. Davidson L. Cheng H.L. Sekiguchi J.M. Frank K. Stanhope-Baker P. Schlissel M.S. Roth D.B. Alt F.W. Immunity. 1997; 7: 653-665Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 13Errami A. Smider V. Rathmell W.K. He D.M. Hendrickson E.A. Zdzienicka M.Z. Chu G. Mol. Cell. Biol. 1996; 16: 1519-1526Crossref PubMed Scopus (160) Google Scholar, 14Singleton B.K. Priestley A. Steingrimsdottir H. Gell D. Blunt T. Jackson S.P. Lehmann A.R. Jeggo P.A. Mol. Cell. Biol. 1997; 17: 1264-1273Crossref PubMed Scopus (165) Google Scholar), suggesting that the heterodimerization is, in part, required for the stabilization of each Ku subunit. On the other hand, there are some differences in the phenotype between Ku70- and Ku80-knockout mice (4Nussenzweig A. Chen C. da Costa Soares V. Sanchez M. Sokol K. Nussenzweig M.C. Li G.C. Nature. 1996; 382: 551-555Crossref PubMed Scopus (579) Google Scholar, 5Gu Y. Seidl K.J. Rathbun G.A. Zhu C. Manis J.P. van der Stoep N. Davidson L. Cheng H.L. Sekiguchi J.M. Frank K. Stanhope-Baker P. Schlissel M.S. Roth D.B. Alt F.W. Immunity. 1997; 7: 653-665Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 15Li G.C. Ouyang H. Li X. Nagasawa H. Little J.B. Chen D.J. Ling C.C. Fuks Z. Cordon-Cardo C. Mol. Cell. 1998; 2: 1-8Abstract Full Text Full Text PDF PubMed Google Scholar, 16Zhu C. Bogue M.A. Lim D.S. Hasty P. Roth D.B. Cell. 1996; 86: 379-389Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). For example, Ku70-knockout mice have small populations of mature T lymphocytes and a significant incidence of thymic lymphoma, but Ku80-knockout mice do not. Ku70 has been reported to show Ku80-dependent and -independent DNA binding, whereas Ku80 requires association with Ku70 for DNA binding (12Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, Ku70 may have unique functions that are independent of Ku80. Ku was originally reported to be a nuclear protein, consistent with its functions as a subunit of DNA-PK. On the other hand, although Ku is thought to be localized and to function only in the nucleus, several studies have revealed the cytoplasmic or cell surface localization of Ku proteins in various cell types (18Prabhakar B.S. Allaway G.P. Srinivasappa J. Notkins A.L. J. Clin. Invest. 1990; 86: 1301-1305Crossref PubMed Scopus (87) Google Scholar, 19Danziel R.G. Mendelson S.C. Quinn J.P. Autoimmunity. 1992; 13: 265-267Crossref PubMed Scopus (36) Google Scholar, 20Reeves W.H. Rheum. Dis. Clin. N. Am. 1992; 18: 391-414PubMed Google Scholar, 21Grawunder U. Finnie N. Jackson S.P. Riwar B. Jessberger R. Eur. J. Biochem. 1996; 241: 931-940Crossref PubMed Scopus (48) Google Scholar). The subcellular localization of Ku70 and Ku80 changes during the cell cycle (22Koike M. Awaji T. Kataoka M. Tsujimoto G. Kartasova T. Koike A. Shiomi T. J. Cell Sci. 1999; 112: 4031-4039PubMed Google Scholar), and the nuclear translocation of Ku70 precedes that of Ku80 in late telophase/early G1 cells (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar). Furthermore, changes in the subcellular localization of Ku could be controlled by various external growth-regulating stimuli (24Fewell J. Kuff E.L. J. Cell Sci. 1996; 109: 1937-1946PubMed Google Scholar). CD40L treatment of the myeloma cells induces a translocation of Ku from the cytoplasm to the cell surface, and that cell surface Ku can mediate both homotypic and heterotypic adhesion (25Teoh G. Urashima M. Greenfield E.A. Nguyen K.A. Lee J.F. Chauhan D. Ogata A. Treon S.P. Anderson K.C. J. Clin. Invest. 1998; 101: 1379-1388Crossref PubMed Google Scholar). Morio et al. (26Morio T. Hanissian S.H. Bacharier L.B. Teraoka H. Nonoyama S. Seki M. Kondo J. Nakano H. Lee S.K. Geha R.S. Yata J. Immunity. 1999; 11: 339-348Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) have reported that DNA-PK activity of human B cells is, at least in part, regulated by the nuclear translocation of Ku. These data suggest that the control mechanism for subcellular localization of Ku70 and Ku80 plays, at least in part, a key role in regulating the physiological function of Kuin vivo, although the mechanism is poorly understood. We have recently identified nuclear localization signals (NLSs) of Ku70 and Ku80 (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). The structures of the NLSs of the two Ku protein subunits are different. NLSs of Ku80 and Ku70 belong to the single-basic type and the variant bipartite-basic type, respectively (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). We have also shown that both Ku70 and Ku80 can translocate to the nucleus without forming a heterodimer using their own NLS (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 28Koike M. Shiomi T. Koike A. Biochem. Biophys. Res. Commun. 2000; 276: 1105-1111Crossref PubMed Scopus (21) Google Scholar). Moreover, the subcellular localization of Ku70 is affected by somatostatin treatment in CV-1 cells, but that of Ku80 is not (24Fewell J. Kuff E.L. J. Cell Sci. 1996; 109: 1937-1946PubMed Google Scholar). These results suggest that the nuclear translocation of Ku70 and Ku80 may be independently regulated. In the present study, we examined the subcellular localization of chimeric constructs of green fluorescent protein (GFP) color variants, and Ku proteins to which mutations were introduced by the site-directed mutagenesis technique, and found that Ku70 and Ku80 translocate to the nucleus not only through their own NLS but also through the heterodimerization, suggesting that the heterodimerization of Ku is important for their nuclear entry and functional regulation. Cells of established hamster cell lines of CHO-K1 and xrs-6 (derived from the CHO-K1 cell line on the basis of their sensitivity to ionizing radiation) were cultured in Ham's F-12 (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibiotics. The cell line of human tumor, HeLa-S3, has been described in previous studies (11Koike M. Miyasaka T. Mimori T. Shiomi T. Biochem. Biophys. Res. Commun. 1998; 252: 679-685Crossref PubMed Scopus (30) Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). The cells were maintained in a humidified incubator at 37 °C under 5% CO2. The xrs-6 cells defective in Ku80 were kindly provided by Dr. P. Jeggo (14Singleton B.K. Priestley A. Steingrimsdottir H. Gell D. Blunt T. Jackson S.P. Lehmann A.R. Jeggo P.A. Mol. Cell. Biol. 1997; 17: 1264-1273Crossref PubMed Scopus (165) Google Scholar, 29Singleton B.K. Torres-Arzayus M.I. Rottinghaus S.T. Taccioli G.E. Jeggo P.A. Mol. Cell. Biol. 1999; 19: 3267-3277Crossref PubMed Scopus (151) Google Scholar). Cells were plated on a six-well dish (Falcon, Lincoln Park, NJ) at a density of 2 × 105 cells/well the night before transfection. Transient transfections were performed in these cells using Effectene (Qiagen Inc., Chatworth, CA) according to the manufacturer's protocol. DNA was stained with 50 μg/ml propidium iodide (PI) (Sigma) containing 200 μg/ml RNase (Sigma). After incubation, the cells were fixed in 0.2 m phosphate buffer (pH 7.4) with 4% paraformaldehyde and then examined under an Olympus IX 70 fluorescence microscope (Olympus, Tokyo, Japan) to determine localization, as described previously (27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). Images were acquired with a Hamamatsu chilled 3-chip color charge-coupled-device camera (C5810-01) driven by the IP Lab imaging software (Signal Analitics Corp., Vienna, VA). cDNA for human Ku70 andKu80 was derived from pEGFP-Ku70(1–609) or pEGFP-Ku80(1–732) (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). Full-length Ku70 orKu80 was cloned in pECFP-C1 or pEYFP-C1 (CLONTECH, Palo Alto, CA) using a previously described method (pECFP-Ku70(1–609) and pEYFP-Ku80(1–732), respectively) (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). The junctions of both constructs were verified by sequencing. Ku70- or Ku80-site-specific mutants were formed by incorporating mutant oligonucleotides by strand extension reactions. The QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) was used according to the manufacturer's recommendations (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar). Following the application of the mutagenesis strategy, each mutant was identified by DNA sequencing as described previously (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar). The immunoblotting analysis was performed as described previously (24Fewell J. Kuff E.L. J. Cell Sci. 1996; 109: 1937-1946PubMed Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). In brief, total lysates from cells were boiled and cleared by centrifugation, and the supernatants were electrophoresed on 5–15% SDS-polyacrylamide gels. The fractionated products were electrotransferred onto Immobilon-P membranes (Millipore, Bedford, MA). After blocking of nonspecific binding sites with 1% bovine serum albumin, the membranes were incubated with a goat anti-Ku70 polyclonal antibody (C-19), goat anti-Ku80 polyclonal antibody (M-20), and antisera of a Japanese patient OM (which contained both anti-Ku70 and -Ku80 antibodies). The corresponding proteins were visualized using a ProtoBlot Western blot AP system (Promega, Madison, WI) according to the manufacturer's instructions (30Koike M. Ishino K. Ikuta T. Huh N. Kuroki T. Oncogene. 1995; 10: 117-122PubMed Google Scholar). Immunoprecipitation was performed as described previously (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar). The Ku products were immunoprecipitated with an anti-Ku70 monoclonal antibody (N3H10), anti-Ku80 polyclonal antibody (AHP317), or anti-Ku70/Ku80 monoclonal antibody (162) in combination with protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). They were subjected to 5–15% SDS-polyacrylamide gel electrophoresis, and the fractionated products were transferred to membranes and immunoblotted as described above. There are some lines of evidence that the control mechanism for subcellular localization of the two Ku subunits may play a key role in regulating the physiological function of Ku (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 24Fewell J. Kuff E.L. J. Cell Sci. 1996; 109: 1937-1946PubMed Google Scholar, 25Teoh G. Urashima M. Greenfield E.A. Nguyen K.A. Lee J.F. Chauhan D. Ogata A. Treon S.P. Anderson K.C. J. Clin. Invest. 1998; 101: 1379-1388Crossref PubMed Google Scholar, 26Morio T. Hanissian S.H. Bacharier L.B. Teraoka H. Nonoyama S. Seki M. Kondo J. Nakano H. Lee S.K. Geha R.S. Yata J. Immunity. 1999; 11: 339-348Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), although the mechanism is poorly understood. Previously, we identified each NLS of human Ku70 (amino acids 539–556) and human Ku80 (amino acids 561–569), suggesting that each Ku translocates to the nucleus through its own NLS (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar, 27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). To further investigate the nuclear translocation mechanism of Ku subunits, we first evaluated whether the EGFP fusion Ku proteins were produced and able to associate with the other Ku subunits in the CHO-K1 mutantxrs-6 cells, which have no Ku80 protein. Whole-cell extracts prepared from CHO-K1, xrs-6, and three xrs-6transfectants contain pEGFP-C2, pEGFP-Ku70(1–609), or pEGFP-Ku80(1–732), respectively. In the lysate of xrs-6cells transformed with pEGFP-Ku70(1–609), a signal of EGFP-Ku70 with the expected molecular weight was detected by immunoblotting using an anti-Ku70 polyclonal antibody (C-19), but not in the other lysates (Fig. 1 A). Expectedly, EGFP-Ku80 was detected by immunoblotting with an anti-Ku80 polyclonal antibody (M-20) in the only lysate of xrs-6 cells transformed with pEGFP-Ku80(1–732) (Fig. 1 B, lane 5). These results indicated that the expression of EGFP-Ku70 or -Ku80 fusion proteins is successful in xrs-6 cells (Fig. 1). As observed previously, each Ku subunit is required to stabilize each other, and the absence of the Ku80 protein in the xrs-6cells has been shown to result in loss of the Ku70 protein (14Singleton B.K. Priestley A. Steingrimsdottir H. Gell D. Blunt T. Jackson S.P. Lehmann A.R. Jeggo P.A. Mol. Cell. Biol. 1997; 17: 1264-1273Crossref PubMed Scopus (165) Google Scholar, 31Taccioli G.E. Gottlieb T.M. Blunt T. Priestley A. Demengeot J. Mizuta R. Lehmann A.R. Alt F.W. Jackson S.P. Jeggo P.A. Science. 1994; 265: 1442-1445Crossref PubMed Scopus (593) Google Scholar). In addition, reintroduction of the Ku80 gene restores the expression of the Ku70 protein in xrs-6 cells (14Singleton B.K. Priestley A. Steingrimsdottir H. Gell D. Blunt T. Jackson S.P. Lehmann A.R. Jeggo P.A. Mol. Cell. Biol. 1997; 17: 1264-1273Crossref PubMed Scopus (165) Google Scholar, 31Taccioli G.E. Gottlieb T.M. Blunt T. Priestley A. Demengeot J. Mizuta R. Lehmann A.R. Alt F.W. Jackson S.P. Jeggo P.A. Science. 1994; 265: 1442-1445Crossref PubMed Scopus (593) Google Scholar). We confirmed that hamster Ku70 was detected in extracts prepared from the pEGFP-Ku80(1–732) transfectants and CHO-K1 cells (Fig. 1 A,lanes 1 and 5), suggesting that the exogenous human Ku80 tagged with EGFP, as well as hamster Ku80, also stabilizes hamster Ku70. Expectedly, hamster Ku80 was not detected in thexrs-6 and their transfectants, whereas it was detected in the CHO-K1 cells (Fig. 1 B). The Ku70 NLS (amino acids 539–556) contains a cluster of basic amino acids (Fig. 2, A andC) (27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). When the fusion products of the Ku70 NLS fragment-substituted single amino acid (K553A), GST and GFP (GST-Ku70(539–556, K553A)-GFP), were microinjected into the cytoplasm of HeLa cells, the purified recombinant proteins of this mutant completely lost their nuclear localization activity (27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). On the other hand, the same mutation in the full-length Ku70 (EGFP-Ku70(1–609, K553A)) affected its nuclear localization activity in a transient expression assay, confirming that Ku70 NLS is important in the nuclear translocation of Ku70. However, this construct showed significant residual nuclear localization (28Koike M. Shiomi T. Koike A. Biochem. Biophys. Res. Commun. 2000; 276: 1105-1111Crossref PubMed Scopus (21) Google Scholar). On the basis of these findings, we considered the possibility that the mutation in the full-length Ku70 may not completely abolish the NLS function and/or that Ku80 may contribute to the localization. To address this possibility, we examined the subcellular localization of chimeric constructs of EGFP and human Ku proteins to which mutations were introduced by the site-directed mutagenesis technique in the xrs-6 cells, which have no Ku80 protein and markedly depressed levels of Ku70. We first confirmed that the wild-type Ku70 fusion proteins (EGFP-Ku70(1–609)) accumulated within the nucleus in thexrs-6 cells, which have undetectable Ku80 (Fig.2 E, panel a), as shown in our previous report (28Koike M. Shiomi T. Koike A. Biochem. Biophys. Res. Commun. 2000; 276: 1105-1111Crossref PubMed Scopus (21) Google Scholar). Then, the expression vectors of the NLS-less deletion mutants, pEGFP-Ku70(1–609, Y534*) or pEGFP-Ku70(1–609, Y530*), were separately transfected into xrs-6 cells. Both mutant proteins have a severely decreased nuclear localization activity, but not completely lost (Fig. 2 E, panels b and c). The expression vectors of the two Ku70 NLS mutants, pEGFP-Ku70(1–609, K553A/K556A) and pEGFP-Ku70(1–609, K542A/R543A/K553A), were separately transfected into xrs-6 cells. The EGFP-Ku70(1–609, K542A/R543A/K553A) lost its nuclear localization activity, whereas EGFP-Ku70(1–609, K553A/K556A) has a severely decreased nuclear localization activity but not completely lost (Fig.2 E, panels f and g). These results support the idea that a mutation in the full-length Ku70 (K553A) did not completely abolish the NLS function. Moreover, these results indicated that Ku70 has a functional NLS. pEGFP-Ku70(1–609, L385R) and pEGFP-Ku70(1–609, L413R) were separately transfected intoxrs-6 cells. As the normal fusion protein EGFP-Ku70(1–609) did (Fig. 2 E, panel a), the two mutant fusion proteins, EGFP-Ku70(1–609, L385R) and EGFP-Ku70(1–609, L413R), accumulated within the nuclei (Fig. 2 E, panels d ande). In contrast, when pEGFP-Ku70(1–609, L385R/K542A/R543A/K553A) was introduced, the double mutant lost its nuclear localization activity (Fig. 2 E, panel h) as the NLS mutant EGFP-Ku70(1–609, K542A/R543A/K553A) did (Fig.2 E, panel g). These results indicate that a single L385R or L413R mutation does not affect the nuclear localization activity in xrs-6 cells. Using the same methods, we examined whether mutations within the NLS site (amino acids 561–569) of Ku80 impair nuclear translocation of Ku80 (Fig. 2, B and D) (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar). The expression vectors of the four Ku80 NLS mutants, pEGFP-Ku80(1–732, P562A), pEGFP-Ku80(1–732, K568A), pEGFP-Ku80(1–732, P562A/K565A/K566A), and pEGFP-Ku80(1–732, K565A/K566A/K568A), were separately transfected intoxrs-6 cells. Two mutant proteins (EGFP-Ku80(1–732, P562A/K565A/K566A) and EGFP-Ku80(1–732, K565A/K566A/K568A)), which have triple mutations, lost their nuclear localization activity (Fig.2 F, panels d and e), indicating that the NLS was functional in Ku80. In contrast, EGFP-Ku80(1–732, K568A) was detected in both the nucleus and the cytoplasm (Fig. 2 F, panel c), suggesting that the single mutation did not completely abolish the NLS function of Ku80. Moreover, EGFP-Ku80(1–732, P562A), as well as the wild-type Ku80 fusion protein (EGFP-Ku80(1–732)), was detected in the nucleus (Figs. 2 F, panels a and b), suggesting that the single mutation did not affect the NLS function of Ku80. These results suggest that the nuclear transport of exogenous human Ku80 is not affected by dimerization with endogenous hamster Ku70, although the exogenous human Ku80 is also involved in stabilizing hamster Ku70 (Fig. 1). Next, pEGFP-Ku80(1–732, A453H/V454H) was transfected into xrs-6 cells. As the normal fusion protein EGFP-Ku80(1–732) did (Fig. 2 F, panel a), the mutant proteins accumulated within the nuclei (Figs.2 F, panel f). In contrast, when pEGFP-Ku80(1–732, A453H/V454H/K565A/K566A/K568A) was introduced, the double mutant lost its nuclear localization activity (Fig.2 F, panel g) as the NLS mutant EGFP-Ku80(1–732, K565A/K566A/K568A) did (Fig. 2 F, panel e). These results indicate that the A453H and V454H mutations do not affect the nuclear localization activity of Ku80 in xrs-6 cells. When pEGFP-Ku80(1–732, P410L) or pEGFP-Ku80(1–732, P410L/H411Y) was separately transfected into xrs-6 cells, both mutant proteins accumulated within the nuclei, suggesting that the P410L and H411Y mutations do not also affect the nuclear localization activity of Ku80 in xrs-6 cells (Fig. 2 F, panels hand i). On the other hand, when the empty vector (pEGFP) was transfected into xrs-6 cells, EGFPs were localized throughout the cell (Fig. 2 E, panel i), because they have a small molecular mass, which enables them to enter the nucleus by passive diffusion. As described above, each Ku has a functional NLS. We examined whether mutations specifically within their NLS sites impair nuclear translocation in human cells, which express Ku70 and Ku80 proteins. We first confirmed that the wild-type Ku70 fusion proteins (EGFP-Ku70(1–609)) accumulated within the nucleus (Fig.3 A, panel a) as shown in our previous report (27Koike M. Ikuta T. Miyasaka T. Shiomi T. Exp. Cell Res. 1999; 250: 401-413Crossref PubMed Scopus (50) Google Scholar). Next, the expression vectors of the two Ku70 NLS mutants, pEGFP-Ku70(1–609, K553A/K556A) and pEGFP-Ku70(1–609, K542A/R543A/K553A), were separately transfected into HeLa cells. Interestingly, both mutant proteins accumulated mainly in the nucleus in a large number of cells unlike the results in the xrs-6cells, although as expected the mutant proteins accumulated mainly in the cytoplasm in some cells (Fig. 3 A, panels dand e) (data not shown). In contrast, when the expression vectors of the NLS-less deletion mutants, pEGFP-Ku70(1–609, Y534*) or pEGFP-Ku70(1–609, Y530*), were separately transfected, the two mutant fusion proteins were localized to both the nucleus and the cytoplasm (Fig. 3 A, panels b and c), suggesting that these proteins, at least a part of them, can still translocate to the nucleus. Taken together, these results suggest that Ku70 can translocate to the nucleus-independent of NLS (amino acid 539–556), although Ku70 has a functional NLS. Using the same methods, we examined the role of NLS in the nuclear translocation of Ku80. The expression vectors of the four Ku80 NLS mutants, pEGFP-Ku80(1–732, P562A), pEGFP-Ku80(1–732, K568A), pEGFP-Ku80(1–732, P562A/K565A/K566A), and pEGFP-Ku80(1–732, K565A/K566A/K568A), were separately transfected into HeLa cells. Unexpectedly, all mutant proteins accumulated within the nucleus (Fig.3 B, panels b–e) as the wild-type Ku80 fusion proteins (EGFP-Ku80(1–732)) did (Fig. 3 B, panel a) (23Koike M. Ikuta T. Miyasaka T. Shiomi T. Oncogene. 1999; 18: 7495-7505Crossref PubMed Scopus (64) Google Scholar). These results suggest that Ku80 can translocate to the nucleus independent of its own NLS (amino acids 561–569). In general, it is known that Ku70 and Ku80 exist as a tight complex. Previously, we confirmed that most of the Ku70 and Ku80 also form heterodimers in the HeLa-S3 cells used in this study (11Koike M. Miyasaka T. Mimori T. Shiomi T. Biochem. Biophys. Res. Commun. 1998; 252: 679-685Crossref PubMed Scopus (30) Google Scholar,
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