Dimerization and Nuclear Localization of Ku Proteins*
Manabu
Koike
,
Tadahiro
Shiomi, and
Aki
Koike
From the Genome Research Group, National Institute of Radiological
Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
Received for publication, December 4, 2000, and in revised form, January 5, 2001
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ABSTRACT |
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 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.
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INTRODUCTION |
Ku is a complex composed of two protein subunits of 70 and 80 kDa,
hereafter designated as Ku70 and Ku80, respectively (1). 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 (2, 3).
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 (4, 5). 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 (4, 5). However,
this appears not to be the case for cs DNA-PK-knockout mice (6).
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 repair
in vivo and is also important in activating DNA-PK, which is
one of the main functions of Ku (7). The interacting regions of Ku70
and Ku80 have been identified by many research groups (8-12), 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 (5, 13, 14), 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 (4, 5, 15, 16). 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 (12). 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 (18-21). The subcellular
localization of Ku70 and Ku80 changes during the cell cycle (22),
and the nuclear translocation of Ku70 precedes that of Ku80 in
late telophase/early G1 cells (23). Furthermore, changes in
the subcellular localization of Ku could be controlled by various
external growth-regulating stimuli (24). 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 (25). Morio et al. (26) 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 Ku
in vivo, although the mechanism is poorly understood.
We have recently identified nuclear localization signals (NLSs) of Ku70
and Ku80 (23, 27). 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
(23, 27). We have also shown that both Ku70 and Ku80 can translocate to
the nucleus without forming a heterodimer using their own NLS (23, 28).
Moreover, the subcellular localization of Ku70 is affected by
somatostatin treatment in CV-1 cells, but that of Ku80 is not (24).
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.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Cultures--
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 (11, 27). 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 (14, 29).
Transfection of DNAs into HeLa and xrs-6 Cells--
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 (27). 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).
Plasmid Construction--
cDNA for human Ku70 and
Ku80 was derived from pEGFP-Ku70(1-609) or
pEGFP-Ku80(1-732) (23, 27). Full-length Ku70 or
Ku80 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) (23, 27). 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 (23). Following the application of the mutagenesis strategy, each
mutant was identified by DNA sequencing as described previously (23).
Immunoblotting and Immunoprecipitation--
The immunoblotting
analysis was performed as described previously (24, 27). 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 (30). Immunoprecipitation was performed as described
previously (23). 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.
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RESULTS |
The Sole NLS of Each Ku Is Functional--
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 (23-26), 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 (23, 27). 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 mutant
xrs-6 cells, which have no Ku80 protein. Whole-cell extracts
prepared from CHO-K1, xrs-6, and three xrs-6
transfectants contain pEGFP-C2, pEGFP-Ku70(1-609), or
pEGFP-Ku80(1-732), respectively. In the lysate of xrs-6
cells 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. 1A). 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. 1B, 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-6
cells has been shown to result in loss of the Ku70 protein (14, 31). In
addition, reintroduction of the Ku80 gene restores the
expression of the Ku70 protein in xrs-6 cells (14, 31). We
confirmed that hamster Ku70 was detected in extracts prepared from the
pEGFP-Ku80(1-732) transfectants and CHO-K1 cells (Fig. 1A,
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 the
xrs-6 and their transfectants, whereas it was detected in
the CHO-K1 cells (Fig. 1B).

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Fig. 1.
Immunoblot analysis of xrs-6
cells. Whole-cell extracts from CHO-K1 cells (lane
1), xrs-6 cells (lane 2), and transiently
transfected xrs-6 cells (lanes 3-5) were
examined by Western blotting using antibodies against Ku70 (C-19)
(A) and Ku80 (M-20) (B). The xrs-6
cells were transfected with pEGFP (lane 3),
pEGFP-Ku70(1-609) (lane 4), or pEGFP-Ku80(1-732) cDNA
(lane 5) as indicated. hamKu70, wild-type hamster
Ku70; hamKu80, wild-type hamster Ku80; Ku70*,
Ku70-EGFP fusion protein; Ku80*, Ku80-EGFP fusion
protein.
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The Ku70 NLS (amino acids 539-556) contains a cluster of basic amino
acids (Fig. 2, A and
C) (27). 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 (27). 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 (28). 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 the
xrs-6 cells, which have undetectable Ku80 (Fig.
2E, panel a), as shown in our previous report
(28). 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. 2E, 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.
2E, 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 into
xrs-6 cells. As the normal fusion protein EGFP-Ku70(1-609) did (Fig. 2E, panel a), the two mutant fusion
proteins, EGFP-Ku70(1-609, L385R) and EGFP-Ku70(1-609, L413R),
accumulated within the nuclei (Fig. 2E, panels d and
e). In contrast, when pEGFP-Ku70(1-609, L385R/K542A/R543A/K553A) was introduced, the double mutant lost its
nuclear localization activity (Fig. 2E, panel h)
as the NLS mutant EGFP-Ku70(1-609, K542A/R543A/K553A) did (Fig.
2E, panel g). These results indicate that a
single L385R or L413R mutation does not affect the nuclear localization
activity in xrs-6 cells.

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Fig. 2.
Nuclear entry of site-specific mutants of
Ku70 or Ku80 in xrs-6 cells. Schematic
diagrams of wild-type Ku70-EGFP (A) and Ku80-EGFP
(B) fusion proteins. Amino acid sequence of human Ku70 NLS
(539) (C) and Ku80 NLS (561) (D). The
subcellular localization of the transiently expressed site-specific
mutants of Ku70 (E) or Ku80 (F) in the
xrs-6 cells. Site-specific mutants of Ku70 or Ku80 were
constructed in pEGFP-C2 as described under "Experimental
Procedures." Each mutant was transfected and expressed in the
xrs-6 cells. For the same cells, EGFP images
(green) and PI-staining DNA images (red) are
shown alone or merged as indicated. E: panel a,
pEGFP-Ku70(1-609); panel b, pEGFP-Ku70(1-609, Y534*);
panel c, pEGFP-Ku70(1-609, Y530*); panel d,
pEGFP-Ku70(1-609, L385R); panel e, pEGFP-Ku70(1-609,
L413R); panel f, pEGFP-Ku70(1-609, K553A/K556A);
panel g, pEGFP-Ku70(1-609, K542A/R543A/K553A); panel
h, pEGFP-Ku70(1-609, L385R/K542A/R543A/K553A); panel
i, pEGFP (Control). F: panel a,
pEGFP-Ku80(1-732); panel b, pEGFP-Ku80(1-732, P562A);
panel c, pEGFP-Ku80(1-732, K568A); panel
d, pEGFP-Ku80(1-732, P562A/K565A/K566A); panel
e, pEGFP-Ku80(1-732, K565A/K566A/K568A); panel f,
pEGFP-Ku80(1-732, A453H/V454H); panel g, pEGFP-Ku80(1-732,
A453H/V454H/K565A/K566A/K568A); panel h, pEGFP-Ku80(1-732,
P410L); panel i, pEGFP-Ku80(1-732, P410L/H411Y). Amino acid
changes are designated by listing the wild-type residue, the amino acid
positions, and then the introduced mutant amino acid.
Asterisks denote the introduction of a stop codon.
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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) (23). 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
xrs-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.
2F, 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. 2F, 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. 2F, 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. 2F, panel a), the
mutant proteins accumulated within the nuclei (Figs.
2F, 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.
2F, panel g) as the NLS mutant EGFP-Ku80(1-732,
K565A/K566A/K568A) did (Fig. 2F, 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. 2F, panels h
and i). On the other hand, when the empty vector (pEGFP) was
transfected into xrs-6 cells, EGFPs were localized
throughout the cell (Fig. 2E, panel i), because
they have a small molecular mass, which enables them to enter the
nucleus by passive diffusion.
Nuclear Entry of the Ku Mutants Lacking the Functional NLS in HeLa
Cells--
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.
3A, panel a) as shown in our
previous report (27). 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-6
cells, although as expected the mutant proteins accumulated mainly in
the cytoplasm in some cells (Fig. 3A, panels d
and 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. 3A, 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.

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Fig. 3.
Subcellular localization of EGFP-tagged
wild-type Ku and their NLS mutant proteins in HeLa cells.
Site-specific mutants of Ku70 or Ku80 were constructed in pEGFP-C2 as
described under "Experimental Procedures." Each mutant was
transfected and expressed in HeLa cells. For the same cells, EGFP
images (green) and PI-stained DNA images (red)
are shown alone or merged as indicated. A: panel
a, pEGFP-Ku70(1-609); panel b, pEGFP-Ku70(1-609,
Y534*); panel c, pEGFP-Ku70(1-609, Y530*); panel
d, pEGFP-Ku70(1-609, K553A/K556A); panel e,
pEGFP-Ku70(1-609, K542A/R543A/K553A). Arrowheads indicate
mainly nuclear localization (panels d and e);
arrows indicate mainly cytoplasmic localization
(panels d and e). B: panel
a, pEGFP-Ku80(1-732); panel b, pEGFP-Ku80(1-732,
P562A); panel c, pEGFP-Ku80(1-732, K568A); panel
d, pEGFP-Ku80(1-732, P562A/K565A/K566A); panel e,
pEGFP-Ku80(1-732, K565A/K566A/K568A). Amino acids changed are
designated by listing the wild-type residue, the amino acid positions,
and then the introduced mutant amino acid. Asterisks denote
the introduction of a stop codon.
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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.
3B, panels b-e) as the wild-type Ku80 fusion proteins
(EGFP-Ku80(1-732)) did (Fig. 3B, panel a) (23).
These results suggest that Ku80 can translocate to the nucleus
independent of its own NLS (amino acids 561-569).
Nuclear Entry of the Ku Mutants Lacking the Functional NLS and the
Ability of Heterodimerization--
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 (11, 22). On the basis of this and the above findings, we speculated two possibilities that 1) both Ku subunits contain additional NLS sequences that work in HeLa cells but not in
xrs-6 cells or 2) each Ku subunit contributes to the
localization of each. Recently, we have shown that a mutation in the
EGFP-tagged human Ku70 corresponding to amino acid 385 or 413 significantly impairs its ability to interact with Ku80, whereas
mutations in the EGFP-tagged human Ku80 corresponding to amino acids
453 and 454 significantly impair its ability to interact with Ku70 (23, 28). We first examined whether the same mutations in the Ku70- or
Ku80-NLS-dysfunctional mutant abrogated their interaction with each
other. The expression vector, pEGFP-Ku70(1-609, K542A/R543A/K553A) or
pEGFP-Ku70(1-609, L385R/K542A/R543A/K553A), was separately transfected
into HeLa cells. We determined the amount of Ku70·Ku80 complexes in lysates of the transformed HeLa cells by reciprocal immunoprecipitation using anti-Ku70, -Ku80, and -Ku70/Ku80 antibodies and then performed by Western blotting (Fig.
4). As reported previously (28), when
extracts of pEGFP-Ku70(1-609) transformants were immunoprecipitated,
wild-type Ku70, Ku80, and EGFP-Ku70(1-609) fusion proteins
immunoprecipitated with both the anti-Ku70 antibody and the anti-Ku80
antibody (Fig. 4, lane 1) (data not shown). When extracts of
pEGFP-Ku70(1-609, K542A/R543A/K553A) transformants were
immunoprecipitated, wild-type Ku70, Ku80, and EGFP-Ku70(1-609, K542A/R543A/K553A) fusion proteins immunoprecipitated with the anti-Ku80 antibody (Fig. 4, lane 4). In contrast, the
EGFP-Ku70(1-609, L385R/K542A/R543A/K553A) fusion proteins were hardly
detectable by immunoprecipitation with the anti-Ku80 antibody (Fig. 4,
lane 7). As shown in Fig. 4 (lanes 3 and
6), when the extracts were immunoprecipitated with the
anti-Ku70 antibody, wild-type Ku70, Ku80, and EGFP-Ku70(1-609,
K542A/R543A/K553A) or EGFP-Ku70(1-609, L385R/K542A/R543A/K553A) fusion
proteins coprecipitated. In addition, the EGFP-Ku70(1-609,
K542A/R543A/K553A) fusion proteins, as well as the EGFP-Ku70(1-609)
fusion proteins, immunoprecipitated with monoclonal antibody 162 (directed against the Ku70/Ku80 dimer and not against free Ku70 or
Ku80) (Fig. 4, lanes 2 and 5). These results
indicate that mutations of Ku70 at amino acids 385, 542, 543, and 553, but not at amino acids 542, 543, and 553, significantly impaired the
interaction of Ku70 with Ku80. When extracts of pEGFP-Ku80(1-732, K565A/K566A/K568A) transformants were immunoprecipitated, wild-type Ku70, Ku80, and EGFP-Ku80(1-732, K565A/K566A/K568A) fusion proteins immunoprecipitated with the anti-Ku70 antibody (Fig. 4, lane
8). In contrast, the EGFP-Ku80(1-732,
A453H/V454H/K565A/K566A/K568A) fusion proteins were hardly detected by
immunoprecipitation with the anti-Ku70 antibody (Fig. 4, lane
10). As shown in Fig. 4 (lanes 9 and 11),
when the extracts were immunoprecipitated with the anti-Ku80 antibody,
wild-type Ku70, Ku80, and EGFP-Ku80(1-732, K565A/K566A/K568A) or
EGFP-Ku80(1-732, A453H/V454H/K565A/K566A/K568A) fusion proteins
coprecipitated. These results indicate that mutations of Ku80 at amino
acids 453, 454, 565, 566, and 568, but not at amino acids 565, 566, and
568, significantly impaired the interaction of Ku80 with Ku70.
Recently, Singleton et al. (29) reported that a mutation in
hamster Ku80 corresponding to amino acid 410 abolished its ability to
interact with Ku70 in the reticulocyte lysate in vitro
translation system. We examined whether the same and other mutations in
the EGFP-tagged human Ku80 abrogated its interaction with human Ku70 in
human cells. When extracts of pEGFP-Ku80(1-732, P410L) or
pEGFP-Ku80(1-732, P410L/H411Y) transformants were immunoprecipitated, wild-type Ku70, Ku80, and EGFP-Ku80(1-732, P410L) or EGFP-Ku80(1-732, P410L/H411Y) fusion proteins immunoprecipitated with the anti-Ku80 antibody (Fig. 4, lanes 13 and 15). In contrast,
the two fusion proteins were hardly detected by immunoprecipitation
with the anti-Ku70 antibody (Fig. 4, lanes 12 and
14). These results indicate that the mutations of human Ku80
significantly impaired its interaction with Ku70. On the other hand,
when the empty vector (pEGFP) was transfected, only the wild-type Ku
proteins immunoprecipitated as expected (Fig. 4, lanes 16 and 17).

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Fig. 4.
Interaction of wild-type Ku and EGFP-tagged
Ku mutant proteins in HeLa cells. Immunoblotting anti-Ku antiserum
(Japanese patient OM) from immunoprecipitation with antibodies
against Ku70 (N3H10) (lanes 1, 3, 6,
8, 10, 12, 14, and
16), Ku80 (AHP317) (lanes 4, 7,
9, 11, 13, 15, and
17), and Ku70/Ku80 (162) (lanes 2 and
5). Each Ku protein is indicated (Ku70, wild-type
Ku70; Ku80, wild-type Ku80; Ku70*, wild or mutant
Ku70-EGFP fusion protein; Ku80*, mutant Ku80-EGFP fusion
protein). Lanes 1 and 2,
pEGFP-Ku70(1-609)-transfected cells; lanes 3-5,
pEGFP-Ku70(1-609, K542A/R543A/K553A)-transfected cells; lanes
6 and 7, pEGFP-Ku70(1-609,
L385R/K542A/R543A/K553A)-transfected cells; lanes 8 and
9, pEGFP-Ku80(1-732, K565A/K566A/K568A)-transfected cells;
lanes 10 and 11, pEGFP-Ku80(1-732,
A453H/V454H/K565A/K566A/K568A)-transfected cells; lanes 12 and 13, pEGFP-Ku80(1-732, P410L)-transfected cells;
lanes 14 and 15, pEGFP-Ku80(1-732,
P410L/H411Y)-transfected cells; lanes 16 and 17,
pEGFP (Control)-transfected cells.
|
|
We examined whether the Ku70 double mutant lacking both the functional
NLS and the ability of heterodimerization is localized in the nucleus.
When transfected into HeLa cells, the double mutant EGFP-Ku70(1-609,
L385R/K542A/R543A/K553A) was detected in the cytoplasm (Fig.
5b). In contrast, the
EGFP-Ku70(1-609, L385R) accumulated within the nucleus (Fig.
5a) as shown in our previous report (28). In addition,
EGFP-Ku70(1-609, K542A/R543A/K553A) accumulated mainly in the nucleus
in a large number of cells (Fig. 3A, panel e)
(data not shown). These results suggest that the EGFP-Ku70(1-609,
K542A/R543A/K553A), at least a part of it, can translocate to the
nucleus through the heterodimerization with Ku80 in HeLa cells.

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Fig. 5.
Analysis of the correlation between Ku70-Ku80
heterodimerization and their nuclear entry in HeLa cells.
The subcellular localization of the transiently expressed fusion
proteins was examined in HeLa cells as described in the legend to Fig.
3. For the same cells, EGFP images (green) and PI-stained
DNA images (red) are shown alone or merged as indicated.
a, pEGFP-Ku70(1-609, L385R); b,
pEGFP-Ku70(1-609, L385R/K542A/R543A/K553A); c,
pEGFP-Ku80(1-732, A453H/V454H); d, pEGFP-Ku80(1-732,
A453H/V454H/K565A/K566A/K568A); e, pEGFP-Ku80(1-732,
P410L); f, pEGFP-Ku80(1-732, P410L/H411Y); g,
pEGFP (Control). Amino acids changed are designated by
listing the wild-type residue, the amino acid positions, and then the
introduced mutant amino acid.
|
|
We next examined whether the Ku80 double mutant lacking both the
functional NLS and the ability of heterodimerization is localized in
the nucleus. EGFP-Ku80(1-732, A453H/V454H/K565A/K566A/K568A), when
transfected into HeLa cells, was detected in the cytoplasm (Fig.
5d), whereas the EGFP-Ku80(1-732, K565A/K566A/K568A) and EGFP-Ku80(1-732, A453H/V454H) accumulated within the nucleus (Figs. 3B, panel e, and 5c) (23), suggesting that Ku80
can translocate to the nucleus through the heterodimerization with Ku70
independent of its NLS. On the other hand, when pEGFP was transfected
into HeLa cells, EGFPs were distributed throughout the cell (Fig.
5g) as described previously (27).
Previously, we reported that a Ku80 mutant lacking the ability of
heterodimerization is localized in the nucleus, suggesting that Ku80
can translocate to the nucleus without binding with Ku70 (23). We
confirmed whether other Ku80 mutants lacking the ability of
heterodimerization are localized in the nucleus (Fig. 4, lanes
12-15, and Fig. 5). When transfected into HeLa cells, the two
mutants, EGFP-Ku80(1-732, P410L) and EGFP-Ku80(1-732, P410L/H411Y),
were detected in the nucleus (Fig. 5, e and f), supporting the idea that Ku80 can translocate to the nucleus without binding with Ku70.
The Role of Heterodimerization in Nuclear Entry--
As mentioned
above, our findings suggest the possibility that Ku70 and Ku80 can
translocate to the nucleus not only through their own NLS but also
through heterodimerization. To further confirm this, the nuclear
distribution of Ku70 and Ku80 was compared by coexpression of the two
proteins. A yellow variant GFP (EYFP)-Ku80 and a cyan variant GFP
(ECFP)-Ku70 were transiently expressed in xrs-6 cells.
Fluorescence images of the GFP color variants were captured separately
using appropriate filter sets (Fig. 6). First, the ECFP-tagged wild-type Ku70 (ECFP-Ku70(1-609)), the EYFP-tagged wild-type Ku80 (EYFP-Ku80(1-732)), or the EYFP-tagged Ku80
mutant (EYFP-Ku80(1-732, A453H/V454H)) transfected alone in the
xrs-6 cells. As each EGFP-tagged fusion protein did in the
xrs-6 cells (Fig. 2: E, panel a; F, panel
a; and Fig. 5c), each variant GFP fusion protein
accumulated within the nucleus (data not shown). Next, the
NLS-dysfunctional mutant (ECFP-Ku70(1-609, K542A/R543A/K553A) or
EYFP-Ku80(1-732, K565A/K566A/K568A)) or double mutant lacking both
nuclear targeting and dimerization functions (ECFP-Ku70(1-609,
L385R/K542A/R543A/K553A) or EYFP-Ku80(1-732, A453H/V454H/K565A/K566A/K568A)) transfected alone in the
xrs-6 cells. As each EGFP-tagged fusion protein did (Fig. 2:
E, panel g; E, panel h; F, panels e
and g), each variant GFP fusion protein lost its nuclear
localization activity (data not shown). These results indicate that
their distribution was not due to the tag. When EYFP-Ku80(1-732) and
ECFP-Ku70(1-609) were coexpressed, the ECFP-Ku70(1-609) accumulated
within the nucleus (Fig. 6, a and a'). On the other hand,
when EYFP-Ku80(1-732) and the NLS-dysfunctional Ku70 mutant were
coexpressed, the ECFP-Ku70(1-609, K542A/R543A/K553A) accumulated
within the nucleus (Fig. 6, b and b'), supporting the idea that Ku70 can traslocate to the nucleus independent of its own
NLS. In addition, when the EYFP-Ku80(1-732) and the double mutant of
Ku70 were coexpressed, ECFP-Ku70(1-609, L385R/K542A/R543A/K553A) did
not accumulate within the nucleus (Fig. 6, c and
c'), indicating that Ku70 can translocate to the nucleus
through the heterodimerization with Ku80. We further examined the role
of Ku80 NLS in the nuclear translocation of Ku70. When both
NLS-dysfunctional mutants (ECFP-Ku70(1-609, K542A/R543A/K553A) and
EYFP-Ku80(1-732, K565A/K566A/K568A)) were coexpressed,
ECFP-Ku70(1-609, K542A/R543A/K553A) did not accumulate within the
nucleus (Fig. 6, d and d'), suggesting that the
nuclear translocation of Ku70 through the heterodimerization with Ku80 is dependent on Ku80 NLS.

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Fig. 6.
Nuclear entry of the NLS-disrupted Ku70 or
Ku80 mutant in xrs-6 cells. Wild-type and
site-specific mutants of Ku70 or Ku80 were constructed in pECFP-C1 or
pEYFP-C1 as described under "Experimental Procedures." The
xrs-6 cells were transiently cotransfected with
pEYFP-Ku80(1-732) and pECFP-Ku70(1-609) (a and
a'), pEYFP-Ku80(1-732) and pECFP-Ku70(1-609,
K542A/R543A/K553A) (b and b'), pEYFP-Ku80(1-732)
and pECFP-Ku70(1-609, L385R/K542A/R543A/K553A) (c and
c'), pEYFP-Ku80(1-732, K565A/K566A/K568A) and
pECFP-Ku70(1-609, K542A/R543A/K553A) (d and d'),
pEYFP-Ku80(1-732, K565A/K566A/K568A) and pECFP-Ku70(1-609)
(e and e'), pEYFP-Ku80(1-732,
A453H/V454H/K565A/K566A/K568A) and pECFP-Ku70(1-609,
L385R/K542A/R543A/K553A) (f and f'), or pEYFP
(control) and pECFP (control) (g and
g'). The cotransfected cells were examined by fluorescence
microscopy and images captured using filters specific for EYFP
(a-g), ECFP (a'-g'), or PI (g"). For
the same cells, EYFP images (green), ECFP images
(cyan), and PI-stained DNA images (red) are shown
as indicated.
|
|
We next examined the role of Ku70 in the nuclear translocation of Ku80.
When ECFP-Ku70(1-609) and the NLS-dysfunctional Ku80 mutant were
coexpressed, the EYFP-Ku80(1-732, K565A/K566A/K568A) accumulated
within the nucleus (Fig. 6, e and e'), supporting the idea that Ku80 can traslocate to the nucleus independent of its own
NLS. Moreover, when both NLS-dysfunctional mutants
(ECFP-Ku70(1-609, K542A/R543A/K553A) and EYFP-Ku80(1-732,
K565A/K566A/K568A)) were coexpressed, EYFP-Ku80(1-732,
K565A/K566A/K568A) did not accumulate within the nucleus (Fig. 6,
panels d and d'), suggesting that the nuclear
translocation of Ku80 through the heterodimerization with Ku70 is
dependent on Ku70 NLS. On the other hand, when the Ku70 and Ku80 double
mutants were coexpressed, both proteins localized in the cytoplasm as
expected (Fig. 6, f and f'). In control
experiments, when pEYFP and pECFP were cotransfected into
xrs-6 cells, both GFP-variant proteins localized throughout
the cell (Fig. 6, g, g', and g"). Taken together,
these results suggest that the heterodimerization between Ku70 and Ku80
plays an important role in the nuclear translocation of these Ku subunits.
 |
DISCUSSION |
In general, it is known that nuclear proteins containing an
intrinsic NLS enter the nucleus associated with NLS receptors (e.g. importin
/
) through their own NLSs (32). We had
previously reported that Ku70 and Ku80 have an intrinsic NLS and that
the NLS receptor can recognize the NLSs of Ku70 and Ku80 (23, 27). In
this study, we have further examined the molecular basis of the
regulation of Ku70 and Ku80 subcellular localization using site-directed mutagenesis. In agreement with our previous observations, we have found that each Ku subunit can translocate to the nucleus through its own NLS (23, 28). Our data have shown that each Ku subunit
can also translocate to the nucleus independent of its own NLS and that
this translocation is dependent on its interaction with the other subunit.
Ku has been generally considered to always form and function as a
heterodimer. In studies using knockout mice, however, inactivation of
Ku70 resulted in some phenotype distinct from that of Ku80-knockout mice (4, 5, 15, 16), suggesting the possibility that Ku70 and Ku80 have
unique functions that are independent of each other. Recently, we
identified the NLSs of human Ku70 (amino acids 539-556) and Ku80
(amino acids 561-569) (23, 27). In this study, the EGFP-Ku80 fusion
protein accumulated within the nuclei of xrs-6 cells (Fig.
2F, panel a). Furthermore, the NLS-dysfunctional mutant
(EGFP-Ku80(1-732, K565A/K566A/K568A) or EGFP-Ku80(1-732, P562A/K565A/K566A)) lost its nuclear localization activity in the
xrs-6 cells (Fig. 2F, panels d and e),
indicating that Ku80 contains a classical NLS and that this NLS is
functional. In this and previous reports, we also showed that the
EGFP-Ku70 fusion protein accumulated within the nuclei of
xrs-6 cells (Fig. 2E, panel a) (28). On the other
hand, the NLS-dysfunctional mutant, EGFP-Ku70(1-609,
K542A/R543A/K553A), lost its nuclear localization activity in the
xrs-6 cells (Fig. 2E, panel g). These
findings support the idea that both Ku70 and Ku80 can translocate from the cytoplasm to the nucleus without forming a heterodimer using their
own NLS. Irradiation resulted in an up-regulation of the cellular level
of Ku70, but not that of Ku80, and Ku70 accumulated within the
nucleus (33). Moreover, the subcellular localization of Ku70 was
affected by somatostatin treatment in CV-1 cells, but that of Ku80 was
not (24). In addition, the nuclear translocation of Ku70 preceded that
of Ku80 at the late telophase/early G1 phase during the
cell cycle (23). Each Ku subunit may have a functional NLS to perform
unique functions, which are independent of each other, although further
studies will be necessary to confirm this. As described above, nuclear
translocation of Ku70 and Ku80 can be independently regulated in
vivo. The structures of NLSs of both Ku protein subunits are quite
different, and NLSs of Ku80 and Ku70 are of the single-basic type and
the variant bipartite-basic type, respectively (23, 27). Thus, we
speculate that the nuclear translocation of Ku proteins is controlled
at least in part at the NLS recognition step and that this is regulated
by NLS receptors with various specificities in vivo.
We have also found that the heterodimerization between Ku70 and Ku80
plays an important role in their nuclear translocation. When two
NLS-dysfunctional Ku80 mutants were transfected into HeLa cells, both
EGFP-Ku80(P562A/K565A/K566A) and EGFP-Ku80(K565A/K566A/K568A) translocated to the nucleus (Fig. 3B, panels d and
e). However, EGFP-Ku80(A453H/V454H/K565A/K566A/K568A), which
was a double mutant lacking both nuclear targeting and dimerization
functions, did not translocate to the nucleus (Fig. 5d).
These results suggest that endogenous Ku70 transports the
NLS-dysfunctional Ku80 mutants into the nucleus in HeLa cells. In
cotransfection analysis using xrs-6 cells, the Ku80 mutant
lacking only the nuclear targeting function was localized in the
nucleus by the exogenous wild-type Ku70 (Fig. 6, e and
e'). In addition, when the same Ku80 mutant and
NLS-dysfunctional Ku70 mutant (ECFP-Ku70(1-609, K542A/R543A/K553A) were coexpressed, EYFP-Ku80(1-732, K565A/K566A/K568A) did not accumulate within the nucleus (Fig. 6, d and d').
These results indicate that Ku70 transport Ku80 into the nucleus via
its own NLS in xrs-6 cells. In this manner, one role of Ku70
may be to regulate the nuclear translocation of Ku80. On the other
hand, the NLS-dysfunctional Ku70 mutant, but not the double mutant
lacking both nuclear targeting and dimerization functions, was
localized into the nucleus due to the presence of exogenous wild-type
Ku80 (Fig. 6, b, b', c, and c'). When the same
Ku70 mutant and NLS-dysfunctional Ku80 mutant (EYFP-Ku80(1-732,
K565A/K566A/K568A) were coexpressed, ECFP-Ku70(1-609,
K542A/R543A/K553A) did not accumulate within the nucleus (Fig. 6,
d and d'). These results indicate that Ku80 can
transport Ku70 into the nucleus via its own NLS in xrs-6
cells. However, the NLS-dysfunctional Ku70 mutant, which can still
interact with Ku80, did not completely localize to the nucleus despite the presence of endogenous Ku80 in HeLa cells (Fig. 3A, panels d and e). The half-life of the Ku80 monomeric form is
less than 2 h (34), whereas that of the EGFP-tagged protein was
more than 24 h (17). Although the discrepancy between the
results in HeLa and in xrs-6 cells remains unclear, this may
be due to the differences in half-life between endogenous Ku80 in HeLa
cells and exogenous EGFP-tagged Ku80 in xrs-6 cells.
Alternatively, in HeLa cells, the Ku70 localization may be dependent
not only on the nuclear import mechanism but also on the nuclear export
mechanism. Further studies will be necessary to confirm that Ku80
transport Ku70 into the nucleus in vivo.
Ku70 and Ku80 play an important role in DNA DSB repair and V(D)J
recombination in vivo (4, 5, 15, 16). Heterodimerization between Ku70 and Ku80 is essential for Ku-dependent DNA DSB
repair in vivo (7), but the role of this interaction in Ku
functions remains unknown. It is also reported that the
heterodimerization is required for the stabilization of each Ku subunit
(5, 13, 14). On the other hand, Morio et al. (26) reported
that the DNA-PK activity of human B cells is, at least in part,
regulated by the nuclear translocation of Ku. Recently, we have
generated cell lines stably expressing the wild-type Ku80 (EGFP-Ku80)
or NLS-dysfunctional Ku80 mutant tagged with EGFP
(EGFP-Ku80(K565A/K566A/K568A)). We have found that the tagged wild-type
Ku80 protein can complement a deficiency of the DNA DSB repair of
xrs-6 cells (data not shown). In contrast, the tagged
NLS-dysfunctional Ku80 mutant protein cannot complement a deficiency of
the DNA DSB repair of xrs-6 cells, although the Ku80 mutant
protein is stabilized by tagged with EGFP (data not shown).
In conclusion, we have shown a novel role of the heterodimerization of
Ku70 and Ku80. Ku70 and Ku80 appear to have multiple functions as a
monomeric form and a heterodimeric form. 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 hetrerodimerization to perform the same functions dependent on each
other. The control mechanism for nuclear localization of Ku70 and Ku80
appear to play, 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.
 |
ACKNOWLEDGEMENT |
We thank Dr. T. Mimori for providing us with
human anti-Ku serum (Japanese patient OM).
 |
FOOTNOTES |
*
This work was supported in part by grants from the Science
and Technology Agency and from the Ministry of Education, Science, Sports, and Culture, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Genome Research Group,
National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku,
Chiba 263-8555, Japan. Tel.: 81-43-251-2111 (ext. 333); Fax:
81-43-251-9818; E-mail: m_koike@nirs.go.jp.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M010902200
 |
ABBREVIATIONS |
The abbreviations used are:
DNA-PK, DNA-dependent protein kinase;
DSB, DNA double-strand break;
NLS, nuclear localization signal;
GFP, green fluorescent protein;
PI, propidium iodide;
EYFP, yellow variant GFP;
ECFP, cyan variant GFP;
EGFP, enhanced GFP;
GST, glutathione
S-transferase.
 |
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