From the Departments of Medicine, ¶ Cellular and
Molecular Medicine, and
Biochemistry, Microbiology and
Immunology, The Loeb Health Research Institute at the Ottawa Hospital,
University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada
Received for publication, January 26, 2001, and in revised form, February 12, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Ku antigen (70- and 80-kDa
subunits) is a regulatory subunit of DNA-dependent
protein kinase (DNA-PK) that promotes the recruitment of the catalytic
subunit of DNA-PK (DNA-PKcs) to DNA ends and to
specific DNA sequences from which the kinase is activated. Ku and
DNA-PKcs plays essential roles in double-stranded
DNA break repair and V(D)J recombination and have been implicated in
the regulation of specific gene transcription. In a yeast two-hybrid screen of a Jurkat T cell cDNA library, we have identified a
specific interaction between the 70-kDa subunit of Ku heterodimer and
the homeodomain of HOXC4, a homeodomain protein expressed in the
hematopoietic system. Unexpectedly, a similar interaction with Ku was
observed for several additional homeodomain proteins including octamer transcription factors 1 and 2 and Dlx2, suggesting that specific binding to Ku may be a property shared by many homeodomain proteins. Ku-homeodomain binding was mediated through the extreme C terminus of
Ku70 and was abrogated by amino acid substitutions at
Lys595/Lys596. Ku binding allowed the
recruitment of the homeodomain to DNA ends and dramatically enhanced
the phosphorylation of homeodomain-containing proteins by DNA-PK. These
results suggest that Ku functions as a substrate docking protein for
signaling by DNA-PK to homeodomain proteins from DNA ends.
The Ku antigen
(Ku70/Ku80)1 plays important
roles in multiple nuclear processes including DNA repair, V(D)J
recombination, telomere maintenance, and the regulation of specific
gene transcription (1-6). Ku is a prodigious DNA-binding protein that
recognizes DNA ends, structural transitions in DNA, and specific DNA
sequences (4, 5). Ku-deficient mice are proportional dwarfs that are immunodeficient and sensitive to DNA-damaging agents (7-10). They also
suffer from genomic instability, undergo chromosome rearrangements, and
develop T cell lymphoma (7, 11-14), while fibroblasts from Ku Ku is an integral regulatory component of DNA-dependent
protein kinase (DNA-PK), and many of its actions in the nucleus,
including DNA repair, recombination, and transcriptional regulation,
occur in concert with the catalytic subunit of DNA-PK
(DNA-PKcs) (4, 16). DNA-PKcs is one of a family
of large phosphatidylinositol 3-kinase-related nuclear kinases that
also includes the ataxia telangiectasia gene product, ATM, and the
TRAPP/PAF400 transcriptional cofactor (4, 17, 18).
DNA-PKcs binds nonspecifically to DNA and is activated from
the ends of double-stranded DNA (19, 20). The interaction of
DNA-PKcs with Ku promotes recruitment of the kinase to DNA ends, thereby enhancing its activation (4, 21). Ku also promotes the
recruitment and activation of DNA-PKcs from specific DNA
sequences (3, 4, 22). However, even when associated with Ku,
DNA-PKcs is an inefficient kinase with a
km for peptide substrates in excess of 200 µM (21, 23, 24). DNA-PK also displays only a modest
specificity for substrates in vitro, with a preference for
(S/T)Q motifs. How these in vitro properties of
DNA-PK translate to what is presumed to be the specific and targeted
phosphorylation of substrates in vivo, remains to be elucidated.
At least two mechanisms offer the potential for increasing the
specificity and efficiency of substrate phosphorylation by DNA-PK.
First, since the affinity of Ku/DNA-PKcs for DNA ends and
sequences is several orders of magnitude higher than the
km of DNA-PK for substrates, the colocalization of
substrates with DNA-PK in cis on DNA can dramatically
enhance their phosphorylation (3, 21, 22, 25). In one specific example,
the glucocorticoid receptor is efficiently phosphorylated by
DNA-PK in the presence of DNA molecules containing binding sites for
both the receptor and Ku/DNA-PKcs. However, when the
receptor binding sites are transferred to a second, covalently closed
circular DNA molecule to which Ku and DNA-PKcs are
not attracted, glucocorticoid receptor phosphorylation is strongly
decreased (3, 22). Phosphorylation was also abrogated by introduction
of a point mutation that disrupts the binding of glucocorticoid
receptor to DNA (22). The consequence of this DNA sequence-specific
phosphorylation is the specific regulation of promoters containing
sequences from which the DNA-PK is activated (22, 26).
A second possibility is that substrates may be recruited to
Ku/DNA-PKcs through specific protein-protein interactions
with components of the DNA-PK complex. Recently, several proteins have been identified as interacting with Ku antigen. For the most
part the functional consequences of these interactions remain to be established. At least some of these Ku binding factors
(e.g. hGCN5, the progesterone receptor, XRCC4, and c-Abl)
are phosphorylated by DNA-PK in vitro (27-32). However, the
contribution of Ku binding to their phosphorylation has not been evaluated.
In the present study, we have identified a specific
protein-protein interaction between the C terminus of the Ku antigen
and the homeodomains of a series of homeodomain proteins that occurs in
solution and leads to the recruitment of homeodomain proteins to
DNA ends. This interaction contributed to a greater than 50-fold enhancement of the phosphorylation of homeodomain-containing proteins by DNA-PK. These results demonstrate that Ku serves a molecular scaffold for the recruitment of homeodomain proteins to DNA ends for phosphorylation by DNA-PK and suggests that DNA-PK-mediated phosphorylation regulates the function of at least some homeodomain proteins in response to DNA damage.
DNA Constructs--
Full-length human Ku70 cDNA and Ku80
cDNA were cloned in vector pAS1-Tet (CLONTECH).
The two-hybrid Ku70 mutant clones Ku70mt595-600-pAS1 and
Ku70mtK595,6N-pAS1 were obtained by cloning the mutated Ku70 cDNA
generated by PCR into pAS1. The Jurkat T-cell cDNA library in
pGAD-10 was purchased from CLONTECH. Plasmid
HOXC4-pACT2 was generated by cloning HOXC4 cDNA (nt 600-1421)
isolated from a Yeast Two-hybrid Screening--
Plasmids were transformed
as described (34) by the lithium acetate method in yeast strain Y190
(MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3,
112, gal4d, gal8d, cyh2, LYS2::GAL1UAS,
-HIS3TATA-HIS3,
URA3::GAL1UAS-GAL1TATA-lacZ) and selected on SD medium lacking Trp, Leu, and His and supplemented with
20 mM 3-aminotriazol (Sigma) to limit spurious activity
from the His promoter. After 10-12 days of incubation at 30 °C,
transformants were tested for Cloning of HOXC4 Full-length cDNA--
Human HOXC4
cDNA was obtained by screening a Protein Expression and in Vitro Translations--
Glutathione
S-transferase (GST) fusion proteins were expressed according
to standard protocols (Amersham Pharmacia Biotech). For EMSA purposes,
GST-Oct2 POU was eluted from the GST beads. Ku was expressed from
recombinant baculovirus-infected insect cells and purified as described
(26). All in vitro translated proteins were obtained by
using the TnT rabbit reticulocyte lysate kit (Promega) according to the
manufacturer's conditions. In vitro translated deletion
mutants were obtained by restricting the plasmids by the appropriate
restriction enzyme, or mutant cDNAs were produced by PCR with
oligonucleotides containing a T7 promoter fused to the 5'-end of the
forward primer. For in vitro translation of cDNAs cloned
in pGAD-10 or pACTII, inserts were amplified by PCR using an
oligonucleotide complementary to aa 116-124 of the Gal4 activation
domain fused to the T7 promoter and an oligonucleotide complementary to
the 3'-end of the multiple cloning site of both vectors. To generate
C-terminal Ku70 truncation or point mutants from the Ku70-pACT2
plasmid, the T7-Met Gal4 AD primer described above was used as forward
primer in combination with Ku70-specific primers.
Co-immunoprecipitations and Binding Assays--
Ku used in
binding assays was immunoprecipitated from Jurkat T cell whole cell
extracts prepared essentially as described (35). Immunoprecipitation of
Ku was performed in WCE buffer (150 mM NaCl, 50 mM Hepes (pH 7.4), 1 mM EDTA, 10% glycerol,
0.5% Nonidet P-40, 0.5 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride) with 0.5 µg/ml of Ku70
antibody (clone N3H10; NeoMarkers) followed by incubation with protein
A-Sepharose. To release Ku80 from the complex, beads were incubated in
100 µl of BC0 buffer (20 mM Tris (pH 8.0), 0.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin)
containing 1 M KCl and 1% sodium deoxycholate for 20 min
and washed extensively in binding buffer (25 mM Hepes (pH
7.9), 60 mM KCl, 0.5 mM EDTA, 0.2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, 0.1% Nonidet P-40, 12% glycerol). Binding of
35S-labeled in vitro translated proteins to Ku
dimer or Ku70 immobilized on beads was performed in binding buffer for
2 h at 4 °C. Complexes were washed three times in WCE buffer
with 0.1% Nonidet P-40, resuspended in SDS sample buffer, and resolved
on SDS-polyacrylamide gel electrophoresis.
For co-immunoprecipitation experiments, 35S-labeled
in vitro translated proteins (typically 0.2-2 µl of TnT
reactions) were added to 100 µg of Jurkat whole cell extract diluted
to 60 mM NaCl, and 0.1% Nonidet P-40 and extracts were
incubated at 4 °C for 2 h before the addition of the Ku70
antibody. Samples were then processed as above. To test Ku binding to
the homeodomain, 0.5-1 µg of GST-Oct-1 POU or GST-HOXC4 HD (as
indicated in the figure legends) was mixed with 35S-labeled
in vitro translated Ku in binding buffer and processed as above.
HeLa cell nuclear extracts were prepared essentially as described (36).
For co-immunoprecipitation, about 1 mg of nuclear extract was diluted
down to 100 mM KCl with binding buffer and incubated with
either Oct-1 antibody (YL15; Upstate Biotechnology, Inc., Lake Placid,
NY) or nonspecific antibody (anti-glucocorticoid receptor (BuGR2)).
Proteins were transferred on membrane and hybridized successively with
antibodies to Ku70 (N3H10; NeoMarkers), Ku80 (clone 111; NeoMarkers)
and Oct-1 (C-21; Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
EMSA--
EMSA was performed as described (26) using
32P-labeled oligonucleotides corresponding to the H2B Oct-2
binding site (5'-AGCTTGCTTATGCAAATAAGGTGGATC-3') and a nonspecific
oligonucleotide (39-mer NS (26)). The preparation of the recombinant Ku
and GST-Oct2 POU peptide employed in these assays was as previously
described (26, 33). Antibodies 111 (NeoMarkers) and YL123 (Upstate
Biotechnology) were added at the beginning of the incubations.
DNA-protein complexes were resolved on 4% polyacrylamide gels in 0.5×
Tris borate buffer.
DNA-PK Phosphorylation Assay--
Phosphorylation of recombinant
proteins by purified DNA-PK (Promega) was performed essentially as
previously described (26). 0.05-2 µg of protein substrate was
incubated for 20 min at 30 °C in kinase buffer (50 mM
HEPES, pH 7.5, 100 mM KCl, 10 mM
MgCl2, 0.2 mM Na2EGTA) in the
presence of 20 ng of HaeIII-restricted calf thymus DNA, 5 µCi of [ Ku Antigen Interacts Physically with Several Homeodomain Proteins
through Determinants within the 70-kDa Subunit--
To identify
proteins that interact with the Ku70, we screened a Jurkat T cell
cDNA library using full-length Ku70 fused to the Gal4 DNA-binding
domain as the bait for interacting peptides. Expression of the
GalDBDKu70 construct alone or together with Gal4 activation
domain was able to activate transcription sufficiently to induce
detectable
Approximately 300 strongly positive clones were isolated in the
original library screen. 80% of these induced
Expression of full-length HOXC4 with the Gal activation domain did not
induce appreciable
To begin to probe the interaction between Ku70 and the homeodomain of
HOXC4 in greater detail, we examined the specific binding between Ku70
and the homeodomain of HOXC4 in an immunoprecipitation binding assay
(Fig. 2A). For these assays,
Ku immunoprecipitates were prepared from whole cell extracts from
Jurkat T cells with a Ku70-specific antibody, N3H10 (lanes 1 and 2). Ku80 was subsequently stripped from Ku70 monomer by
extraction of the immunoprecipitate with a buffer including 1%
deoxycholate and 1 M KCl (lane 3). The HOXC4
homeodomain peptide from clone 18-27 produced by in vitro
translation interacted efficiently with the immunoprecipitated Ku70
monomer (lane 5) and also with the Ku heterodimer
(lane 6), illustrating that Ku70 binding was not impeded by
Ku dimerization. By contrast, the in vitro translated HOXC4
peptide did not interact with precipitates prepared from Jurkat cell
extracts by incubation with protein A-Sepharose alone (lane
7).
Homeodomain proteins comprise a large superfamily of transcription
factors that regulate multiple aspects of development and are also key
regulators of cell homeostasis (37, 38). The 60-amino acid homeodomain
DNA binding motif exhibits an extremely high degree of conservation and
serves as a platform for many protein-protein interactions that are
crucial to homeodomain protein function (39, 40). Since the Ku70-HOXC4
interaction mapped to the HOXC4 homeodomain, we sought to determine
whether Ku70 could interact with other homeodomain proteins.
Strikingly, immunoprecipitated Ku70 was bound specifically by all four
of the additional homeodomain proteins tested (Fig. 2B):
HOXD4 (41), a close orthologue to HOXC4 (lanes 1-3);
zebrafish Dlx2 (42), a more distant family member (lanes
3-6); the POU homeodomain factor Oct-2 (43) (lanes
7-9); and the divergent homeodomain factor Oct-60 (44)
(lanes 10-12). By contrast, CREB (45), a basic
region/leucine zipper (bZip) transcription factor, did not interact
with Ku70 (lanes 13-15). These homeodomain proteins interacted similarly with the Ku heterodimer, while CREB and a regulatory subunit of protein phosphatase 2A (PP2AA) did
not interact with Ku dimer (data not shown). Last, the stringency of
the washes employed in preparing the Ku70 for binding, which was
sufficient to strip away Ku80 from Ku70, suggested that the interaction
between Ku and the homeodomain proteins was likely to be direct.
To test the specificity of binding of Ku to these homeodomain proteins
under conditions that more closely resembled the cellular milieu, we
mixed the in vitro translated homeodomain proteins with the
Jurkat whole cell extract prior to immunoprecipitation with the Ku70
antibody (Fig. 2C). Under these more stringent conditions, Oct-60 binding to Ku was no longer detected, demonstrating that at
least some degree of specificity existed for Ku binding within the
homeodomain protein family (lanes 10-12). However, HOXD4, Dlx2, and Oct-2 binding remained (lanes 1-9). Further,
Ku-homeodomain binding occurred independently from the binding of
either factor to DNA, since ethidium bromide treatments sufficient to
disrupt protein-DNA interactions had no discernible effect on the
Ku-homeodomain interaction (data not shown).
Last, to confirm directly that Ku could interact with
homeodomain proteins in vivo, we tested whether Ku could
be co-immunoprecipitated from HeLa cell extracts with the endogenous
octamer transcription factor 1 (Oct-1) (Fig.
3). Oct-1 is a ubiquitous POU homeodomain protein that is highly homologous to Oct-2 within the POU-specific domain and homeodomain but unrelated to Oct-2 outside these
domains (46). Endogenous Ku heterodimer was efficiently
co-immunoprecipitated with Oct-1 (lane 2) but was not
observed in immunoprecipitates prepared with an unrelated antibody
(lane 3). Thus, these results indicate that several
homeodomain proteins may be expected to interact with the 70-kDa
subunit of the Ku antigen heterodimer in vivo.
To begin to examine the requirements within Oct-1 for Ku binding, we
tested the ability of in vitro translated C-terminally truncated Oct-1 peptides (Fig.
4A) to bind the
immunoprecipitated Ku heterodimer. Full-length in vitro
translated Oct-1 and an N-terminal Oct-1 peptide truncated to the edge
of the homeodomain bound specifically to immunoprecipitated Ku (Fig.
4B, lanes 1-6). However, truncation of the Oct-1
by a further 28 amino acids into the homeodomain abrogated binding
(lanes 7-9), demonstrating the requirement of the
homeodomain for Ku binding by Oct-1.
Ku-Homeodomain Binding Is Abrogated by Site-directed Mutations in
the Extreme C Terminus of Ku70--
To delimit the determinants of Ku
required for homeodomain binding, we examined the binding of in
vitro translated Ku to a GST-HOXC4 homeodomain fusion protein in
pull-down assays (Fig. 5A).
Full-length Ku70, but not Ku80, bound efficiently to the immobilized
GST-HOXC4 homeodomain (lanes 2 and 5), while
neither peptide bound to GST alone (lanes 3 and
6). Truncation of only 26 amino acids from the C terminus of
Ku70 eliminated HOXC4 binding (lanes 7-9 and
13-15). By contrast, binding was unaffected by truncation
of Ku70 from the N terminus as far as amino acid 538 (lanes
10-12 and 16-21).
Alignment of the Ku70 C-terminal peptide sequence from several species
revealed a six-amino acid motif, KKQELL, that was highly conserved
between vertebrate Ku70s (Fig. 5B). Further, although deletion of Ku70 C-terminal peptide to amino acid 603 had no effect on
binding to GST-HOXC4, truncation to amino acid 594 eliminated Ku70
binding (Fig. 5C). Replacement of the KKQELL motif with
NNMAHH abrogated the binding of full-length Ku70 to GST-HOXC4 (Fig.
5D, lanes 1-6). Substitution of
Lys595 and Lys596 with Asn also eliminated
binding of the C terminus of Ku70 to GST-HOXC4 (lanes
7-12), whereas E598Q had no effect on binding (lanes
13-15).
To confirm that Lys595 and Lys596 were required
for the binding of Ku70 to homeodomain proteins, we compared the
interaction of WTKu70 and Ku70595N/596N with full-length
HOXC4 and Oct-2 in a yeast two-hybrid assay (Fig.
6). Both full-length Oct-2 and HOXC4 when
expressed as fusion proteins with the Gal4 activation domain interacted
strongly with the Gal4DBDKu70 construct to induce
Ku Binding Promotes the Phosphorylation of Homeodomain Proteins by
DNA-PK from DNA Ends--
To begin to examine the influence of DNA
binding on the interaction between the homeodomain proteins, we
compared the association of recombinant Ku with a GST-Oct-2 peptide
containing the complete DNA binding domain of Oct-2 in EMSA (Fig.
7). GST-Oct-2 bound specifically to the
octamer motif of the histone H2B gene but did not associate with a
nonspecific oligonucleotide (lanes 2 and 10). By
contrast, recombinant Ku bound to both oligonucleotides in the presence
of 1 µg of calf thymus competitor DNA but did not bind when the
competitor DNA was increased to 2.5 µg (lanes 3,
8, 11, and 16-18). Co-incubation of
GST-Oct-2 with Ku resulted in the appearance of an additional complex
on the nonspecific DNA that was specifically competed by an Oct-2
antibody (lanes 4-7), demonstrating that Oct-2 could
associate with DNA end-bound Ku. Similarly, on the octamer motif, a
Ku-Oct-2 complex was detected under conditions where Ku also bound to
DNA ends (lanes 12-15). However, at 2.5 µg of competitor
DNA, the Oct-2-octamer motif complex remained, but no additional
Ku-containing complex was detected (lanes 16-18),
indicating that Ku did not associate with the DNA-bound Oct-2 fusion
protein under these conditions. Last, the inclusion of 10 mM Mg2+, which has previously been shown to
induce changes in the interaction between Ku and DNA (26), had no
further influence the effect of DNA on the Ku-Oct-2 interaction (lanes
5, 13, and 17). These results were
consistent with our subsequent observation that the presence of Ku did
not significantly affect the activation of transcription by Oct-1 or
Oct-2 from octamer motifs in transient transfection experiments (data
not shown).
These results suggested that the basis for the functional consequence
of Ku-homeodomain binding was likely to lie in the recruitment of
homeodomain proteins to DNA ends. One possibility was that Ku-homeodomain binding could promote the phosphorylation of homeodomain proteins by DNA-PK. Full-length Oct-1 and Oct-2 are phosphorylated by
DNA-PK in vitro, and Oct-1 contains at least two
phosphorylation sites for DNA-PK, one of which is in the POU-specific
domain (data not shown). To assess whether the interaction of Oct-1 to
Ku bound to DNA ends could promote its phosphorylation by DNA-PK, we
evaluated the contribution of the Oct-1 homeodomain for phosphorylation within the POU-specific domain. This was first tested by comparing the
phosphorylation of GST-Oct-1 POU wild-type protein and a mutant lacking
the POU homeodomain that can no longer interact with Ku (Fig.
8A). At a 1:1 molar ratio,
inclusion of the homeodomain increased phosphorylation within the
POU-specific domain by 55-fold (Fig. 8B, lanes 1 and 2). Increasing the concentration of the POU-specific
domain peptide resulted in a proportional increase in phosphorylation
(lanes 3-7). However, even at a 40-fold higher concentration, phosphorylation of this peptide remained 2.3-fold lower
than obtained when the homeodomain was included.
To provide additional evidence that this difference in phosphorylation
was due to recruitment of the POU-specific domain of Oct-1 to Ku by the
homeodomain, we repeated the in vitro phosphorylation experiment with chimeric proteins in which we substituted the homeodomain of HOXC4 into Oct-1 (Fig. 8A). The HOXC4
homeodomain peptide was not appreciably phosphorylated by DNA-PK (Fig.
8C, lane 1), while the POU-specific peptide
of Oct-1 was weakly phosphorylated (lanes 2-4). However,
fusion of the homeodomain of HOXC4 to the N terminus of the
POU-specific domain of Oct-1 resulted in a strong induction of
phosphorylation within the POU-specific domain to a level equivalent to
that observed with the WT Oct-1 peptide (lanes 5-8). Thus,
Ku-homeodomain binding appears to strongly potentiate protein
phosphorylation by DNA-PK.
In this study, we provide insight into two aspects of the
mechanism of action of DNA-dependent protein kinase. First,
our results demonstrate that the Ku antigen may serve as a scaffold or
adapter protein for the attraction of DNA-PK substrates. Second, our
results suggest that Ku binding may be a property of many homeodomain
proteins. Together these results highlight the potential for the
regulation of homeodomain protein function by DNA-PK in vivo.
We have determined in immunoprecipitation and two-hybrid experiments
that several homeodomain proteins interact through their homeodomains
to Ku antigen through a motif contained within the extreme C terminus
of Ku70. Binding was unaffected by dimerization of Ku70 with Ku80 and
the binding of Ku to DNA ends but appeared to be exclusive of the
sequence-specific DNA binding of the homeodomain protein.
Docking proteins that function as scaffolds to promote the
assembly of protein complexes for phosphorylation are a common feature
of phosphorylation signaling cascades. For example, SH2 and SH3 domains
and specific adapter proteins determine the phosphorylation of
downstream effector proteins by signal-dependent tyrosine
kinases (47). Similar types of interactions have also been shown to be
important for signaling through Ser/Thr phosphorylation (48). Our
results indicate that substrate phosphorylation by DNA-PK may
also be regulated through related protein-protein interactions employing Ku as the attractant or adapter protein for DNA-PK substrates.
The affinity and specificity of DNA-PK for its substrates described to
date is modest. While a large number of proteins have been shown to
have the potential to be phosphorylated by DNA-PK in vitro
when incubated at high concentration with the kinase (21), the search
for in vivo phosphorylation targets for DNA-PK continues
unsatisfied. Previously it has been demonstrated that colocalization of
DNA-PK and potential substrates to the same double-stranded DNA
molecule as a result of high affinity DNA binding can dramatically
enhance their phosphorylation (3, 22, 25). We have recently extended
these results to demonstrate that DNA-PK can also efficiently
phosphorylate substrates from single-stranded DNA structures when these
proteins are co-localized to the same DNA
molecules.3 Our results here
demonstrate that specific binding to Ku can also dramatically promote
substrate phosphorylation by DNA-PK. This mechanism of promoting
substrate phosphorylation alleviates the requirement for adjacent
substrate DNA-binding sites and broadens the potential for DNA-PK
phosphorylation to non-DNA-binding proteins.
The homeodomain is a 60-amino acid helix-turn-helix DNA binding domain
whose primary sequence and secondary structure is highly conserved
within the extended homeodomain gene family (37, 39, 40). In addition
to participating in DNA binding, homeodomains have also been reported
to participate extensively in protein-protein interactions with
transcriptional modifiers and other factors (49-51). However, there is
enough variation within the homeodomain to allow for discrimination in
protein-protein interactions between even closely related family
members. For example, although the homeodomains of Oct-1 and Oct-2
differ by only seven amino acids, including four conservative
substitutions, only Oct-1 is targeted by the herpes simplex virus
protein 16 and host cell factor (52). Nonetheless, there are also at
least two precedents for the broadly based specific interactions
between nuclear factors and the homeodomain. It has been reported that
the transcriptional repressor N-CoR can interact with a diverse series
of homeodomain proteins (53, 54). Second, a motif within the
glucocorticoid hormone receptor has also been reported to interact with
a series of homeodomain proteins in transfected cells, and ectopic
expression of this motif in one-cell stage embryos dramatically
affected embryonic development (55).
On the basis of the results reported here, it would be premature to
extrapolate Ku70 binding and DNA-PK phosphorylation throughout the
homeodomain protein family, particularly since Oct-60 binding was lost
under stringent conditions. However, our results have demonstrated
specific interactions between full-length HOXC4, Oct-1, Oct-2, and
either the Ku heterodimer or the Ku70 monomer in vivo and
have shown that the homeodomain is sufficient for this interaction
in vitro. The apparent conservation of the ability to
interact with Ku that was observed between representatives of the HOX,
Dlx, and POU subfamilies of homeodomain proteins highlights the need
for a more extensive investigation of Ku-homeodomain protein interactions.
How might Ku binding and DNA-PK phosphorylation affect homeodomain
protein function? Homeodomain proteins are transcriptional regulators
that direct specific gene expression programs (38, 40, 56, 57), whereas
DNA-PK is activated from DNA ends in response to DNA damage (4). DNA
damage induces multiple changes in cellular regulation including many
changes in specific gene transcription. These changes contribute to
arresting cell growth until the DNA damage is repaired or to directing
irretrievably damaged cells into apoptosis (58, 59). Thus, our results
suggest that one way in which DNA-PK may act from DNA ends subsequent to DNA damage is to modify the transcriptional regulatory potential of
at least some homeodomain proteins.
Continuing experiments suggest that this may be the case at least for
Oct-1. Oct-1 is a ubiquitous homeodomain protein that is required for
the expression of histone H2B and other constitutively expressed genes.
Recent reports have indicated that Oct-1 protein levels and binding
activity are modified following DNA damage (60, 61).2 In
normal cells, Oct-1 is dispersed within the nucleus and is also
localized to the nuclear periphery (62-64). We have determined that
following exposure of cells to ionizing radiation, Oct-1 redistributes
to the cytoplasm over a period of hours. Intriguingly, this
redistribution was not observed in cells lacking Ku70.2
Whether this alteration in the response of Oct-1 to radiation is
sensitive to the Asn595/Asn596 substitutions
that abrogate Oct-1-Ku binding and whether it is mediated through
phosphorylation of Oct-1 by DNA-PK remains to be determined. It will be
interesting to determine whether the relocalization of Oct-1 to the
cytoplasm occurs upon DNA damage in the presence of the
K595N/K596N-substituted Ku70 and whether the relocalization reflects
Oct-1 phosphorylation by DNA-PK at one of the sites that we have identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice undergo premature senescence in
culture (8, 15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11 cDNA library (see below) into pACT2. The
pGEX2T-HOXC4 HD construct was obtained by inserting the HOXC4 cDNA
fragment 1048-1290 (aa 148-227) isolated from clone 18-27 into
pGEX2T. Oct-2-pACT2 was constructed by inserting full-length human
Oct-2 cDNA in pACT2. To obtain pGEX3X-Oct-1POU, a PCR fragment
containing the Oct-1 POU domain (nt 860-1398, aa 268-446) was
inserted in pGEX-3X. This construct was used to generate the GST-Oct-1
POU-specific domain mutant (POUsp, aa 268-369) by removing
the POU homeodomain by EcoRI digestion and religating the
vector. pGEX2T-HOXC4 HD-Oct-1 POUsp was generated by
cloning the Oct-1 POU-specific domain (aa 268-369) in the
SmaI site of plasmid PGEX2T-HOXC4 HD. pGSTOct-2 POU used in
bandshift has been described (33).
-galactosidase expression by filter
lift assay. About 2.5 × 106 transformants were
screened. Positives were restreaked on plates lacking Trp, Leu, and His
and retested for
-galactosidase activity. pGAD-10 prey plasmids from
positive colonies were then recovered and transformed in
Escherichia coli DH5
' by electroporation, selected on
ampicillin plates, and amplified. Purified plasmid DNA was then
retransformed in yeast in parallel with Ku70-pAS1 or control Rab3A
plasmid (34).
gt11 T cell/Jurkat cDNA
library (CLONTECH) with a HOXC4 oligonucleotide
spanning nt 871-900. All procedures for screening, isolation, and
preparation of phage DNA were performed as described in the
CLONTECH protocol. After three rounds of screening,
positive phages were grown, and the inserts were analyzed by PCR using
primers designed to amplify the full-length cDNA (nt 600-1421) to
determine full-length clones. Positive clones were then amplified by
PCR using the same primers, cloned into vector pACT2, and sequenced
entirely using a LICOR automated sequencer.
-32P]ATP (6000 Ci/mmol; Amersham Pharmacia
Biotech), and 8 units of DNA-PK (Promega).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity upon overnight incubation (Fig.
1B). This low level of
transcriptional activity was consistent with previous reports of a weak
acidic activation domain within the N terminus of Ku70 and was
abrogated by deletion of the first 57 amino
acids.2 However, this low
level of activation did not prevent the detection of interactions with
peptides expressed from the Jurkat cDNA library that led to a
strong induction of
-galactosidase activity through a specific
interaction with Ku70.
View larger version (30K):
[in a new window]
Fig. 1.
Identification of HOXC4 as a Ku70-binding
protein by yeast two-hybrid screening. A, schematic
presentation of HOXC4 and the HOXC4 peptide (18-27)
identified in the two-hybrid screen. B, yeast two-hybrid
assay testing the interaction of clone 18-27 containing a partial
cDNA of HOXC4 and full-length HOXC4 (fl) with Ku70,
Ku80, and Rab3A. -Galactosidase expression was tested by filter lift
assay, and blue colonies were scored as follows: +++, over 90% blue by
3 h; +, 50% blue by 6 h; (+), light blue by 24 h;
,
white after 24 h.
-galactosidase activity in the absence of the Ku70 bait, while a further 15% were
completely negative. DNA sequence analysis of one of the remaining
positive clones identified it as encoding a 94-amino acid peptide
encompassing the homeodomain of HOXC4 (Fig. 1A). Interestingly, this HOXC4 peptide also displayed a significant ability
to activate
-galactosidase expression in the absence of Ku70, with
blue color being detectable after 6-7 h (Fig. 1B). Thus,
this clone was almost missed in the secondary screen. Nevertheless, co-expression of this HOXC4 peptide with GalDBDKu70 led to
a strongly enhanced activity, with the blue color detectable within
3 h, suggesting the existence of a significant interaction between the two proteins. By contrast, the HOXC4 peptide did not interact with
the Rab GTPase expressed from the same vector as a GalDBD fusion protein.
-galactosidase expression, indicating that the
nonspecific effect of the homeodomain was suppressed within the
full-length protein (Fig. 1B). Nonetheless,
HOXC4GalAct interacted strongly with GalDBDKu70
to induce
-galactosidase activity. This result confirmed the
observation of a specific interaction between Ku70 and HOXC4 in yeast.
Further, the interaction was specific for Ku70 alone, since no
interaction was observed with the Rab or with the Ku80 subunit in the
same assay.
View larger version (48K):
[in a new window]
Fig. 2.
Ku interacts with homeodomain
proteins. A (left), Western blot analysis
showing the preparation of Ku70 and Ku70/80 dimer used in binding
assays. Lane 1, 25 µg of Jurkat T cell whole cell extract.
Lane 2, the Ku dimer immunoprecipitated from Jurkat T cell
extracts with Ku70 antibody N3H10. Lane 3, removal of Ku80
subunit following KCl/deoxycholate treatment of the immunoprecipitate.
A (right), 35S-labeled in
vitro translated HOXC4 homeodomain (clone 18-27) was tested for
binding to immunoprecipitated Ku70 (lanes 5), the
immunoprecipitated Ku dimer (lane 6), or protein-Sepharose
beads alone (lane 7) and compared with 10% of input proteins
(lane 4). B, 35S-labeled in
vitro translated proteins HoxD4 (lanes 1-3), Dlx2
(lanes 4-6), Oct-2 (lanes 7-9), Oct-60
(lanes 10-12), and CREB (lanes 13-15) were
tested for binding to protein A-Sepharose-bound Ku70 (Ku70) or
protein-Sepharose alone ( ). I, 10% of input proteins.
C, 35S-labeled in vitro translated
proteins (HoxD4, Dlx2, Oct-2, Oct-60, and CREB) were incubated in whole
cell extract prepared from Jurkat T cells and immunoprecipitated with
Ku70 N3H10 antibody (Ku) or with protein A-Sepharose alone
(
). Lanes I show 5% of input proteins.
View larger version (46K):
[in a new window]
Fig. 3.
Co-immunoprecipitation of Ku and Oct-1 in
HeLa cells. Proteins from HeLa nuclear extracts were incubated
with an Oct-1 antibody (lane 2) or a nonspecific antibody
(lane 3), precipitated with protein A-Sepharose and resolved
on a 10% SDS-polyacrylamide gel. Proteins were transferred on membrane
and hybridized with a Ku70 antibody (top). The blot was
successively stripped and rehybridized with antibodies to Ku80
(middle) and Oct-1 (bottom). Immunoprecipitates
are compared with Ku and Oct-1 found in 15 µg of HeLa nuclear extract
(lane 1).
View larger version (48K):
[in a new window]
Fig. 4.
The integrity of the
homeodomain is required for Ku interaction. A,
schematic representation of human Oct-1 indicating the position of the
restriction sites used to prepare the Oct-1 homeodomain deletion
mutants. B, binding of full-length 35S-labeled
in vitro translated Oct-1 (lanes 1-3) or Oct-1
deletion mutants (lanes 4-9) to immunoprecipitated Ku dimer
from Jurkat T cell extracts (Ku) or to protein A-Sepharose ( ) is
compared with 10% of input (I).
View larger version (37K):
[in a new window]
Fig. 5.
Ku70 determinants for homeodomain
interaction. A, 35S-labeled in
vitro translated Ku70 (lanes 1-3), Ku80 (lanes
4-6), and deletion mutants of Ku70 (lanes
7-21) were tested for binding to GST-HOXC4 homeodomain
(GST-HD) or to GST alone (GST) by a pull-down
assay. 10% of input was loaded on the gel to compare the levels of
binding. The arrows indicate the position of the complete
in vitro translated proteins. B, alignment of the
C-terminal amino acid sequence of Ku70 from several species. Amino
acids displayed in capital letters are conserved
with respect to the human sequence. Amino acids in boldface
letters outline the highly conserved motif targeted for
mutation. C, 35S-labeled in vitro
translated Ku70 C-terminal peptides with C-terminal deletions of six
amino acids (301-603; lanes 1-3) or 15 amino
acids (301-594; lanes 4-6) were tested for
binding to GST-HOXC4 HD as described for A. D,
point mutations in the C-terminal domain of Ku70 abrogate homeodomain
binding. GST pull-down assay using GST-HOXC4 HD was performed to test
the binding of 35S-labeled in vitro translated
full-length wild-type Ku70 (lanes 1-3) compared with Ku70
containing mutations of amino acids 595-600 (KKQELL to NNMAHH;
lanes 4-6). Binding of the C-terminal Ku70 peptide
(301-609, lanes 7-9) was compared with Ku70 peptides
bearing a double point mutation of aa 595 and 596 (KK to
NN, lanes 10-12) or a single point mutation of
aa 598 (E to Q, lanes 13-15).
In vitro translated products bound to GST-HOXC4 HD
(GST-HD) or to GST alone (GST) were compared with
10% of input proteins (Input). Proteins were run on a 10%
polyacrylamide gel.
-galactosidase activity, while Gal4DBDRab was again
negative. In contrast to WTKu70, however, both the mt595-600 and the
K595N/K596N substituted Ku70 Gal-DBD construct failed to interact with
either HOXC4 or Oct-2. Thus, the interaction of Ku70 with
homeodomain proteins is mediated through a conserved element and
can be disrupted by a substitution of only two amino acids.
View larger version (28K):
[in a new window]
Fig. 6.
A two-amino acid substitution in Ku70
C-terminal domain disrupts Ku-homeodomain interaction in yeast.
Rab3A, Ku70, Ku70mt595-600, and
Ku70mtK595,596N expressed in yeast as Gal4 DBD fusion
proteins (bait) were tested for binding to the Gal4 activation
domain alone (pACTII) or expressed as a fusion protein with full-length
Oct-2 or HOXC4 (prey). The strength of the interaction between two
proteins, which is reflected by the -galactosidase activity, was
analyzed by filter lift assay and was scored as follows: +++, over 90%
colonies blue by 3 h;
, all white by 8 h. The expression of
the proteins was verified by Western blot (data not shown).
View larger version (51K):
[in a new window]
Fig. 7.
Ku-homeodomain complexes form on DNA
ends. The formation of Ku-homeodomain complexes was analyzed by
EMSA using a nonspecific oligonucleotide (lanes 1-8) or an
oligonucleotide containing an octamer motif (lanes 9-18).
The 32P-labeled oligonucleotides were incubated with
purified recombinant Ku (about 10 ng), GST-Oct-2 POU (about 50 ng), or
with GST alone (about 50 ng) as indicated at the top, in the
presence of 1 or 2.5 µg of competitor calf thymus DNA (CT)
as indicated at the bottom. The specificity of the complexes
was assessed by including an Oct-2 antibody (Oct-2Ab) or a
nonspecific antibody (NS Ab). 10 mM of
MgCl2 was included as indicated. The arrows
indicate the composition of the complexes.
View larger version (36K):
[in a new window]
Fig. 8.
Phosphorylation within the POU-specific
domain of Oct-1 is strongly enhanced by interaction of Ku with the
homeodomain. A, representation of the GST fusion
constructs used as substrate for DNA-PK in the phosphorylation assays.
B, DNA-PK-mediated phosphorylation of a GST fusion protein
containing the Oct-1 POU-specific domain and homeodomain (Oct-1
POUsp + HD, lanes 1) is compared
with phosphorylation of increasing amounts of a GST fusion protein with
the Oct-1 POU-specific domain alone (Oct-1 POUsp,
lanes 2-7). After completion of the reactions, the products
were separated on a 10% SDS-polyacrylamide gel and stained with
Coomassie Blue (top panel). Then the gels were dried, and
32P-incorporation was quantified by a Typhoon 8600 (Molecular Dynamics, Sunnyvale, CA) (bottom panel).
Protein concentration was confirmed by quantification of the
Coomassie-stained gel on a Bio-Rad Gel Doc system. The
arrowhead shows the position of the full-length GST
Oct-1POUsp + HD peptide. C, DNA-PK
phosphorylation of GST fusion proteins containing the homeodomain of
HOXC4 (HOXC4 HD, lane 1), Oct-1 POUsp
(lanes 2-4), a HOXC4 HD-Oct-1 POUsp fusion
peptide (lanes 5 and 6), and
Oct-1POUsp + HD (lanes 7 and 8).
Lanes 1, 2, 6, and 8 contain similar amounts of proteins. Resolution and quantification were
as in A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Marc Ekker, Marie-Andrée Akimenko, Marc Mumby, Paolo Sassone-Corsi, and Madeleine Lemieux for materials used in this work.
![]() |
FOOTNOTES |
---|
* This work was supported by an operating grant from the Arthritis Society of Canada (to R. J. G. H.).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.
§ Recipient of a Postdoctoral Fellowship from the Canadian Institutes of Health Research and the Arthritis Society of Canada.
** An investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: The Loeb Health Research Institute at the Ottawa Hospital, 725 Parkdale Ave., Ottawa, Ontario, Canada K1Y 4K9. Tel.: 613-798-5555 (ext. 16283); Fax: 613-761-5036; E-mail: rhache@lri.ca.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100768200
2 C. Schild-Poulter and R. J. G. Haché, unpublished observation.
3 S. Soubeyrand, H. Torrance, W. Giffin, W. Gong, C. Schild-Poulter, and R. J. G. Haché, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Ku70, Ku antigen 70-kDa subunit; Ku80, Ku antigen 80-kDa subunit; DNA-PK, DNA-dependent protein kinase, DNA-PKcs; DNA-dependent protein kinase catalytic subunit, Oct-1 and -2, octamer transcription factor 1 and 2, respectively; DBD, DNA binding domain. GST, glutathione S-transferase; HD, homeodomain; EMSA, electromobility shift assay; PCR, polymerase chain reaction; aa, amino acid; nt, nucleotide; CREB, cAMP-response element-binding protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Critchlow, S. E., and Jackson, S. P. (1998) Trends Biochem. Sci. 23, 394-398[CrossRef][Medline] [Order article via Infotrieve] |
2. | Haber, J. E. (1999) Cell 97, 829-832[Medline] [Order article via Infotrieve] |
3. | Giffin, W., Torrance, H., Rodda, D. J., Prefontaine, G. G., Pope, L., and Haché, R. J. (1996) Nature 380, 265-268[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Smith, G. C.,
and Jackson, S. P.
(1999)
Genes Dev.
13,
916-934 |
5. |
Tuteja, R.,
and Tuteja, N.
(2000)
Crit. Rev. Biochem. Mol. Biol.
35,
1-33 |
6. | Weaver, D. T. (1998) Curr. Biol. 8, R492-R494[CrossRef][Medline] [Order article via Infotrieve] |
7. | Gu, 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., and Alt, F. W. (1997) Immunity 7, 653-665[Medline] [Order article via Infotrieve] |
8. | Nussenzweig, A., Chen, C., da Costa Soares, V., Sanchez, M., Sokol, K., Nussenzweig, M. C., and Li, G. C. (1996) Nature 382, 551-555[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Ouyang, H.,
Nussenzweig, A.,
Kurimasa, A.,
Soares, V. C.,
Li, X.,
Cordon-Cardo, C.,
Li, W.,
Cheong, N.,
Nussenzweig, M.,
Iliakis, G.,
Chen, D. J.,
and Li, G. C.
(1997)
J. Exp. Med.
186,
921-929 |
10. | Zhu, C., Bogue, M. A., Lim, D. S., Hasty, P., and Roth, D. B. (1996) Cell 86, 379-389[Medline] [Order article via Infotrieve] |
11. | Difilippantonio, M. J., Zhu, J., Chen, H. T., Meffre, E., Nussenzweig, M. C., Max, E. E., Ried, T., and Nussenzweig, A. (2000) Nature 404, 510-514[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Ferguson, D. O.,
Sekiguchi, J. M.,
Chang, S.,
Frank, K. M.,
Gao, Y.,
DePinho, R. A.,
and Alt, F. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
6630-6633 |
13. |
Hsu, H. L.,
Gilley, D.,
Galande, S. A.,
Hande, M. P.,
Allen, B.,
Kim, S. H.,
Li, G. C.,
Campisi, J.,
Kohwi-Shigematsu, T.,
and Chen, D. J.
(2000)
Genes Dev.
14,
2807-2812 |
14. | Li, G. C., Ouyang, H., Li, X., Nagasawa, H., Little, J. B., Chen, D. J., Ling, C. C., Fuks, Z., and Cordon-Cardo, C. (1998) Mol. Cell 2, 1-8[Medline] [Order article via Infotrieve] |
15. |
Vogel, H.,
Lim, D. S.,
Karsenty, G.,
Finegold, M.,
and Hasty, P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10770-10775 |
16. | Jin, S., Inoue, S., and Weaver, D. T. (1997) Cancer Surv. 29, 221-261[Medline] [Order article via Infotrieve] |
17. | Hartley, K. O., Gell, D., Smith, G. C., Zhang, H., Divecha, N., Connelly, M. A., Admon, A., Lees-Miller, S. P., Anderson, C. W., and Jackson, S. P. (1995) Cell 82, 849-856[Medline] [Order article via Infotrieve] |
18. | Zakian, V. A. (1995) Cell 82, 685-687[Medline] [Order article via Infotrieve] |
19. |
Hammarsten, O.,
and Chu, G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
525-530 |
20. |
Yaneva, M.,
Kowalewski, T.,
and Lieber, M. R.
(1997)
EMBO J.
16,
5098-5112 |
21. | Lees-Miller, S. P. (1996) Biochem. Cell Biol. 74, 503-512[Medline] [Order article via Infotrieve] |
22. |
Giffin, W.,
Kwast-Welfeld, J.,
Rodda, D. J.,
Prefontaine, G. G.,
Traykova-Andonova, M.,
Zhang, Y.,
Weigel, N. L.,
Lefebvre, Y. A.,
and Haché, R. J.
(1997)
J. Biol. Chem.
272,
5647-5658 |
23. | Anderson, C. W., and Lees-Miller, S. P. (1992) Crit. Rev. Eukaryot. Gene Exp. 2, 283-314[Medline] [Order article via Infotrieve] |
24. | Anderson, C. W., Connelly, M. A., Lees-Miller, S. P., Lintott, L. G., Zhang, H., Sipley, J. A., Sakagushi, K., and Appella, P. (1995) in Methods in Protein Structure and Analysis (Atassi, M. Z. , and Appella, E., eds) , pp. 395-406, Plenum Press, New York |
25. | Jackson, S. P., MacDonald, J. J., Lees-Miller, S., and Tjian, R. (1990) Cell 63, 155-165[Medline] [Order article via Infotrieve] |
26. |
Giffin, W.,
Gong, W.,
Schild-Poulter, C.,
and Haché, R. J.
(1999)
Mol. Cell. Biol.
19,
4065-4078 |
27. |
Barlev, N. A.,
Poltoratsky, V.,
Owen-Hughes, T.,
Ying, C.,
Liu, L.,
Workman, J. L.,
and Berger, S. L.
(1998)
Mol. Cell. Biol.
18,
1349-1358 |
28. |
Leber, R.,
Wise, T. W.,
Mizuta, R.,
and Meek, K.
(1998)
J. Biol. Chem.
273,
1794-1801 |
29. | Kharbanda, S., Pandey, P., Jin, S., Inoue, S., Bharti, A., Yuan, Z. M., Weichselbaum, R., Weaver, D., and Kufe, D. (1997) Nature 386, 732-735[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Jin, S.,
Kharbanda, S.,
Mayer, B.,
Kufe, D.,
and Weaver, D. T.
(1997)
J. Biol. Chem.
272,
24763-24766 |
31. |
Modesti, M.,
Hesse, J. E.,
and Gellert, M.
(1999)
EMBO J.
18,
2008-2018 |
32. |
Sartorius, C. A.,
Takimoto, G. S.,
Richer, J. K.,
Tung, L.,
and Horwitz, K. B.
(2000)
J. Mol. Endocrinol.
24,
165-182 |
33. |
Préfontaine, G. G.,
Lemieux, M. E.,
Giffin, W.,
Schild-Poulter, C.,
Pope, L.,
LaCasse, E.,
Walker, P.,
and Haché, R. J.
(1998)
Mol. Cell. Biol.
18,
3416-3430 |
34. |
Martincic, I.,
Peralta, M. E.,
and Ngsee, J. K.
(1997)
J. Biol. Chem.
272,
26991-26998 |
35. | Romero, F., Dargemont, C., Pozo, F., Reeves, W. H., Camonis, J., Gisselbrecht, S., and Fischer, S. (1996) Mol. Cell. Biol. 16, 37-44[Abstract] |
36. | Andrews, N. C., and Faller, D. V. (1991) Nucleic Acids Res. 19, 2499[Medline] [Order article via Infotrieve] |
37. |
Kornberg, T. B.
(1993)
J. Biol. Chem.
268,
26813-26816 |
38. | Manak, J. R., and Scott, M. P. (1994) Dev. Suppl. 1994, 61-71 |
39. | Gehring, W. J., Qian, Y. Q., Billeter, M., Furukubo-Tokunaga, K., Schier, A. F., Resendez-Perez, D., Affolter, M., Otting, G., and Wuthrich, K. (1994) Cell 78, 211-223[Medline] [Order article via Infotrieve] |
40. | Gehring, W. J., Affolter, M., and Burglin, T. (1994) Annu. Rev. Biochem. 63, 487-526[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Phelan, M. L.,
and Featherstone, M. S.
(1997)
J. Biol. Chem.
272,
8635-8643 |
42. | Akimenko, M. A., Ekker, M., Wegner, J., Lin, W., and Westerfield, M. (1994) J. Neurosci. 14, 3475-3486[Abstract] |
43. | Clerc, R. G., Corcoran, L. M., LeBowitz, J. H., Baltimore, D., and Sharp, P. A. (1988) Genes Dev. 2, 1570-1581[Abstract] |
44. | Hinkley, C. S., Martin, J. F., Leibham, D., and Perry, M. (1992) Mol. Cell. Biol. 12, 638-649[Abstract] |
45. | Hoeffler, J. P., Meyer, T. E., Yun, Y., Jameson, J. L., and Habener, J. F. (1988) Science 242, 1430-1433[Medline] [Order article via Infotrieve] |
46. | Tanaka, M., and Herr, W. (1990) Cell 60, 375-386[Medline] [Order article via Infotrieve] |
47. | Pawson, T. (1995) Nature 373, 573-580[CrossRef][Medline] [Order article via Infotrieve] |
48. | Faux, M. C., and Scott, J. D. (1996) Cell 85, 9-12[Medline] [Order article via Infotrieve] |
49. | Chariot, A., Gielen, J., Merville, M. P., and Bours, V. (1999) Biochem. Pharmacol. 58, 1851-1857[CrossRef][Medline] [Order article via Infotrieve] |
50. | Scott, M. P. (1999) Nature 397, 649-651[CrossRef][Medline] [Order article via Infotrieve] |
51. | Vershon, A. K. (1996) Curr. Opin. Biotechnol. 7, 392-396[CrossRef][Medline] [Order article via Infotrieve] |
52. | Lai, J. S., Cleary, M. A., and Herr, W. (1992) Genes Dev. 6, 2058-2065[Abstract] |
53. | Laherty, C. D., Billin, A. N., Lavinsky, R. M., Yochum, G. S., Bush, A. C., Sun, J. M., Mullen, T. M., Davie, J. R., Rose, D. W., Glass, C. K., Rosenfeld, M. G., Ayer, D. E., and Eisenman, R. N. (1998) Mol. Cell 2, 33-42[Medline] [Order article via Infotrieve] |
54. | Xu, L., Lavinsky, R. M., Dasen, J. S., Flynn, S. E., McInerney, E. M., Mullen, T. M., Heinzel, T., Szeto, D., Korzus, E., Kurokawa, R., Aggarwal, A. K., Rose, D. W., Glass, C. K., and Rosenfeld, M. G. (1998) Nature 395, 301-306[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Wang, J. M.,
Prefontaine, G. G.,
Lemieux, M. E.,
Pope, L.,
Akimenko, M. A.,
and Haché, R. J.
(1999)
Mol. Cell. Biol.
19,
7106-7122 |
56. |
Edelman, G. M.,
and Jones, F. S.
(1993)
J. Biol. Chem.
268,
20683-20686 |
57. | Ryan, A. K., and Rosenfeld, M. G. (1997) Genes Dev. 11, 1207-1225[CrossRef][Medline] [Order article via Infotrieve] |
58. | Lowndes, N. F., and Murguia, J. R. (2000) Curr. Opin. Genet. Dev. 10, 17-25[CrossRef][Medline] [Order article via Infotrieve] |
59. | Weinert, T. (1998) Cell 94, 555-558[Medline] [Order article via Infotrieve] |
60. | Meighan-Mantha, R. L., Riegel, A. T., Suy, S., Harris, V., Wang, F. H., Lozano, C., Whiteside, T. L., and Kasid, U. (1999) Mol. Cell. Biochem. 199, 209-215[CrossRef][Medline] [Order article via Infotrieve] |
61. |
Zhao, H.,
Jin, S.,
Fan, F.,
Fan, W.,
Tong, T.,
and Zhan, Q.
(2000)
Cancer Res.
60,
6276-6280 |
62. |
Grande, M. A.,
van der Kraan, I.,
de Jong, L.,
and van Driel, R.
(1997)
J. Cell Sci.
110,
1781-1791 |
63. |
Imai, S.,
Nishibayashi, S.,
Takao, K.,
Tomifuji, M.,
Fujino, T.,
Hasegawa, M.,
and Takano, T.
(1997)
Mol. Biol. Cell
8,
2407-2419 |
64. | Kim, M. K., Lesoon-Wood, L. A., Weintraub, B. D., and Chung, J. H. (1996) Mol. Cell. Biol. 16, 4366-4377[Abstract] |