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INTRODUCTION |
Nuclear receptor coactivators mediate gene activation though
interactions with nuclear receptors and components in the
transcriptional apparatus. The interactions subsequently permit the RNA
polymerase II complexes to access target genes (1, 2). Studies of
coactivators in the past several years have significantly refined our
model of ligand-induced interaction between the receptor ligand-binding domains and the coactivator LXXLL motifs. However, the role
of receptor-bound coactivators in their interactions with the
transcriptional complex is less clear, and it remains a current
research focus. Coactivator actions appear to involve multiple
cooperative mechanisms. The functional properties of coactivators
include the following: direct protein-protein interactions with
transcriptional complexes (1); enzymatic activities of certain
coactivators, such as histone acetyltransferase in
CBP1 and steroid receptor
coactivator-1 (SRC-1) family or arginine transmethylase in
coactivator-associated arginine methyltransferase 1 (CARM1) (3, 4); RNA
interactions, with RNA recognition motifs such as in peroxisome
proliferator-activated receptor-
-coactivator-1 (PGC-1), CoAA, and
SMRT/HDAC1-associated repressor protein (SHARP); and RNA alone such as
steroid receptor RNA activator (SRA) (5-8). It is becoming
increasingly important to understand how coactivator-targeted molecules, such as interacting proteins or enzyme substrates, are
regulated by coactivators.
We previously cloned and characterized thyroid hormone receptor-binding
protein (TRBP) as a nuclear receptor coactivator (9). TRBP, designated
as NcoA6 by the National Center for Biotechnology Information
(NCBI) nomenclature committee, was concurrently identified by
several groups as AIB3/ASC-2/RAP250/PRIP/TRBP/NRC (1, 2). TRBP is a
high molecular weight, ubiquitously expressed coactivator. A single
LXXLL motif is required for the ligand-dependent
interaction with a number of nuclear receptors and subsequent
transcriptional activation. The Ser-884 residue adjacent to the TRBP
LXXLL motif was shown to regulate the selectivity of TRBP
for different nuclear receptors (10). TRBP also coactivates multiple
transcriptional factors including AP-1 and NF-
B (1). In addition,
gene amplification was observed for TRBP in human breast cancers (11).
Furthermore, the C terminus of TRBP was shown to interact with
coactivator CoAA (6), CBP/p300, and DRIP complexes (9).
In a search for additional nuclear factors that might be targeted by
TRBP, we identified a distinct set of TRBP-bound proteins using a mass
spectrometric protein sequencing approach. Proteins identified include
the DNA-dependent protein kinase (DNA-PK) components. DNA-PK is a nuclear serine/threonine protein kinase that belongs to the
PI3K family (12-16). Previous biochemical and genetic studies revealed
DNA-PK to be a heterotrimeric enzyme composed of a catalytic subunit,
DNA-PKcs, and two regulatory subunits, Ku86 and Ku70. Although
DNA-PK is known to be activated by DNA ends, recent compelling evidence
suggests that its kinase activity can also be stimulated by protein
interactions (17-19). Supported by a large body of evidence, DNA-PK
has been shown to be involved in transcriptional regulation as well as
in recombination and DNA repair (12, 13, 19-21). DNA-PK is unique
among nuclear protein kinases because it associates with DNA templates
and phosphorylates a variety of protein factors that are important for
transcriptional regulation. The identified in vitro
substrates of DNA-PK include DNA-binding transcriptional factors such
as c-Myc, c-Jun, Sp1, Oct-1, and nuclear receptors GR,
progesterone receptor, tumor suppressor p53, HMG proteins, Ku
subunits, as well as DNA-PKcs itself through autophosphorylation (12).
The close relationship of DNA-PK with transcriptional regulation is
also illustrated by its ability to phosphorylate the C-terminal domain
(CTD) of RNA polymerase II (22-24), which might be important for
coupled transcription and pre-mRNA processing (25). In addition,
mouse cells with severe combined immune deficiency (scid)
that lack functional DNA-PK showed the defects not only in DNA
recombination and DNA repair (26) but also in Ku phosphorylation and
transcriptional activation (21, 27). Thus, DNA-PK kinase activity might
be essential for transcriptional control.
We report here the identification of DNA-PK components as
TRBP-interacting proteins. Through its C-terminal region, TRBP
interacts with DNA-PK via a direct interaction with the regulatory
subunit, Ku70. TRBP can be phosphorylated by DNA-PK in
vitro. Our results also suggest that TRBP stimulates DNA-PK kinase
activity in the absence of DNA ends. This DNA-independent activity may
result in potentially altered substrate phosphorylation specificity. In
addition, DNA-PK-deficient cells have altered TRBP nuclear localization
and exhibit a defect in TRBP-mediated transcription activation. Our
studies suggest a novel connection between coactivator-stimulated DNA-PK phosphorylation and transcriptional regulation.
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EXPERIMENTAL PROCEDURES |
Plasmids and Antibodies--
Human TRBP in pcDNA3 vector and
its derived GST fusion fragments have been described previously (9).
Briefly, GST-TRBPs were subcloned into SmaI/XhoI
or EcoRI/XhoI sites of pGEX-4T-2 (Amersham
Biosciences), as indicated in Fig. 1. Full-length human Ku70 and Ku80
were cloned by RT-PCR and inserted into the
HindIII/XhoI sites of pcDNA3. Mouse mammary
tumor virus (MMTV) promoter luciferase reporter was generated by
inserting MMTV promoter into PXP2 vector, and pCMV-GR was generated by
inserting human GR into the BamHI/XhoI sites of
pcDNA3 as described previously (6). Monoclonal anti-Ku 70 (Ab-4,
clone N3H10), anti-Ku86 (Ab-2, clone 111), and anti-DNA-PKcs (Ab-4)
antibodies were obtained from NeoMarkers. Polyclonal anti-TRBP antibodies were produced from two rabbits (TRBP-1652 and TRBP-1653) using the GST-TRBP-6 (aa 1641-2063) as antigen (Covance). The TRBP
antibody was affinity-purified by Affi-Gel 10 according to the
manufacturer's protocol (Bio-Rad).
Isolation of DNA-PK Complexes--
The GST and GST fusion TRBPs
were produced in Escherichia coli BL21(DE3) and purified by
glutathione-Sepharose resin (Amersham Biosciences). Nuclear extracts
were isolated from a large quantity of HeLa or GH3 cells,
approximately six 15-cm plates. Briefly, cells were lysed in buffer A
(20 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.1% Triton X-100, 1 mM DTT) with addition of 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 10 µg/ml trypsin inhibitor for 15 min on ice. The
nuclei were then collected by centrifugation at 4 °C and extracted
with buffer B (20 mM HEPES, pH 7.4, 420 mM
NaCl, 10 mM KCl, 1 mM EDTA, 1 mM
EGTA, 0.5 mM MgCl2, 1 mM DTT) with
the above protease inhibitors for 30 min on ice. After centrifugation,
the supernatants containing nuclear extracts were further filtered with
a 0.65 µm spin column (Millipore) to remove completely any insoluble
cellular debris. This step is important to prevent nonspecific protein
contamination in the subsequent binding assay. The binding assays were
carried out with 5-10 µg of GST fusion proteins on beads plus
600-800 µg of freshly prepared nuclear extracts in 12 ml of binding
buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Triton X-100, 10%
glycerol, 1 mM DTT, and protease inhibitors) with shaking at 4 °C overnight. Bound resins were washed three times with binding buffer and resolved by SDS-PAGE, followed by Coomassie Blue R-250 staining (Bio-Rad).
Protein Microsequencing--
Protein bands of interest
identified by preparative SDS-PAGE were carefully excised and washed
with 50% acetonitrile according to the Harvard Microchemistry Facility
protocol (28, 29). Sequence analysis performed at the Harvard
Microchemistry Facility included the proteolytic digestion of peptides,
analysis by microcapillary high pressure liquid chromatography
nano-electrospray tandem ion trap mass spectrometry, and mass
spectrometry/mass spectrometry peptide sequence interpretation, which
was facilitated with the Algorithm Sequest and programs developed at
the Harvard Microchemistry Facility. Multiple peptides determined by
sequence analysis were matched to the known GenBankTM entries.
Recombinant Protein Binding Assays--
In vitro
binding assays were performed by incubating GST resin (20 µl, 2 µg)
and [35S]methionine-labeled, in vitro
translated proteins (5 µl) produced by rabbit reticulocyte lysate
(Promega). Proteins were incubated at room temperature for 1 h in
the binding buffer (20 mM HEPES, pH 7.4, 50 mM
NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Triton
X-100, 10% glycerol, 1 mM DTT). Bound proteins were washed
3 times with binding buffer and subjected to SDS-PAGE and autoradiography.
Immunoblotting--
GST-TRBP resins (20 µl, 2-5 µg) were
incubated with 200 µl of HeLa or GH3 nuclear extracts at
4 °C overnight in the binding buffer as above with additional
protease inhibitors. Bound proteins were separated on SDS-PAGE and
detected by Western blotting (ECL). Anti-DNA-PKcs and anti-Ku70
monoclonal antibodies were obtained from NeoMarkers.
DNA-PK Kinase Assay--
Phosphorylation of TRBP recombinant
fragments in vitro by DNA-PK was assayed using the SigmaTECT
DNA-dependent Protein Kinase Assay System from Promega with
modifications. Briefly, DNA-PK enzyme purified from HeLa nuclear
extract (Promega) was incubated with [
-32P]ATP, with
or without linear double strand (ds) DNA as activator, and with
GST-TRBP fusion proteins as substrates. Labeled GST proteins were
washed and resolved by SDS-PAGE and autoradiography. Full-length GST-p53 from Santa Cruz Biotechnology was used as a positive control substrate for the DNA-dependent phosphorylation, and GST
alone as an unphosphorylated protein was used as a negative control. When TRBP was measured for its stimulating activity, p53 peptide was
used as a substrate. The amount of purified DNA-PK used (5-10 units)
in each assay was titrated and determined with a biotinylated p53
peptide substrate prior to use. Alternatively, when endogenous DNA-PK
from HeLa cell nuclear extracts was assayed, GST-TRBP-5 and -6 were
preincubated with HeLa nuclear extracts, and washed beads containing
TRBP bound DNA-PK were used in the assays. DNA-PK kinase activities
were measured using the same SigmaTECT system in the presence or the
absence of DNA, except a biotinylated p53 peptide was used as
substrate. Phosphorylated p53 peptides were captured by the
streptavidin-cellulose papers, and the papers were washed and counted
in a scintillation counter. GST alone and GST-TRBP-3 were used as
negative controls.
Immunofluorescence--
TRBP antibodies were affinity-purified
by Affi-Gel 10 chromatography according to the manufacturer's protocol
(Bio-Rad). Mouse cell lines SCSV3 (DNA-PK-deficient, scid) and SCH8-1
(scid plus human DNA-PK) were methanol-fixed and double-stained with
affinity-purified polyclonal anti-TRBP (1:50) and monoclonal anti-Ku70
(1:100, clone N3H10, NeoMarkers). Anti-rabbit Cy3- and anti-mouse
fluorescein isothiocyanate-conjugated secondary antibodies (Jackson
ImmunoResearch) were applied at a dilution of 1:200. Images were
obtained using a Nikon E800 fluorescent microscope.
Cell Culture and Transient Transfection--
HeLa cells, mouse
SCSV3 (DNA-PK
/
), and SCH8-1 (DNA-PK +/+) cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 5 µg/µl penicillin/streptomycin in 5%
CO2 at 37 °C. Cells in 24-well plates were transfected
with MMTV-luc (0.1 µg), GR (0.01 µg), and TRBP (0.2 µg) plasmids
per well using Lipofectin (Invitrogen). Cells were incubated with fresh
medium containing the indicated concentrations of ligands and
wortmannin 16-24 h after transfection. After another 24 h, cells
were harvested, and luciferase activities were assayed. Total amounts
of DNA for each well were equalized by adding vector pcDNA3
(Invitrogen). Data are shown as means of triplicate transfections ± S.E.
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RESULTS |
DNA-PK Complexes Interact with TRBP C Terminus--
Several lines
of evidence support the identification of the C terminus of coactivator
TRBP (aa 1641-2063) as a binding domain for the interactions with
multiple nuclear proteins, including coactivator CBP/p300, DRIP complex
component DRIP130 (9), and coactivator activator CoAA (6). In addition,
the majority of the transcriptional activity of TRBP is mediated
through its C terminus (9). To identify in vivo protein
complexes in close contact with the TRBP C-terminal region, we carried
out mass spectrometric analysis of nuclear proteins precipitated with
immobilized recombinant TRBP fragments (Fig.
1). HeLa nuclear extracts were incubated with selected TRBP regions including its C terminus, and binding proteins were identified by SDS-PAGE (Fig.
2). Two overlapping recombinant TRBP-C
fragments with different sizes (TRBP-5, aa 1237-2063, and TRBP-6, aa
1641-2063) were used, in order to detect the binding proteins in same
size range of TRBP-C that might otherwise be obscured by size
overlap.

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Fig. 1.
Schematic representation of coactivator
TRBP. Schematic diagram illustrates the regions of TRBP used for
detecting TRBP-interacting proteins. Numbers indicate amino
acids. The LXXLL motif is shown. Protein factors that
interact with TRBP C terminus are indicated.
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Fig. 2.
Identification of DNA-PK complex components
that associate with TRBP C terminus. A, analytical
SDS-PAGE for TRBP-associated proteins. Selected GST-TRBP fragments were
incubated with or without HeLa nuclear extracts as indicated. Bound
proteins were washed and resolved by SDS-PAGE followed by Coomassie
Blue staining. Open arrows indicate GST-TRBP-5 and
GST-TRBP-6. Small open circles denote the TRBP-interacting
proteins. B, the preparative gels from which the
TRBP-interacting proteins were excised for sequence analysis.
Open arrows indicate GST-TRBP fragments; closed
arrows indicate the multiple protein bands before excision for
protein sequencing. Proteins were designated according to their
molecular sizes, and their revealed identities are shown in
parentheses.
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A number of proteins ranging from 70 to 400 kDa were observed. However,
only the predominant bands were isolated for further microsequence
analysis. The identified TRBP-associated proteins are p70, p80, p90,
p100, p200, and p400. These proteins were carefully isolated from the
preparative gel (Fig. 2B), and their identities were
determined by proteolytic peptide sequencing (Table
I). Remarkably, among the six analyzed
proteins, four of them belong to the DNA-PK complex, including DNA-PK
catalytic subunit (p400), DNA-PK regulatory subunits, Ku 70 (p70) and
Ku 86 (p80), and PARP (p100). The other two proteins were DNA
topoisomerase I (p90) and NuMA (p200). The profiles of associated
proteins for TRBP-5 and TRBP-6 were overlapping, particularly regarding
DNA-PKcs, Ku70 and Ku86. PARP was more closely associated with TRBP-6,
although the reason is unclear. DNA-PK subunits were not found to be
associated with other TRBP regions, suggesting that the binding of
these proteins was specific to the C-terminal region of TRBP (Fig.
2A). The stoichiometry of Ku and TRBP-5 interaction is very
high (Fig. 2B), indicating Ku subunits might have higher
affinities toward TRBP. In general, the number of the peptides
sequenced to match to a GenBankTM accession number
correlates with the reliability of the identity of a given protein
(Table I). Data also revealed the presence of contaminating GST-TRBP,
which was shown with a small number of peptides. It should be noted
that proteins present in lower abundance may not be identified by this
sequencing approach, yet may still bind to TRBP either directly or
indirectly. Other approaches, such as the yeast-two hybrid screen, may
be more suitable for the isolation of these proteins (6). Nevertheless,
we conclude from our data that DNA-PK complexes interact with TRBP at
its C terminus.
TRBP Directly Interacts with DNA-PK Ku70 in Vitro--
We decided
to focus our current studies on DNA-PK and investigate its relationship
with TRBP because the role of DNA-PK in transcriptional regulation is
becoming increasingly appreciated (19, 30). To confirm the interaction
of the DNA-PK complex with TRBP observed during peptide sequencing, we
performed Western blot analysis using anti-DNA-PKcs and anti-Ku70
antibodies. As shown in Fig.
3A, recombinant GST fusion
TRBPs were incubated with HeLa nuclear extract, and bound proteins were
probed with indicated antibodies. These results suggest that DNA-PKcs
and Ku70 interact with TRBP, but only at its C terminus. Similar
results were obtained with anti-Ku86 antibody and with GH3
nuclear extracts (not shown).

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Fig. 3.
TRBP-interacts with DNA-PK complex through
Ku70. A, TRBP C terminus interacts with endogenous
DNA-PK complex. GST-TRBP fusion protein fragments were incubated with
HeLa nuclear extracts, and TRBP-bound proteins were subjected to
Western blot analysis. Anti-DNA-PKcs and anti-Ku70 antibodies are
indicated. B, GST, GST-TRBP-5, or TRBP-6 was incubated
with in vitro translated,
[35S]methionine-labeled full-length Ku70 or Ku86.
Luciferase was a negative control. Bound proteins were resolved by
SDS-PAGE and detected by autoradiography.
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Because regulatory subunits Ku70 and Ku86 often interact with proteins
that regulate DNA-PK kinase activity (17, 19), an in vitro
GST pull-down assay was carried out to compare the potential direct
interactions of Ku subunits with TRBP. Interestingly, as shown in Fig.
3B, whereas interaction of TRBP with Ku 70 was very strong,
interaction with Ku86 was absent. Negative controls including
luciferase and GST alone did not yield signals. Notably, the strongest
binding of TRBP was not to the full-length Ku70 but rather to fragments
of Ku70. This may due to a regulated binding of full-length Ku70 with
TRBP. These data confirm that TRBP interacts with DNA-PK, possibly
through its Ku70 subunit.
DNA-independent Phosphorylation of TRBP C Terminus by
DNA-PK--
The data presented above indicated that TRBP might have a
close relationship with DNA-PK. To understand this interaction further, we first examined the possibility that TRBP is a substrate of the
kinase. Recombinant TRBP fragments were used as substrates in a DNA-PK
kinase assay, as shown in Fig.
4A. Whereas the control GST
alone was not phosphorylated, GST-TRBP C terminus was phosphorylated by
DNA-PK. The GST-p53 was used as a positive control for
DNA-dependent phosphorylation. Remarkably, the
phosphorylation of TRBP-5 or TRBP-6, which overlaps at C terminus, was
DNA-independent. The TRBP middle region (TRBP-3) can be phosphorylated
but in a DNA-dependent manner. Other TRBP regions appeared
not to be good substrates for DNA-PK (data not shown). Together, these
results suggest that TRBP-C can be phosphorylated by DNA-PK in a
DNA-independent manner. This differs from the phosphorylation of p53 or
TRBP-3, which is largely DNA-dependent.

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Fig. 4.
DNA-independent phosphorylation of TRBP C
terminus by DNA-PK. A, GST-TRBP fusion proteins
were used as substrates in DNA-PK kinase assays in the absence or
presence of linear double strand (ds) DNA as DNA-PK
activator. GST alone and GST-p53 were negative and positive controls.
Phosphorylated GST fusion proteins ([ -32P]ATP-labeled)
were washed and resolved on the gel followed by autoradiography.
B, comparison of TRBP-3 and TRBP-6 in DNA-PK
phosphorylation assays in the absence of DNA ( ) or the presence of
double strand (ds) or supercoiled plasmid (sc)
DNA as activators. Wortmannin (1 µM) was used as a DNA-PK
inhibitor. C, similar phosphorylation assays were
performed using different amounts of DNA-PK (10, 50, and 250 units).
Double strand DNA and wortmannin (1 µM) used are as
indicated. Closed arrows indicate the bands produced by
DNA-dependent phosphorylation, and open arrow
indicates the lower migrating bands produced by DNA-independent
phosphorylation.
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A closer examination of the TRBP phosphorylation patterns revealed that
the DNA-independent phosphorylation produced a slower migrating band
than DNA-dependent phosphorylation (Fig. 4, A
and B). Due to the smaller size of TRBP-6, which was more
obvious for the upper band than TRBP-5, we compared phosphorylation
patterns of TRBP-3 and TRBP-6 in assays shown in Fig. 4, B
and C. The DNA-independent phosphorylation of TRBP-6 had two
phosphorylation bands. Both phosphorylation bands can be inhibited by
wortmannin, a PI3K inhibitor that blocks the kinase activity of DNA-PK,
indicating the phosphorylation was DNA-PK-specific. The data suggest
that the TRBP C terminus may, by itself, function as an activator to
stimulate DNA-independent kinase activity. In addition, the
TRBP-activated kinase may phosphorylate TRBP at different site(s) than
the DNA-activated kinase to yield a band with slower mobility on the
gel. This indicates that there is a potential differential substrate
specificity of DNA-PK and, depending on the activator, is either TRBP
or linear DNA ends.
Compared with DNA ends, TRBP might be a unique stimulator of DNA-PK. We
reached this conclusion by excluding mechanisms that might stimulate
DNA-PK other than TRBP. TRBP itself did not exert any kinase activity,
as a smaller amount of DNA-PK enzyme was not able to produce the signal
(Fig. 4C). Similar to ds DNA, supercoiled plasmid DNA did
not produce a lower mobility band (Fig. 4B), suggesting that
the phosphorylation site(s) might be different for the protein activator. We also washed GST-TRBP resin with high salt to prevent any
protein contamination in the assay. In addition, we compared assays
with pretreatment of TRBP resin with DNase or RNase to prevent
potential bacterial nucleic acid contamination during the isolation of
GST fusion protein. In any case, however, nonspecific DNA contamination
would not produce a lower mobility band. Thus, these results are all
consistent with our conclusion that TRBP protein is responsible, at
least in part, for the DNA-independent activation of DNA-PK.
TRBP C Terminus Stimulates DNA-independent Activity of
DNA-PK--
To this end, we sought further evidence for
TRBP-stimulated activity of DNA-PK. In TRBP-stimulated kinase assays, a
biotinylated p53 peptide was used as a substrate, which can be
separated from TRBP on streptavidin-cellulose paper before
quantitation. As shown in Fig.
5A, the TRBP C
terminus, including TRBP-5 and TRBP-6, was able to stimulate kinase
activities in the absence of DNA, although the kinase was also
stimulated in the presence of DNA. When anti-TRBP antibodies, but not
when anti-Ku70 or anti-Ku86 antibodies, were included in the assay,
DNA-independent activities were significantly blocked (Fig.
5B). However, the antibodies had much less or almost no
effect on the DNA-dependent activities in the same assay.
Furthermore, in assays comparing the preimmune serum derived from the
same rabbit with two anti-TRBP antibodies (1652 and 1653), we
consistently showed that anti-TRBPs, but not preimmune sera, blocked
DNA-independent activities, and DNA-dependent activities
were not affected (Fig. 5C). Collectively, these results indicate that TRBP at its C-terminal region may contain a specific domain or motif, which is able to stimulate DNA-PK kinase activity by
interacting with the DNA-PK complex in the absence of DNA as an
activator.

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Fig. 5.
TRBP C terminus stimulates DNA-PK kinase
activity in the absence of DNA ends. DNA-PK kinase assays were
carried out using a biotinylated p53 peptide as substrate and GST-TRBP
proteins as activators as described under "Experimental
Procedures." Briefly, after the reaction, the
[ -32P]ATP-labeled biotinylated p53 peptides were captured by the streptavidin-cellulose papers and separated
from GST-TRBP proteins. The papers were then washed and counted in a
scintillation counter. A, each TRBP fragment was
compared for the activity that stimulates DNA-PK in the absence or the
presence of DNA. B, polyclonal anti-TRBP antibodies
1652 and 1653, prepared using TRBP fragment as antigen (aa 1641-2063),
were compared with anti-Ku antibodies for their capability to block
TRBP-5-stimulated DNA-PK activities. Anti-TRBP antibodies but not
anti-Ku antibodies blocked the DNA-independent activity.
C, anti-TRBP antibody (1652) and its preimmune serum
from the same rabbit were tested with TRBP-5 and TRBP-6 in a similar
kinase assay. GST alone and GST-TRBP-3 were negative controls. Assays
were performed in the absence or the presence of ds DNA.
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Unexpectedly, during the course of studies with the antibodies that
might affect DNA-PK, we discovered that an anti-DNA-PKcs antibody alone
was able to activate DNA-PK in the absence of any activators. Fig.
6 showed that the combination of this
antibody and TRBP protein can produce a robust DNA-independent activity that is comparable with the level of DNA-stimulated activity. The
anti-TRBP again blocked the kinase activity, and preimmune serum and
anti-Ku70 or anti-Ku80 antibodies had no effect, which were used as
controls. Although anti-DNA-PKcs-stimulated DNA-PK does not occur
in vivo, the data indeed indicate that a combination of
protein interactions, TRBP and anti-DNA-PKcs, for example, might result
in a conformational change of DNA-PK and subsequently stimulate the
kinase with activity comparable with DNA-induced activity. These data
suggest that DNA-PK might be regulated in vivo by
interacting proteins as well as linear DNA ends. Because TRBP plays an
important role in transcription, the direct stimulation by TRBP, or
combined with other potential stimulatory proteins, may activate DNA-PK
in the absence of DNA ends in transcriptional regulation.

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Fig. 6.
Synergistic stimulation of DNA-PK activity by
TRBP and anti-DNA-PKcs. DNA-PK kinase assay was performed using a
biotinylated p53 peptide as substrate in the absence of DNA. TRBP-5 was
used as an activator. The phosphorylated biotinylated p53 peptide can
be separated from TRBP using streptavidin-cellulose papers, and kinase
activities were measured by counting in a scintillation counter.
Anti-TRBP, its preimmune serum, anti-Ku, and anti-DN-PKcs antibodies
were added as indicated. Anti-DNA-PKcs antibody alone can stimulate
kinase activity, and the stimulation was synergistic with TRBP
protein.
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Interrelationship of TRBP and DNA-PK in Cells--
Because TRBP
can stimulate the activity of purified DNA-PK in vitro, we
examined whether TRBP might also stimulate endogenous DNA-PK in cells.
As shown in Fig. 7A,
endogenous DNA-PK from HeLa nuclear extracts was isolated using TRBP
resin and the TRBP-bound DNA-PK activity was measured. Whereas DNA-PK
derived from crude HeLa nuclear extract exhibited strict
DNA-dependent activity, the TRBP-bound DNA-PK had
significant activity in the absence of DNA. These data indicated that
TRBP not only interacts with endogenous DNA-PK but is also able to
activate it. To determine whether the activation of DNA-PK is essential
for the transcriptional activity of TRBP in vivo, we
examined TRBP-mediated transcriptional activity in DNA-PK-deficient
cells. A mouse scid cell line, SCSV3, is deficient in DNA-PK
phosphorylation. The human chromosome 8-complemented scid
cell line, SCH8-1, contains the human DNA-PK catalytic subunit and has
restored DNA-PK phosphorylation (26, 27). When TRBP activity was
compared in these two lines, TRBP activity was dramatically decreased
in the DNA-PK-deficient SCSV3 cells compared with that observed in
SCH8-1 cells, which has restored DNA-PK activity (Fig. 7B).
Therefore, these results confirm the idea that TRBP may, in part, exert
its transcriptional activity in vivo through the DNA-PK
function, inasmuch as DNA-PK represents a critical enzymatic activity
in the transcriptional complex.

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Fig. 7.
TRBP stimulates endogenous DNA-PK in
cells. A, DNA-PK kinase assay was carried out
using GST-TRBP-bound DNA-PK from HeLa nuclear extracts. GST-TRBP beads
were incubated with HeLa nuclear extracts at 4 °C for 4 h and
washed three times with the binding buffer prior to assay. A
biotinylated p53 peptide was used as substrate in the presence or the
absence of DNA. GST and GST-TRBP-3 were negative controls, and HeLa
nuclear extract (NE, 2 µg) was the positive control.
Whereas DNA-PK from HeLa nuclear extract showed
DNA-dependent activity, TRBP-bound DNA-PK showed
DNA-independent activity. B, DNA-PK is required for
TRBP activity. Mouse SCSV3 (DNA-PK / , scid) and SCH8-1 (DNA-PK +/+)
cells were cotransfected with MMTV luciferase reporter (100 ng), GR (10 ng), and full-length TRBP (200 ng). After transfection, cells were
grown overnight in the presence or the absence of 100 nM
dexamethasone (DEX). Total amounts of DNA for each well were
equalized with additional vector pcDNA3. Data are shown as means of
triplicate transfections ± S.E.
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We also performed immunofluorescent studies to analyze the nuclear
localization of TRBP in DNA-PK-deficient cells. Mouse SCSV3 (DNA-PK
/
, scid) and SCH8-1 (DNA-PK +/+) cells were
methanol-fixed and double-stained with affinity-purified polyclonal
anti-TRBP and monoclonal anti-Ku70 antibodies. It appeared that the
distribution of TRBP as well as Ku complex were much more localized in
SCSV3 cells when compared with SCH8-1 cells, in which TRBP was more evenly spread out in the nucleus (Fig.
8). However, when the two images of TRBP
and Ku70 were merged as shown in the right panels of Fig. 8,
TRBP and Ku were seen to be more colocalized in SCH8-1 cells than in
SCSV3 cells. In DNA-PK-deficient SCSV3 cells, TRBP appeared to have a
more punctate pattern, which did not always match the Ku70 pattern.
Hence, TRBP had less colocalization with Ku70 in DNA-PK-deficient
cells. Although complex mechanisms in vivo may lie behind
the differences reflected in these localization patterns, it is
possible that a defect of DNA-PK activation leads to a defect of TRBP
phosphorylation as well as Ku phosphorylation (27). The absence of
DNA-PK may also affect the appropriate nuclear localization of its
interaction partners. In sum, the defect of DNA-PK function resulted in
diminished TRBP action in transcription (Fig. 7B), which
suggests that DNA-PK may be essential for TRBP activity in
vivo.

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Fig. 8.
TRBP has altered nuclear localization
in DNA-PK-deficient cells. Mouse cell lines SCSV3 (DNA-PK / ,
scid) and SCH8-1 (DNA-PK +/+) were methanol-fixed and double-stained
with affinity-purified polyclonal anti-TRBP (red) and
monoclonal anti-Ku70 (green). Secondary antibodies were
anti-rabbit Cy3- and anti-mouse fluorescein
isothiocyanate-conjugated antibodies. The merged images of the
two panels for TRBP and Ku70 are shown on the right.
DNA-PK-deficient cells exhibit a more restricted distribution of TRBP
and Ku70. Thus, TRBP exhibits less colocalization with Ku70 in
DNA-PK-deficient cells.
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Inhibition of DNA-PK Represses TRBP-mediated Transcription--
To
examine further whether DNA-PK may influence TRBP transcriptional
function, transient transfection assays, using HeLa cells, were carried
out with wortmannin, a PI3K inhibitor that inhibits DNA-PK. Fig.
9 shows that increasing amounts of
wortmannin inhibit TRBP-stimulated transcription when both full-length
TRBP and TRBP fragments containing the C terminus are tested. Although
wortmannin also inhibits other PI3K family numbers in addition to
DNA-PK, DNA-PK is one of the most abundant PI3Ks in the nucleus,
especially in HeLa cells. These data are consistent with the above
finding that DNA-PK may be involved in TRBP-mediated transcriptional
activation.

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Fig. 9.
Inhibition of DNA-PK represses
TRBP-stimulated transcription. HeLa cells were cotransfected with
MMTV luciferase reporter (100 ng), GR (10 ng), and full-length TRBP or
TRBP fragments as indicated (200 ng). TRBP-MC contains the TRBP
C-terminal region (amino acids 714-2063). TRBP-M does not contain the
C-terminal region (amino acid 714-1242), which is used as a control.
After transfection, cells were grown in the presence or the absence of
100 nM dexamethasone (DEX), and with different
amounts of wortmannin (0, 100, 500 nM) as indicated. Total
amounts of DNA for each well were equalized with additional vector
pcDNA3. Data are shown as means of triplicate transfections ± S.E.
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DISCUSSION |
The discovery of the interaction of TRBP with the DNA-PK complex
was initially serendipitous. During the analysis of coactivator CBP and
DRIP130 interactions with TRBP fragments, a 400-kDa abundant protein
from nuclear extracts, later revealed as DNA-PKcs by protein sequencing, was repeatedly seen to interact with the C terminus of
TRBP. This compelled further analysis of additional interacting proteins, which ultimately identified associated components including the DNA-PK complex. It does not, however, diminish the possibility that
other nuclear factors, possibly detected as minor bands on the gel,
could also directly or indirectly associate with TRBP, although these
proteins may be insufficiently abundant to permit appropriate sequence analysis.
DNA-PK has been extensively characterized as a nuclear PI3K involved in
regulating transcriptional activation, DNA repair, and
V(D)J recombination (12, 15, 26). The
two tightly associated regulatory subunits of DNA-PK, Ku70 and Ku80,
were originally identified as the autoantigens in autoimmune diseases.
Ku-deficient mice, phenotypically similar to DNA-PKcs-deficient mice,
are immunodeficient and sensitive to DNA damage (32-34). Ku is a
protein that binds DNA in a non-sequence-specific manner and regulates
DNA-PKcs catalytic activity. Although DNA was originally shown as a
potent activator of DNA-PKcs, recent studies (17, 19) indicate that the
multiple protein factors recruited by the Ku subunit may also serve as activators to stimulate DNA-PK activity. For instance, several homeodomain proteins, including Oct-1, have recently been shown (17) to
interact with Ku70 and enhance DNA-PK phosphorylation. Consistent with
their finding, TRBP interacts with Ku70 but not Ku80 in
vitro, with apparently high affinity (Fig. 3B). This
interaction may consequently result in stimulation of DNA-PK in the
absence of DNA (Figs. 4 and 5). The DNA-independent stimulation of
DNA-PK by protein factors was also observed in other proteins such as in C1D (18, 35). In addition, an anti-DNA-PKcs antibody synergistically activates DNA-PK together with TRBP (Fig. 6), strongly indicating that
DNA-PK can be fully activated in the absence of DNA. It appears that
although DNA-PK can be stimulated by DNA ends (36), or by other kinases
(37, 38), protein interactions via Ku70 may be another more important
but previously less appreciated mechanism for DNA-PK activation,
especially in transcriptional regulation.
In addition to DNA-PK subunits, several other proteins were also found
in the TRBP-interacting complex, including DNA topoisomerase I, NuMA,
PARP, and TCOF1, that have not been investigated in this study. These
factors may also associate with TRBP directly or indirectly. PARP is a
chromatin-associated protein that catalyzes the transfer of ADP-ribose
from NAD+ to nuclear proteins. PARP is the only
nuclear-ribosylating enzyme that can be phosphorylated by DNA-PK (39)
and, in turn, ribosylates DNA-PK. PARP is up-regulated in human tumors
(40, 41) and serves as a death substrate in apoptosis (42). PARP has
also been shown to associate with DNA-PK and regulate transcription including gene activation mediated by nuclear receptors (43-45). It is
possible that PARP associates with TRBP through the DNA-PK complex. In
addition to the DNA-PK complex, the C terminus of TRBP has been shown
previously (6, 9), by different approaches, to interact with
coactivators such as CBP/p300, DRIP components, and coactivator CoAA.
These interactions taken together may provide a mechanism for the
function of TRBP as a coactivator acting, in part, via its C terminus.
The substrate specificity of DNA-PK displays fewer sequence
characteristics. A number of phosphorylation sites, including sites in
the p53 substrate, are at serines or threonines followed by glutamines
(SQ or TQ) (46). However, many other substrates of DNA-PK have
phosphorylation sites other than (S/T)Q. RNA polymerase II
CTD can be heavily phosphorylated in vitro at serines that are not followed by glutamine (12, 23). It is currently unclear whether
the substrate specificity of DNA-PK has any correlation with its
stimulation properties, i.e. whether DNA-activated
phosphorylation results in distinct patterns than those
protein-activated. Interestingly, we observed that the TRBP-stimulated
DNA-independent phosphorylation produced an additional lower mobility
band that is distinguishable from products of DNA-stimulated
phosphorylation (Fig 4). A similar observation was reported using RNA
polymerase II CTD as substrate and Gal4 as an activator (23). The
substrate specificity and phosphorylation sites in these cases may be
altered, and the phosphorylation sites might be worth mapping in future
studies. These results, nevertheless, indicate a possible regulation of
DNA-PK substrate specificity by coactivators, which might be important
for the in vivo function if confirmed.
In addition to DNA repair and recombination, the involvement of DNA-PK
in transcription is evident (15, 19, 21). DNA-PK phosphorylates many
transcriptional factors including glucocorticoid receptor (GR) and
regulating the MMTV promoter (47). The DNA-PKcs- or Ku-deficient cells
are defective in transcription on multiple promoters tested in
vitro, and the deficiency of Ku leads to severe defects in
transcription (20). It is also noteworthy that there are
examples where protein factors are involved in the regulation of DNA-PK
in transcription. A transcriptionally active Gal4 domain was previously
shown to stimulate DNA-PK phosphorylation of RNA polymerase II CTD (22,
23). Homeodomain-containing proteins stimulate DNA-PK via Ku70 (17). A
recent report (30) also suggested that a limited
Ku-dependent protein factor, which is not
template-associated, may be responsible for the reinitiation of
transcription. Together with our current studies, it appears that
DNA-PK is targeted by multiple stimulating factors, such as
coactivators, and functions as a signaling kinase for a variety of
nuclear functions including transcription.
Mice with scid have a nonfunctional immune system due to a
defect of V(D)J recombination and
double strand break repair (16). Genetic analysis of
scid mice revealed the presence of mutations in the
catalytic domain of DNA-PK. Mouse scid cells are also
defective in phosphorylation of DNA-PK substrates such as Ku (27). In scid cells, both TRBP phosphorylation by DNA-PK and
TRBP-stimulated DNA-PK activation may be abolished. This likely
explains the severe defect of TRBP-stimulated transactivation (Fig.
7B), because DNA-PK may synergistically be responsible for
the regulation of transcription. Consistent with this, previous
evidence has suggested that transcription level of multiple genes is
profoundly decreased in scid cells (21). It is also
interesting that the localization of nuclear components, including
TRBP, may be altered in scid cells (Fig. 8). DNA-PK activity
might be required for the phosphorylation of a number of nuclear
proteins including TRBP and Ku70. Hence, the phosphorylation states may
determine nuclear localization, which, in turn, may affect function.
Restored DNA-PK activity in SCH8-1 cells, however, rescues these
defects and promotes the coactivator function of TRBP.
In summary, as the machinery for transcription, recombination, and DNA
repair may functionally overlap and share similar factors (31, 48),
DNA-PK may be one of the critical and versatile components. Consistent
with this view, TRBP may stimulate DNA-PK and, in turn, phosphorylate
proteins in the transcriptional complex.