The DNA-dependent protein kinase
(DNA-PK) is composed of a large catalytic subunit of approximately 470 kDa (DNA-PKcs) and the DNA-binding protein, Ku. Absence of DNA-PK
activity confers sensitivity to x-rays and defects in both DNA
double-strand break repair and V(D)J recombination. However, the
precise function of DNA-PK in DNA double-strand break repair is not
known. Here we show, using electrophoretic mobility shift assays, that
polypeptides in a fraction purified from human cells interact with
DNA-PK and stabilize the formation of a complex containing DNA-PKcs-Ku
and DNA. Five polypeptides in this fraction have been identified by amino-terminal sequence analysis and/or immunoblotting. These proteins
are NF90 and NF45, which are the 90- and 45-kDa subunits of a protein
known to bind specifically to the antigen receptor response element of
the interleukin 2 promoter, and the
,
, and
subunits of
eukaryotic translation initiation factor eIF-2. We also show that NF90,
NF45, and eIF-2
are substrates for DNA-PK in vitro. In
addition, recombinant NF90 promotes formation of a complex between
DNA-PKcs, Ku, and DNA, and antibodies to recombinant NF90 or
recombinant NF45 immunoprecipitate DNA-PKcs in vitro. Together, our data suggest that NF90, in complex with NF45, interacts with DNA-PKcs and Ku on DNA and that NF90 and NF45 may be important for
the function of DNA-PK.
 |
INTRODUCTION |
The DNA-dependent protein kinase,
DNA-PK,1 is composed of a
large catalytic subunit of ~470 kDa (DNA-PKcs) and a DNA binding heterodimer of approximately 70- and 80-kDa subunits called Ku (reviewed in Refs. 1-4). The catalytic subunit, DNA-PKcs, belongs to
the phosphatidyl inositol 3 kinase family of proteins (5) that includes
ATM (the gene product defective in ataxia telangiectasia) and the
FKBP-rapamycin-binding protein, FRAP (reviewed in Refs. 6-8). DNA-PK
acts as a serine/threonine protein kinase, and several in
vitro protein substrates have been identified (reviewed in Refs.
1, 2, and 4). DNA-PK requires ends of double-stranded DNA for activity
(1, 9, 10), although hairpins, dumbbells, and DNA constructs containing
single-stranded to double-stranded transitions also activate DNA-PK
in vitro (11). Absence of any of the DNA-PK protein
components confers sensitivity to ionizing radiation and defects in
both DNA double-strand break repair and V(D)J recombination (reviewed
in Refs. 3, 12, and 13). Although DNA-PK is essential for these
processes in vivo, its precise function is at present
unknown. One possibility is that upon binding of Ku to a suitable DNA
end, DNA-PKcs is recruited to form the active kinase, which may then
phosphorylate itself and/or other molecules that are required for DNA
end rejoining. Alternatively, the DNA-PK complex may act as a scaffold
to which other proteins required for DNA repair may be recruited. In
addition to its role in DNA repair, DNA-PK may also play a role in the regulation of transcription by both RNA polymerases I and II
(14-17).
To determine the role of DNA-PK in these processes, it is essential to
understand how the kinase interacts both with DNA and with other
protein partners. The interaction of Ku with ends of DNA has been
extensively studied. Ku binds with high affinity to free ends of
double-stranded DNA (18-22) as well as to nicked DNA (22, 23),
hairpins, and dumbbell structures in vitro (23). Studies of
the interaction of Ku with DNA ends have relied in large part on the
electrophoretic mobility shift assay (EMSA). Using this assay, Ku forms
multiple protein-DNA complexes, the number of which depends on the
length of the DNA and the amount of Ku protein (19, 22). In contrast,
relatively little is known about the formation of the DNA-PKcs-Ku-DNA
complex. Several observations suggest that the interaction between
DNA-PKcs and Ku in the absence of DNA is transient or weak. For
example, the DNA-PKcs monomer and the Ku dimer purify separately during
chromatography under mild nondenaturing conditions (9, 10, 24, 27). In
addition, DNA is required for co-immunoprecipitation of purified DNA-PKcs and Ku in vitro (26, 27), and antibodies have been reported to stabilize the interaction of DNA-PKcs with Ku (28). Also,
little is known about the interaction of Ku and DNA-PKcs with other
proteins. Two hybrid screens using either Ku70 or Ku80 as bait have
identified the corresponding Ku partner but not DNA-PKcs (29). Other,
similar studies have revealed that the product of the vav
proto-oncogene interacts with Ku (30); however, the significance of
this observation is not known.
Here we used an EMSA to show that the interaction of highly purified
DNA-PKcs with Ku and DNA is weak or transient and that several
polypeptides that partially copurify with DNA-PKcs can stabilize
complex formation among DNA-PKcs, Ku, and DNA. Five polypeptides have
been identified by protein sequence analysis and/or Western blot as
NF45 and NF90, which are the 45- and 90-kDa subunits of a protein
previously shown to bind specifically to the interleukin 2 promoter in
activated T cells (31, 32) and the
,
, and
subunits of
eukaryotic initiation factor eIF-2, a heterotrimeric protein that is
required for the initiation of protein synthesis (reviewed in Ref. 33).
We provide evidence that NF90 and NF45 interact with DNA-PK in
vitro and that NF90, NF45, and the
subunit of eIF-2 are
phosphorylated by DNA-PK in vitro. Our data suggest that
NF90 and NF45 interact with DNA-PKcs and Ku to form a supercomplex at
the ends of DNA and that this interaction may be important for the
function of DNA-PK.
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MATERIALS AND METHODS |
Electrophoretic Mobility Shift Assay--
Conditions for the
electrophoretic mobility shift assay were similar to those of Blier
et al. (22), with the following modifications. The plasmid
vector pGEM 7Zf+ (Promega) was propagated in JM109 and
harvested and purified using Qiagen columns according to the
manufacturer's instructions. DNA probes of 40, 80, and 102 bp were
generated by digestion of plasmid DNA with HaeIII.
Oligonucleotides were separated using MonoQ 5/5HR chromatography using
a linear gradient of 0.61-0.79 M NaCl in 20 mM
Tris-HCl (0.15 ml/min, 0.5 mM NaCl/min) for 360 min
according to the manufacturer's recommendations (Pharmacia FPLC
Application File: Restriction DNA Fragments) and purified using a
MERmaid DNA purification kit (Bio 101 Inc., La Jolla, CA). Also,
oligonucleotides corresponding to the 40-bp HaeIII fragments
were synthesized and gel purified by the DNA sequence facility,
University of Calgary. The sequence of the 40-bp oligonucleotide used
was 5
-CCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGG-3
. Synthesized oligonucleotides were combined in equimolar amounts in TE (10 mM Tris-HCl, 1 mM EDTA), pH 8.0, containing 50 mM NaCl, heated to 95 °C for 10 min and annealed
overnight at room temperature. Oligonucleotides were end labeled with
[
-32P]ATP (Easy-tide, NEN Life Science Products) using
polynucleotide kinase (Life Technologies, Inc.) according to the
manufacturer's instructions. Unincorporated nucleotide was removed
using Microspin-G50 columns (Pharmacia) or Centricon 30 microconcentrators (Amicon) using TE, pH 8.0, as buffer.
DNA binding reactions usually contained purified Ku (5-10 ng) and/or
purified DNA-PKcs (15-30 ng) in a buffer consisting of 25 mM Hepes, pH 7.5, 50 mM KCl, 10% glycerol, 2 mM DTT. Radiolabeled DNA probe (generally about 15 fmol;
i.e. 0.3 ng of DNA, 40,000 dpm) was added, and reactions
(final volume, 20 µl) were incubated for 10 min at RT. Samples were
next subjected to electrophoresis directly or, where indicated,
purified protein fractions or chemical cross-linkers were added and
samples were incubated for a further 5 min at RT prior to
electrophoresis. Bis(sulfosuccinimidyl) suberate (BS3)
(Pierce), is a water-soluble cross-linker of spacer length 11.4 Å.
BS3 is not cleaved by reducing agents. BS3 was
added to 1.2 mM (as per the manufacturer's recommendation of a final concentration of 0.2-5 mM) after preincubation
of proteins and DNA. Glutaraldehyde (E. Merck, Darmstadt, Germany; 25%
solution, electron microscopy grade) was freshly diluted in water to
2.5% and then added to binding reactions containing protein and DNA probe to a final concentration of 0.06% (v/v). No loading dyes were
added to samples prior to electrophoresis. Samples were analyzed on
nondenaturing gels (3.9% acrylamide: 0.1% bisacrylamide) in 50 mM Tris-HCl, 0.382 M glycine, pH 8.0, buffer.
Gels were prerun for 1 h at 200 V immediately prior to loading,
and samples were analyzed after electrophoresis for a further 2 h
at 200 V. Gels were dried and exposed to Fuji x-ray film at
80 °C
with intensifying screens. In early experiments, EDTA was added to 1 mM in binding reactions and 0.5 mM in gels and
running buffer; however, presence or absence of EDTA had no effect on
the formation and migration of gel shift complexes.
For EMSA experiments using antibodies, total IgG was purified from
rabbit preimmune or immune sera as described (35). Protein concentrations were determined using the Bio-Rad dye reagent assay as
described previously (25). Purified proteins were incubated with
DNA-PKcs, Ku, and DNA as described, followed by addition of purified
IgG. Incubations were continued for a further 15 min at RT prior to
electrophoresis as described above.
Purification of Proteins--
DNA-PKcs and Ku were purified from
human placenta over three ion exchange resins followed by
chromatography on dsDNA-cellulose as described previously (25). Using
this methodology, DNA-PKcs and Ku fractionated into two pools, referred
to as pool A and pool B, respectively (25). Pool A contained six other
predominant polypeptides with molecular masses of approximately 90, 75, 52, 50, 45, and 37 kDa. Polypeptides in pool A were further
chromatographed on MonoQ FPLC (Pharmacia) in pH 8.0 buffer containing
50 mM Tris-HCl, 5% glycerol (v/v), 0.2 mM
EDTA, 1 mM DTT, 0.02% Tween-20 (v/v), and KCl. Proteins
were applied to the column in 0.1 M KCl and eluted with a
linear gradient of 0.1-0.5 M KCl at 4 mM
KCl/min. DNA-PKcs eluted from MonoQ FPLC at approximately 150-200
mM KCl and was further purified over MonoS FPLC as
described (25), followed by hydrophobic interaction chromatography
using a phenyl superose HR5/5 FPLC column (Pharmacia). DNA-PKcs was
applied to the phenyl superose column in Buffer A (50 mM
Tris-HCl, pH 8.0, containing 50 mM KCl, 1 M
NH4SO4, 1 mM DTT) and eluted with a linear gradient of Buffer A to Buffer B (containing 50 mM
Tris-HCl, pH 8.0, 50 mM KCl, 1 mM DTT) at 0.7%
Buffer B/min. The peak of DNA-PKcs protein eluted at approximately 680 mM ammonium sulfate and was homogeneous when analyzed by
silver staining of SDS gels. The 90-, 75-, 52-, 50-, 45-, and 37-kDa
polypeptides eluted from MonoQ FPLC at approximately 250-300
mM KCl and were combined to form pool C (see Fig. 2). Pool
C was dialyzed and applied to MonoS FPLC under exactly the same buffers
and conditions as described above for MonoQ chromatography. The 90-, 75-, 52-, 50-, 45-, and 37-kDa polypeptides eluted from MonoS FPLC as a
broad peak at approximately 200-300 mM KCl (see Fig. 3).
Fractions containing these polypeptides were either used alone or
combined as indicated. Ku was purified as described previously (25).
Purified proteins were dialyzed against 25 mM Hepes, 100 mM KCl, 1 mM DTT, pH 7.5, concentrated using
Centricon 30 microconcentrators (Amicon) and stored in aliquots at
80 °C unless otherwise indicated. All purification steps were
performed at 4 °C. FPLC steps were performed at either RT or 4 °C
with no detectable difference in results. Phenylmethylsulfonyl fluoride, leupeptin, and aprotinin were present at 0.2 mM,
10 µg/ml, and 10 µg/ml, respectively, in all buffers and samples up
to DEAE. Phenylmethylsulfonyl fluoride (0.2 mM) was present in buffers up to and including the FPLC steps.
Immunoprecipitation--
Approximately 20 µg of total protein
in the fraction pre-dsDNA cellulose was incubated under the conditions
described for EMSA, either with or without 40-bp dsDNA at 5 µg/ml.
Antibodies to DNA-PKcs, NF45, NF90, SV40 TAg, or preimmune serum were
added as indicated, and immunoprecipitations were carried out under nondenaturing conditions as described (27, 35) except that protein
G-Sepharose (Pharmacia) was used. Immunoprecipitated proteins were
detected by Western blot as described.
Electrophoresis and Western Blot--
SDS gels and Western blot
for analysis of DNA-PKcs were as described (25) except that transfer
buffer contained 20% methanol. For all other polypeptides,
electrophoretic transfer for Western blot was in 25 mM
Tris, 200 mM glycine, 20% methanol at 100 V for 35 min.
Blots were developed was using Enhanced Chemiluminescence (Amersham
Corp.) according to the manufacturer's instructions. DPK1 serum was as
described (34). Antisera to NF90 and NF45 were as described (32).
Antisera to eIF-2
and
were a generous gift from Dr J. Hershey
(University of California, Davis).
Protein Sequence Analysis--
Approximately 10 µg of each of
the polypeptides eluting from MonoS FPLC corresponding to fractions
44-64 (see above and Fig. 3) was run on an SDS 10% polyacrylamide gel
that had been pre-aged for 24 h and analyzed by electrophoresis
under normal conditions. Polypeptides were then transferred to
polyvinylidene difluoride membrane (Bio-Rad; sequencing grade, 0.2 µm) in 10 mM CAPS, 10% methanol (v/v), pH 11; as
described (36). The polypeptides were visualized briefly in freshly
prepared Coomassie Blue stain and briefly destained in 50% (v/v)
methanol 0.05% (v/v) acetic acid. The appropriate bands were excised
and sent for protein sequence analysis to either the University of
Wisconsin Biotechnology Center (Madison, WI) (p50/52 and p37) or the
University of Victoria Sequencing Center (Victoria, British Columbia,
Canada) (p75 and p45).
Phosphorylation Reactions--
Phosphorylation reactions were as
described previously (27). 0.5-1 µg of purified protein was
incubated in the presence or absence of purified DNA-PKcs, Ku, and DNA
as indicated and analyzed by SDS-PAGE and autoradiography.
Purification of Recombinant Proteins--
Expression and
purification of recombinant His-tagged NF90 (rNF90) and NF45 (rNF45) in
bacteria was as described previously (32) with some modification.
Briefly, proteins were induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside and harvested
using standard methodologies. As found previously (32), both rNF45
and rNF90 were insoluble and were resolubilized from the inclusion
bodies in 8 M urea, 50 mM Tris-HCl, pH 8.0. Resolubilized proteins in 8 M urea were diluted with an
equal volume of 50 mM Tris-HCl, pH 8.0, and loaded onto
Ni2+-NTA resin (Qiagen) that had been equilibrated in the
same buffer. The chelation column was washed with 50 mM
Tris-HCl, pH 8.0, containing 10 mM imidazole, and rNF90 and
rNF45 were eluted with 50 mM Tris-HCl, pH 8.0, containing
200 mM imidazole. Purity was estimated at >95% by
Coomassie Blue staining. Purified proteins were dialyzed into 50 mM Tris-HCl, 50 mM KCl, 5% glycerol, 1 mM DTT and stored in aliquots at
80 °C.
 |
RESULTS |
Interaction of Highly Purified DNA-PKcs with Ku and DNA Is
Stabilized by Chemical Cross-linkers--
The interaction of Ku with
DNA has long been studied using the electrophoretic mobility shift
assay (18-22); however, relatively little is known regarding the
interaction of DNA-PKcs with the Ku-DNA complex. Here we describe an
EMSA that can be used to detect complexes between highly purified
DNA-PKcs, Ku, and DNA. EMSAs were usually carried out using a 40-bp ds
deoxyoligonucleotide probe that was either purified after
HaeIII digestion of pGem plasmid DNA or was synthesized
chemically and annealed prior to labeling with
[
-32P]ATP as described under "Materials and
Methods." The sequence of this oligonucleotide has no known specific
binding sites and therefore is used as a source of free (blunt) dsDNA
ends. Under the conditions used in our assay, the purified Ku
heterodimer forms two complexes with the 40-bp DNA probe (labeled
a and b in Fig. 1,
lane 2). Similar protein-DNA complexes have been observed in
many previous studies and have been interpreted as being due either to
Ku binding to both ends and internal sequences of DNA or to the
formation of Ku multimers on the DNA (18-22).

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Fig. 1.
Ku/DNA-PKcs/DNA complex formation on EMSA is
stabilized by chemical cross-linkers. Highly purified Ku (5 ng)
was incubated with DNA alone (lanes 2, 8, and 14)
or with highly purified DNA-PKcs and DNA (lanes 4-7,
10-13, and 16-19). Samples in lanes 3, 9, and 15 contained DNA-PKcs (30 ng) and DNA. Purified DNA-PKcs was present at 10 ng in lanes 4, 10, and 16; 20 ng in lanes 5, 11, and 17; 30 ng in lanes 6, 12, and 18 or 50 ng in lanes 7, 13, and 19. Lane
1 contained probe alone. All samples were incubated with a
radiolabeled 40-bp DNA probe for 10 min at RT. Samples in lanes
1-7 were analyzed directly on nondenaturing gels as described under "Materials and Methods." Samples in lanes 8-13
were treated with glutaraldehyde (final concentration, 0.06% (v/v))
for an additional 5 min at room temperature prior to electrophoresis. Samples in lanes 14-19 were incubated in the presence of
the chemical cross-linker BS3 at 1.2 mM for an
additional 5 min at room temperature prior to electrophoresis.
Lowercase a and b indicate Ku-DNA complexes
formed in the absence of chemical cross-linker. Ku-DNA complexes formed in the presence of cross-linker are designated A and
B. The putative DNA-PKcs-Ku-DNA complex is designated
C.
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We next used this assay to examine the interaction of highly purified
DNA-PKcs with Ku and DNA. The stoichiometry of the interaction between
Ku and DNA-PKcs is not known; however, roughly equal amounts of
DNA-PKcs monomer (470 kDa) and Ku heterodimer (156 kDa) are required
for maximum catalytic activity (11, 25). For our studies, we therefore
used amounts of purified DNA-PKcs and Ku that gave molar ratios of
approximately 1. In the presence of a 1.3-fold molar excess of DNA-PKcs
to Ku, the predominant bands seen on EMSA migrated in the same position
as the bands formed with Ku alone (Fig. 1, lanes 4-7);
however, a faint band of slower mobility was often observed (labeled
C in Fig. 1, lanes 6 and 7). Addition
of a 2.5-fold molar excess of DNA-PKcs did not result in increased
formation of band C under these conditions (data not shown). Variation
of the salt concentration between 50 and 150 mM and
inclusion of up to 10 mM DTT in the binding reaction had
little or no effect on the formation of complex C under these conditions. Addition of magnesium chloride at 1-5 mM had
no significant effect on complex formation (data not shown). At least
five different preparations of highly purified DNA-PKcs and Ku were
used for EMSA reactions, and in no case was a strong band observed at
position C.
We therefore explored the possibility that protein cross-linking agents
might enhance the formation of a DNA-PKcs-Ku-DNA complex. Recent
studies using EMSA to analyze complexes formed between p53 and its
cognate DNA binding sequence have used low concentrations of
glutaraldehyde to stabilize the protein-DNA complex (37). Under the
conditions used by Tegtmeyer and colleagues (37), protein cross-linking
was specific for p53 and DNA. We therefore investigated the use of
glutaraldehyde on the formation of the DNA-PKcs-Ku-DNA complex.
Purified DNA-PKcs and Ku were incubated with DNA as above, and after 10 min, glutaraldehyde was added to a final concentration of 0.06% (v/v).
After a further 5 min of incubation, the sample was analyzed by
electrophoresis as described above. Addition of glutaraldehyde to Ku
resulted in a slightly increased mobility of the Ku-DNA complexes (Fig.
1, complexes A and B; compare lane 8 to lane 2), consistent with formation of a more compact
structure (37). Significantly, addition of glutaraldehyde to reactions
containing DNA-PKcs and Ku resulted in increased amounts of a complex
with slower mobility that migrated in the approximate position of
complex C (Fig. 1, lanes 10-13). These data suggest that
glutaraldehyde stabilizes or promotes the formation of a complex
between DNA-PKcs-Ku and DNA. Because glutaraldehyde may promote
formation of nonspecific protein-protein and protein-DNA interactions,
we used the chemical cross-linker BS3 in place of
glutaraldehyde. BS3 interacts with free amino groups over a
distance of approximately 11 Å. Like glutaraldehyde, addition of
BS3 to reactions containing DNA-PKcs, Ku, and DNA resulted
in a significant increase in the amount of the slower migrating complex
C (Fig. 1A, lanes 16-19). Similar results were obtained
using a 80-bp dsDNA oligonucleotide but not with a 20-bp DNA molecule
(data not shown), suggesting that about 40 bp is required for stable complex formation in this assay. Chemical cross-linkers did not promote
indiscriminate cross-linking between DNA-PKcs and DNA (Fig. 1) between
BSA and either Ku or DNA-PKcs, or among heat-denatured DNA-PKcs, Ku,
and DNA (data not shown), suggesting that under these conditions, the
observed interactions are specific. Formation of protein-DNA complexes
after cross-linking was greatly reduced by excess linear dsDNA but not
by closed circular plasmid DNA (data not shown). These properties are
consistent with the known properties of Ku-DNA complexes (22) and are
consistent with the known properties of DNA-PK.
Identification of Polypeptides That Interact with DNA-PK--
One
of the proposed functions of DNA-PK is to recruit other proteins to the
site of DNA damage. The inability of highly purified DNA-PKcs to form a
stable complex with Ku and DNA in the absence of chemical cross-linkers
suggested to us that this EMSA might be used to detect proteins that
interact with and stabilize the DNA-PKcs-Ku-DNA complex. We previously
described the purification of DNA-PK from HeLa cells (24) and human
placenta (25). In each case, several polypeptides co-fractionated with
DNA-PKcs on double-stranded DNA-cellulose chromatography, and several
of these polypeptides were phosphorylated by DNA-PK in vitro
(24). Currently, our laboratory purifies DNA-PK from human placenta (25), and because polypeptides of a similar size were found to
co-purify with DNA-PKcs during this purification procedure also, we
were curious as to whether these polypeptides might interact with the
DNA-PKcs-Ku-DNA complex.
Briefly, the purification scheme involves homogenization of whole
placenta in high salt buffer and ammonium sulfate precipitation followed by anion and cation exchange chromatography. These steps are
followed by a second round of chromatography on a weak anion exchange
resin (DEAE-CL6B) in the presence of magnesium. Under these conditions,
DNA-PKcs, Ku, and several other polypeptides no longer bind to the
resin, whereas the majority of proteins are retained on the DEAE resin
as in the initial anion exchange step (24, 25). In the next
purification step, DNA-PK and the associated polypeptides from the flow
through of the magnesium-DEAE column were fractionated by gradient
elution on double-stranded DNA cellulose. Several polypeptides in
addition to DNA-PKcs eluted from dsDNA cellulose chromatography between
0.2 and 0.4 mM KCl, including polypeptides of approximately
90, 75, 52, 50, 45, and 37 kDa. These fractions have previously been
designated as pool A (25). Polypeptides in pool A were further
chromatographed on MonoQ FPLC as described under "Materials and
Methods." DNA-PKcs eluted from MonoQ FPLC at approximately 150-200
mM KCl, and the fraction containing the 90-, 75-, 52-, 50-, 45-, and 37-kDa polypeptides eluted at approximately 250-300
mM KCl (Fig. 2, pool
C). The SDS-PAGE conditions used often failed to resolve the 52- and 50-kDa polypeptides, in which case they appeared as a single band,
which is labeled p50/52 (see, for example, Fig. 2).

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Fig. 2.
SDS-PAGE of fractions from MonoQ FPLC.
Pool A from dsDNA cellulose gradient chromatography was dialyzed and
applied to MonoQ FPLC as described under "Materials and Methods."
10 µl of each fraction was analyzed by SDS 10% PAGE followed by
Coomassie Blue staining. Fractions eluting between approximately 0.1 and 0.3 M KCl are shown. Molecular mass markers (in kDa)
are indicated on the left. Approximate molecular masses of
prominent polypeptides are indicated on the right. Fractions
pooled for subsequent MonoS FPLC are indicated by pool
C.
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Polypeptides in pool C were then further purified over MonoS FPLC as
described under "Materials and Methods." Silver staining of
fractions 42-64 from MonoS chromatography again revealed six major
polypeptides migrating at approximately 90, 75, 50, 52, 45, and 37 kDa
(Fig. 3). No DNA-PKcs or Ku was present
in these MonoS fractions as judged by lack of DNA-PK activity and by
the absence of DNA-PKcs and Ku proteins by Western blot (data not shown). Fractions 44-64 in Fig. 3 were pooled and assayed for their
ability to be phosphorylated by DNA-PK. Fig.
4A shows a Coomassie-stained
SDS-10% acrylamide gel of the phosphorylation reactions, and Fig.
4B shows the corresponding autoradiogram. In the absence of
added DNA-PKcs and Ku, no endogenous phosphorylation was observed, but
addition of DNA-PK resulted in DNA-dependent phosphorylation of the 90-, 50/52-, and 45-kDa polypeptides (Fig. 4,
A and B, lanes 5 and 6). Under the
conditions used, p90 and p75 were separated from phosphorylated Ku80;
however, p50 and p52 co-migrated. A similar sample was therefore run
for a longer period of time to separate the 50- and 52-kDa polypeptides
(Fig. 4, C and D). These data show that p50 is
phosphorylated by DNA-PK, whereas p52 and p37 are not.

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Fig. 3.
SDS-PAGE of fractions from MonoS FPLC.
Pool C (from Fig. 2) was dialyzed and applied to MonoS FPLC
as described under "Materials and Methods," and fractions eluting
between approximately 110 and 350 mM KCl were analyzed by
SDS-PAGE using 10% polyacrylamide gels. A silver-stained gel
representing 5 µl of fractions from 34 to 74 is shown. The positions
of molecular mass markers (in kDa) is shown on the left.
Approximate molecular masses of the prominent polypeptides are
indicated on the right. The poorly resolved 50- and 52-kDa
polypeptides indicated by p50/52 are partially separated in fractions
48-52. The letter X indicates artifacts of the silver
staining process.
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Fig. 4.
p90, p45, and p50 are phosphorylated by
DNA-PK in vitro. A, fractions 44-64 were pooled
and incubated under phosphorylating conditions with purified DNA-PKcs
and Ku. Samples in lanes 1 and 2 contained
purified DNA-PKcs and Ku; lanes 3 and 4 contained a pool of fractions 44-64 from MonoS FPLC; lanes 5 and
6 contained DNA-PKcs, Ku, and the MonoS pool. Samples in
odd-numbered lanes were incubated in the presence of DNA, and
even-numbered lanes were incubated without DNA. Samples were run on
10% polyacrylamide SDS-PAGE and stained with Coomassie Blue. p75 is
shown migrating below Ku80. B, autoradiogram corresponding
to the gel shown in panel A. C, a sample similar
to that shown in A and B was incubated under
phosphorylating conditions as described for A and then run on an SDS-10% polyacrylamide gel for 10 min after the dye had reached
the end of the gel to separate p50 and p52. Samples in lanes
1 and 2 contained the MonoS pool alone, and samples in
lanes 3 and 4 contained the MonoS pool plus
DNA-PK. Samples in odd-numbered lanes were incubated in the presence of
DNA, and samples in even-numbered lanes were incubated without DNA.
Shown is a portion of a silver-stained gel. The sample shown in
C contained a higher proportion of p50, p52, and p37 than
Ku70 and p45; hence, phosphorylation of Ku70 and p45 was diminished
relative to A and B. D, autoradiogram
corresponding to the gel shown in panel C.
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It is perhaps interesting to note that the extent of
autophosphorylation of DNA-PKcs and both Ku subunits is decreased in the presence of the added proteins from MonoS FPLC (Fig.
4B). We have previously shown that DNA-PK loses activity
when autophosphorylated and that loss of kinase activity is reduced in
the presence of a suitable peptide substrate (27). Our data suggest
that the presence of the 90-, 50/52-, and 45-kDa polypeptides may
reduce autophosphorylation and hence inactivation of DNA-PK. This would also suggest that these polypeptides may be better substrates for
DNA-PK than either DNA-PKcs or Ku.
To determine whether the polypeptides present in fractions 44-64 from
MonoS FPLC were able to interact with DNA-PK, these fractions were
added to purified DNA-PKcs and Ku and analyzed using the EMSA. In this
case, however, chemical cross-linkers were not included at
any point. Aliquots of individual fractions from MonoS FPLC were added
to the DNA probe (in the absence of chemical cross-linker) either alone
(Fig. 5A, lanes 2-9) or in the presence of Ku plus DNA-PKcs (Fig. 5A, lanes 12-19). In
the absence of DNA-PKcs and Ku, none of the MonoS fractions supported formation of a complex, although some retardation of the DNA probe was
observed (Fig. 5A, lanes 3-5). Addition of aliquots from
individual fractions 46-62 to reactions containing DNA-PKcs, Ku, and
DNA resulted in dramatic formation of a slower migrating protein-DNA complex at the expense of the Ku-DNA complexes a and b (labeled D in Fig. 5A, lanes 13-17). Addition of the
MonoS fractions to DNA-PKcs alone did not induce formation of
additional complexes (Fig. 5B). However, addition of the
MonoS fractions to Ku and DNA did result in enhanced formation of the
Ku-DNA complexes (Fig. 5B), suggesting that these
polypeptides may induce a modest change in the interaction of Ku with
DNA.

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Fig. 5.
EMSA of MonoS fractions 42-70 in the
presence and absence of DNA-PK proteins. A, fractions
eluting from MonoS FPLC were assayed for interaction with the DNA probe
(nonspecific sequence) in the absence or presence of DNA-PKcs and Ku.
Lane 1 contained free probe. Samples in lanes
2-9 contained 3 µl of fractions 42-70 (containing
approximately 30-45 ng of total protein) as indicated, plus DNA probe
alone. Samples in lanes 12-19 contained 3 µl of fractions
42-70 plus DNA-PKcs (30 ng), Ku (10 ng), and DNA. Lane 10 contained Ku (10 ng), and lane 11 contained Ku (10 ng) plus DNA-PKcs (30 ng). EMSAs were carried out in the absence of chemical cross-linkers, and electrophoresis was performed as described under
"Materials and Methods." B, samples contained the MonoS fractions, DNA, and either Ku (10 ng) or DNA-PKcs (30 ng) as indicated. Lane 1 contained probe alone. Lane 2 contained Ku
alone, and lanes 3-10 contained Ku plus the MonoS FPLC
fractions as indicated. Lane 11 contained DNA-PKcs alone,
and lanes 12-19 contained DNA-PKcs plus the MonoS FPLC
fractions as indicated.
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To confirm that DNA-PKcs was present in complex D, fractions 44-64
from MonoS FPLC were pooled and added to DNA binding reactions containing purified DNA-PKcs and Ku. Purified IgG from either immune
serum (antibody DPK1 raised to amino acids 2018-2135 of DNA-PKcs) or
preimmune serum was added to samples prior to electrophoresis as
described under "Materials and Methods." Addition of IgG from DPK1
serum but not from preimmune serum resulted in abrogation of complex D,
either by preventing complex formation or by supershifting complex D
into the well (Fig. 6, lanes 7 and 8). The presence of Ku in complexes a, b, and D was
confirmed by Western blot (data not shown).

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Fig. 6.
Antibodies to DNA-PKcs alter formation of
complex D on EMSA. Purified DNA-PKcs, Ku, and the pooled MonoS
FPLC fractions were preincubated in DNA binding buffer for EMSA,
followed by incubation with purified IgGs for a further 15 min as
indicated. Lane 1, probe alone; lane 2, purified
Ku (10 ng); lane 3, Ku (10 ng) plus DNA-PKcs (30 ng).
Lanes 4-8 contained Ku and DNA-PKcs as in lane 3 but with 30 ng of the combined MonoS fractions 44-64. Samples in
lanes 4-8 contained 0.1 µg (lane 5) or 0.2 µg (lane 6) of IgG from preimmune rabbit serum or 0.1 µg
(lane 7) or 0.2 µg (lane 8) of IgG from DPK1
serum. All samples were analyzed by electrophoresis without chemical
cross-linking as described in Fig. 5.
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These data strongly suggest that components of fractions 44-64 from
MonoS chromatography can interact with DNA-PKcs and Ku in
vitro and stabilize the formation of a larger protein-DNA complex that contains DNA-PKcs, Ku, and DNA. The polypeptide in these fractions
that most closely tracks with the ability to form the slower migrating
protein-DNA complex, complex D, is p90. However, because this activity
was spread over at least 17 fractions, multiple polypeptides could be
involved. We therefore proceeded to identify each of the major
polypeptides present in the MonoS fraction.
Identification of the Polypeptides in the MonoS
Fraction--
Approximately 10 µg of each of the six predominant
polypeptides found in fractions 44-64 from MonoS FPLC was transferred
to polyvinylidene difluoride membrane and analyzed by amino-terminal sequence analysis as described under "Materials and Methods." Amino-terminal sequence was obtained for four of the polypeptides, and
in each case, the sequence obtained corresponded precisely to known
gene products. In the protein preparation used to obtain the sequence,
considerably more p75 than p90 was obtained; therefore, only p75 was
sequenced. The amino-terminal 10 amino acids from polypeptide p75 were
identical to the amino terminus of a 90-kDa polypeptide known as NF90,
and polypeptide p45 was identified as NF45 (Table
I). NF90 and NF45 are 90- and 45-kDa
proteins, respectively, that were first identified as nuclear factors
that bind to a cis-acting response element of the interleukin 2 promoter, known as the antigen receptor response element, in T cells
that have been stimulated with ionomycin and phorbol myristate acetate (31, 32). Moreover, in vitro transcription assays indicated that NF90 and NF45 modulate expression from the interleukin 2 promoter
(31). However, NF90 and NF45 are also found in the nucleus of
nonstimulated cells (31), suggesting that they may be widely
expressed.
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Table I
Identification of major polypeptides in MonoS fraction
Polypeptides were purified from human placenta and transferred to
polyvinylidene difluoride as described. Results are expressed using the
amino acid single-letter codes. Uppercase letters represent assigned
amino acids, and lowercase letters represent ambiguous sequence
assignments. X refers to unidentified amino acids. In each
case, 10 or 15 amino acids were read, and in each case, all positively
identified amino acids were an exact match to the sequences of the
following known gene products: NF90 (GenBank accession no., U10324);
NF45 (GenBank accession no., U10323); eIF-2 (GenBank accession no.,
J02645; the reported cDNA sequence is MPGLSCRFYQHKFPE, suggesting
that the amino-terminal methionine had been lost and that amino acids
1, 4, 5, and 6 in the sequence obtained correspond to P, S, C, and R,
respectively); and eIF-2- (GenBank accession no., L19161; the
reported cDNA sequence is MAGGEAGVTLGQPHLSR, suggesting that the
amino-terminal methionine has been lost and that the assignments of T
and G at positions 8 and 10, respectively, in the read sequence are
correct).
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p37 was identified as the
subunit of eukaryotic initiation factor
eIF-2. The 50- and 52-kDa polypeptides were not separated in the sample
sent for sequencing; therefore, both polypeptides were present. Only
one sequence was read from these polypeptides, and this was identified
as the
subunit of eukaryotic translation initiation factor, eIF-2.
eIF-2 forms a ternary complex with GTP, initiator methionine-tRNA, and
the 40S ribosomal subunit, which then recruits the 60S ribosomal
subunit and mRNA to initiate protein translation (reviewed in Ref.
38). The
subunit of eIF-2 is phosphorylated by the
dsRNA-dependent protein kinase and possibly other protein
kinases (reviewed in Refs. 38 and 39). We have shown by direct amino
acid sequencing that p37 corresponds to the
subunit of eIF-2 and
that the
subunit of eIF-2 is present in the polypeptide doublet
composed of p50 and p52 (Table I). The predicted molecular mass of the
subunit of eIF-2 is 38 kDa; however, this polypeptide is known to
migrate aberrantly on SDS-PAGE, and it often co-migrates with the
52-kDa
subunit (40). Also, the amino terminus of eIF-2
is
amino-terminally blocked (39, 40). These observations are therefore
consistent with our results and provide an explanation for why only one
sequence was obtained from the p52/p50 polypeptides. Aberrant migration
of p50 on one-dimensional gels is consistent with the behavior of
eIF-2
(39). Also, phosphorylated p50 migrated on two-dimensional gel
electrophoresis close to actin, which has a pI of 5.5 (data not shown),
consistent with the known behavior of eIF-2
, which has a pI of 5.9 (39). We therefore conclude that p50 corresponds to the
subunit of
eIF-2 and is a substrate for DNA-PK in vitro (Fig. 4).
Antibodies to recombinant NF90 and NF45 were provided by one of us
(P. N. K.), and antibodies to purified human eIF-2
and
subunits were obtained from Dr. John Hershey (University of California,
Davis). Antibodies to NF90 cross-reacted with both p75 and p90 across
fractions 44-60, suggesting that p75 is related to NF90, perhaps being
a proteolytic breakdown product (Fig.
7A). The presence of NF45 was
confirmed by Western blot in fractions 44-60 of the MonoS FPLC
fractions (Fig. 7B). Antibodies to eIF-2
cross-reacted
with the 50-kDa polypeptide (p50) that is phosphorylated by DNA-PK and
antibodies to eIF-2
reacted with p52 in fractions 48-58 (Fig.
7C and data not shown). From these experiments, we conclude
that the protein fraction purified from MonoS FPLC that contains
proteins capable of interacting with DNA-PKcs and Ku contains NF90,
NF45, and the
,
, and
subunits of eIF-2.

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Fig. 7.
Western blot of fractions from MonoS
gradient. 10 µl each of fractions 40-64 from MonoS FPLC (see
Fig. 3) was run on SDS-PAGE and transferred to polyvinylidene
difluoride as described under "Materials and Methods." The blot was
incubated with antibodies to NF90 (A), NF45 (B),
or eIF-2 (C) as indicated and detected by enhanced
chemiluminescence.
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NF90 and NF45 Interact with DNA-PK--
Recombinant His-tagged
NF90 and NF45 proteins were purified from bacteria as described and
assayed for their ability to interact with DNA-PKcs, Ku, and DNA in the
EMSA in the absence of chemical cross-linkers. Incubation of
recombinant NF90, recombinant NF45, or recombinant NF90/45 (combined)
with DNA in the absence of DNA-PK, did not result in formation of a
significant protein-DNA complex (Fig. 8,
lanes 4-7). Similarly, no protein-DNA complexes were observed when recombinant NF90/45 proteins were added to DNA-PKcs and
DNA (Fig. 8, lanes 12-15) or to Ku and DNA (Fig. 8,
lanes 8-11), although in some experiments, a modest
increase in the amount of Ku band b relative to Ku band a was observed
(data not shown). Significantly, addition of recombinant NF90 to
DNA-PKcs, Ku, and DNA induced the formation of a slower migrating
complex (Fig. 8, lane 17, labeled D
) that
co-migrated with complex D previously observed using DNA-PKcs-Ku-DNA
and the MonoS fractions. Incubation of DNA-PKcs, Ku, and DNA with
recombinant NF45 alone did not cause significant complex formation
(Fig. 8, lane 18). However, incubation with recombinant NF90
plus recombinant NF45 resulted in complex formation similar to that
observed with recombinant NF90 alone (Fig. 8, lanes 19 and
20). These data suggest that NF90 or NF90 plus NF45 can
interact with or stabilize a complex between DNA-PKcs, Ku and DNA.

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Fig. 8.
Recombinant NF90 supports complex formation
with DNA-PKcs, Ku, and DNA. Recombinant NF90 and NF45 were
expressed and purified as described and incubated either with DNA alone
(lanes 4-7), with purified Ku plus DNA (lanes
8-11), with purified DNA-PKcs plus DNA (lanes 12-15),
or with purified DNA-PKcs, Ku, and DNA (lanes 17-20). Ku
was present at 10 ng (lanes 2, 8-11, and
16-20); DNA-PKcs was present at 30 ng (lanes 3 and 12-20); recombinant NF90 was present at 200 ng in
samples in lanes 4, 8, 12, and 17; and
recombinant NF45 was present at 200 ng in samples in lanes 5, 9, 13, and 18. Lanes 6, 10, 14, and
19 contained 100 ng each of recombinant NF90 and recombinant
NF45; samples in lanes 7, 11, 15, and 20 contained 200 ng each of recombinant NF90 and recombinant NF45.
Lane 1 contained DNA probe alone.
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As further evidence for interaction of DNA-PK with NF90 and NF45,
polyclonal antibodies to recombinant NF90 or recombinant NF45 were used
to immunoprecipitate polypeptides from a partially purified extract
(pre-dsDNA cellulose chromatography) containing DNA-PK and other
polypeptides, including NF90, NF45, and eIF-2. Immunoprecipitates were
analyzed by SDS-PAGE and Western immunoblot using a rabbit polyclonal
antibody to DNA-PKcs (DPK1) or NF90 (Fig.
9). DNA-PKcs was immunoprecipitated with
antibodies to rNF90 and rNF45 (Fig. 9, lanes 1-4) but not
with preimmune mouse serum (Fig. 9, lane 7) or a control
monoclonal antibody (Fig. 9, lane 8). Interestingly,
DNA-PKcs immunoprecipitated with NF90 and NF45 in the presence and
absence of DNA, suggesting that NF90 and NF45 may interact with
DNA-PKcs in the absence of Ku and DNA. Antibodies to NF90 or NF45
immunoprecipitated both p90 and p75 (Fig. 9B), consistent
with NF90 and NF45 being a heterodimer. In the experiment shown,
DNA-PKcs appeared to be preferentially immunoprecipitated with NF90 in
the presence of DNA; however, this was not a consistent finding. The
presence or absence of DNA had no effect on the ability of preimmune
sera or the monoclonal antibody to SV40T antigen to immunoprecipitate
either DNA-PKcs, NF90, or NF45 (data not shown).

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Fig. 9.
Antibodies to recombinant NF90 and
recombinant NF45 immunoprecipitate DNA-PKcs. Approximately 20 µg
of protein from the pre-dsDNA cellulose fraction was incubated as
described for EMSA DNA binding in a volume of 20 µl, plus or minus 5 µg/ml 40-bp DNA probe as indicated. The following antibodies were
added for immunoprecipitation: lanes 1 and 2, a
mouse polyclonal antibody to recombinant NF90; lanes 3 and
4, a mouse polyclonal antibody to recombinant NF45;
lanes 5 and 6, monoclonal antibody 42-27 to
DNA-PKcs; lane 7, preimmune mouse serum; and lane
8, monoclonal antibody to SV40 T antigen. Lane 9 contained 5 µg of pre-DNA cellulose sample alone.
Immunoprecipitations were as described (29) except that protein
G-Sepharose (Pharmacia) was used. Immunoprecipitates were analyzed by
Western blot using a rabbit polyclonal antibody DPK1 to DNA-PKcs
(A). The blot was stripped and probed with a rabbit
polyclonal antibody to recombinant NF90 (B).
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DISCUSSION |
DNA-PK is required for DNA double-strand break repair; however,
its precise function is as yet unknown. The large size of the DNA-PK
complex suggests that DNA-PK may act as a scaffold to which other
proteins are recruited. We have used highly purified proteins in an
EMSA to show that under the conditions of this assay, very highly
purified DNA-PKcs and Ku only form a stable complex on DNA in the
presence of chemical cross-linkers. Furthermore, this protein-DNA
complex has properties similar to those previously reported for Ku and
DNA-PK. Our data also show that DNA-PKcs does not interact with DNA in
the absence of Ku, even in the presence of cross-linkers (Fig. 1,
lanes 3, 9, and 15), consistent with previous
reports using UV cross-linking (10). Double-stranded DNA of 40 and 80 bp readily supported DNA-PK complex formation, whereas complex
formation was less efficient on dsDNA of 20 bp (data not shown),
suggesting a minimum size requirement of about 30 bp. In our
hands, DNA-PKcs-Ku complexes that formed on DNA of 100 bp or greater
did not enter the gel (data not shown).
This assay was then used to identify proteins that interact with DNA-PK
and stabilize complex formation. We have identified a protein fraction
from human cells that stabilizes the formation of a slowly migrating
complex on EMSA in the absence of added chemical cross-linkers. This
protein fraction has been purified extensively through several
chromatographic stages and two FPLC columns in the presence of the
nonionic detergent Tween 20. The major polypeptides in this fraction
have been identified as NF90, NF45, and the
,
, and
subunits
of eIF-2. Fractions containing NF90 and NF45 tracked most closely with
the activity capable of interacting with DNA-PK, and recombinant NF90
induced formation of a DNA-PKcs-Ku-DNA complex on EMSA. Because neither
recombinant NF90, NF45, nor the purified MonoS polypeptides alone had
significant affinity for the Ku-DNA complex, our data suggest that the
interaction with the DNA-PK complex occurs via DNA-PKcs.
Immunoprecipitation data showing that NF90 and NF45 can interact with
the DNA-PKcs in the presence or absence of DNA further support
the notion that the interaction is likely mediated through
DNA-PKcs rather than Ku.
NF90 and NF45 were initially purified as a dimer (31, 32), and
antibodies to NF90 coimmunoprecipitate NF45 and vice versa (Fig. 9 and data not shown). However, recombinant NF45 did not support
formation of complex D
on EMSA (Fig. 8), suggesting that the
interaction with DNA-PKcs is mediated through the NF90 subunit. We
therefore propose a model in which NF90 interacts with DNA-PKcs to form
a complex between NF90-NF45 and DNA-PKcs and that this complex can
interact with Ku in the presence of DNA. The Ku heterodimer forms
multiple protein-DNA complexes on EMSA, the number of which depends
both on the length of DNA and on the protein concentration (19-22).
These protein-DNA complexes may represent Ku bound to either ends of
DNA or bound at internal sites after translocation after end binding.
Alternatively, the complexes may represent different oligomeric forms
of Ku. Our data suggest that the DNA-PKcs-Ku-DNA-containing complexes
form preferentially from Ku complex b (Figs. 1 and 5 and data not
shown). Attempts are currently under way to determine the precise
composition of the different Ku-DNA and DNA-PKcs-Ku-DNA complexes.
Both NF45 and the 90 kDa polypeptide that cross-reacts with antibodies
to NF90 are phosphorylated by DNA-PK in vitro (Fig. 4). We
also show that the amino-terminal 10 amino acids of the 75 kDa
polypeptide (called p75 in Figs. 2-4, 7, and 9) corresponds exactly to
the predicted amino-terminal sequence of NF90 (Table I). Moreover, p75
is recognized by antibodies to recombinant NF90 both in Western blot
and immunoprecipitation and is immunoprecipitated by antibodies to NF45
(Figs. 7 and 9). However, p75 was not phosphorylated by DNA-PK in
vitro, suggesting either that p90 and p75 are variants of NF90 or
that p75 has suffered proteolysis during the purification that results
in loss of a carboxyl-terminal polypeptide of about 15 kDa that
contains the DNA-PK phosphorylation sites. Interestingly, addition of
phosphatase to extracts from stimulated T cells that contain NF90 and
NF45 resulted in reduced binding to the antigen receptor response
element sequence (31), suggesting that NF90 and NF45 may be
phosphoproteins and that their function may be regulated by
phosphorylation in vivo. DNA-PK is the first protein kinase
known to phosphorylate NF90 and NF45 in vitro. Although many
in vitro substrates have been identified for DNA-PK
(reviewed in Refs. 1, 2, and 4), identification of in vivo
substrates for DNA-PK has proved difficult. Potential substrates such
as replication protein A and p53 may be phosphorylated less efficiently in cell lines that lack significant DNA-PK activity (41), although another study found no differences between DNA-PK-negative and normal
cells (42). Because both NF90 and NF45 are likely phosphorylated in vivo (31, 32) and interact with or are part of the DNA-PK complex (this study), these proteins are likely candidates for in
vivo targets of DNA-PK. Attempts are under way to determine whether NF90 and NF45 are phosphorylated in vivo by DNA-PK
and to determine whether DNA-PK plays a role in the regulation of cytokine expression via interaction with or phosphorylation of NF90 and
NF45.
We also show that the
subunit of eIF-2 (p50 in Fig. 4C)
is phosphorylated by DNA-PK in vitro. Phosphorylation occurs
on predominantly serine residues (data not shown). eIF-2
is also phosphorylated in vivo (43) and is a substrate for casein
kinase II and PKC in vitro (44, 45). However, the
physiological effect of phosphorylation of eIF-2
is not known. The
eIF-2 heterotrimer binds to GTP and to Met-tRNAi (reviewed
in Ref. 33), and eIF-2
can interact with RNA in vitro
(46). In our hands, eIF-2 (present as p50/52 plus p37 in the MonoS
fractions) bound to both double-stranded and single-stranded DNA
cellulose resins during purification from human cells, suggesting that
eIF-2 may be capable of interacting with nucleic acid other than RNA.
The sequence of eIF-2
reveals several polylysine regions (39) that
could conceivably contribute to nuclear localization and/or be
important for binding to nucleic acids. The significance of the three
subunits of eIF-2 in the polypeptide fraction that can interact with
DNA-PK is not clear. Although these proteins are known to be involved
in translation, we cannot exclude the possibility that eIF-2 may be
involved in complex formation with DNA-PK and NF90/45. The lack of
suitable antibodies has so far prevented us from exploring this
possibility further. Interestingly, Lobo et al. (47)
recently reported that eIF-2 localizes to the nucleus as well as the
cytoplasm, in keeping with a role for eIF-2 in the nucleus in addition
to its role in translation. Also, it has recently been shown that the
yeast TOR1 and TOR 2 proteins, which are related to DNA-PKcs by virtue
of the carboxyl-terminal phosphatidylinositol-3 kinase domain, play a
role in translation initiation (48), and TOR2 signals to the GTP
exchange/GTPase proteins RHO and ROM to control cytoskeletal reorganization in response to nutrient availability (49). These observations set a precedent for a functional relationship between phosphatidylinositol 3-kinase homologues, guanine nucleotide exchange proteins, and translational initiation.
We thank Dr. J. B. Hershey for
antibodies to eIF-2 subunits, Dr. Tom Shenk for monoclonal antibody
42-27 to DNA-PKcs, and Drs. Jane Walent (University of Wisconsin
Biotechnology Center) and Sandy Keilland (University of Victoria,
Victoria, British Columbia, Canada) for protein sequence analysis. We
also thank Drs. J. P. Lees-Miller, M. F. Lohka, L. W. Browder, P. Lee (University of Calgary) and N. Sonnenburg (McGill
University) for helpful discussions and Ruiqiong Ye for excellent
technical assistance.