Mol Cell Biol, July 1998, p. 4221-4234, Vol. 18, No. 7
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
B
and I
B
Regulates NF-
B DNA Binding
Properties
Divisions of Molecular Virology and Hematology-Oncology1 and Departments of Medicine and Microbiology,2 University of Texas Southwestern Medical Center, Dallas, Texas 75235, and Harvard Microchemistry Facility, Cambridge, Massachusetts 021383
Received 27 October 1997/Returned for modification 18 December 1997/Accepted 28 April 1998
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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Regulation of the I
B
and I
B
proteins is critical for
modulating NF-
B-directed gene expression. Both I
B
and I
B
are substrates for cellular kinases that phosphorylate the amino and carboxy termini of these proteins and regulate their function. In this
study, we utilized a biochemical fractionation scheme to purify a
kinase activity which phosphorylates residues in the amino and carboxy
termini of both I
B
and I
B
. Peptide microsequence analysis by capillary high-performance liquid chromatography ion trap
mass spectroscopy revealed that this kinase was the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK phosphorylates serine residue 36 but not serine residue 32 in the amino terminus of
I
B
and also phosphorylates threonine residue 273 in the
carboxy terminus of this protein. To determine the biological relevance of DNA-PK phosphorylation of I
B
, murine severe combined
immunodeficiency (SCID) cell lines which lack the DNA-PKcs gene were
analyzed. Gel retardation analysis using extract prepared from these
cells demonstrated constitutive nuclear NF-
B DNA binding activity, which was not detected in extracts prepared from SCID cells
complemented with the human DNA-PKcs gene. Furthermore, I
B
that
was phosphorylated by DNA-PK was a more potent inhibitor of NF-
B
binding than nonphosphorylated I
B
. These results suggest that
DNA-PK phosphorylation of I
B
increases its interaction with
NF-
B to reduce NF-
B DNA binding properties.
INTRODUCTION
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NF-
B comprises a family of
proteins including p50, p52, p65 or RelA, p100, p105, and c-Rel which
regulate the expression of a variety of cellular and viral genes
(reviewed in references 7, 75, and
79). Each of these proteins contains a region known
as the Rel homology domain which is critical for the DNA binding and
dimerization properties of these proteins. One of the major regulatory
mechanisms which control NF-
B activity is the unique cellular
localization of different members of this family. In unstimulated
cells, p65 or RelA is nearly exclusively localized in the cytoplasm
(4-6, 13, 34), but it translocates to the nucleus upon
treatment of the cells with a variety of inducers such as phorbol
esters, interleukin 1, and tumor necrosis factor alpha (TNF-
)
(43, 73). RelA dimerizes with other NF-
B family members (7, 75, 79) and activates gene expression via its potent transactivation domain (8, 67, 70). Thus, cellular proteins which regulate the nuclear translocation of NF-
B are critical for the control of NF-
B activation of viral and
cellular genes.
The I
B proteins constitute a group of cytoplasmic proteins that bind
to NF-
B and sequester these proteins in the cytoplasm by
preventing their nuclear localization. A number of different I
B
proteins have been identified including I
B
, I
B
, I
B
(reviewed in reference 79), and I
B
(80). I
B
(41) and I
B
(76) are the best studied of these regulatory proteins. Treatment of cells
with a variety of agents such as phorbol esters, TNF-
, and UV
irradiation results in the degradation of I
B
and I
B
and the
nuclear translocation of NF-
B (12, 17, 43, 73). I
B
present in the nucleus terminates the induction process in response to
TNF-
and other activators (2, 3, 60).
I
B
and I
B
have distinct functional domains. For example,
the N terminus and the ankyrin repeats of I
B
are required for the
cytoplasmic regulation of NF-
B while C-terminal sequences are
required to regulate NF-
B function in the nucleus
(60). The activity of I
B is regulated by its
phosphorylation state. The C termini of the I
B
and I
B
proteins contain PEST domains with serine and threonine residues that
are phosphorylated by cellular kinases which regulate the intrinsic
stability of these proteins (10, 11, 25, 57, 61, 66, 81). In
addition, the amino termini of these proteins each contain two closely
spaced serine residues that are also capable of being phosphorylated by
cellular kinases (16, 17, 28, 32, 77). Serine residues at
positions 32 and 36 of I
B
(16, 17, 28, 32, 77) and 19 and 23 of I
B
(62) are phosphorylated when cells are treated with various agents such as TNF-
and phorbol esters. Phosphorylation of these residues leads to their ubiquitination and
proteasome-mediated degradation (1, 23, 24, 28, 32, 58, 69,
77). Mutations of these amino-terminal serine residues in
I
B
and I
B
prevent the degradation of these proteins upon treatment of cells with TNF-
or phorbol esters and inhibit the nuclear translocation of NF-
B (16, 28, 62, 77).
Biochemical fractionation has been performed to identify
cellular kinases that are capable of phosphorylating
I
B
. A protein complex migrating at approximately 700 kDa
is capable of phosphorylating I
B
on serine residues 32 and
36, resulting in I
B
degradation by the proteasome (24,
51). Two related kinases isolated from a
similar-size complex, IKK
and IKK
, phosphorylate serine
residues 32 and 36 in I
B
(27, 63, 65, 83, 85). Another
kinase, RSK1, also phosphorylates the amino terminus of
I
B
(71). In contrast to IKK
and IKK
, RSK1
phosphorylates I
B
exclusively on serine residue 32. Cellular
kinases are also capable of phosphorylating the carboxy
terminus of I
B
. For example, casein kinase II phosphorylates serine and threonine residues in the carboxy terminus of I
B
(10, 20, 58, 59, 82). Mutation of these carboxy-terminal serine and threonine residues increases the steady-state levels of I
B
(57, 58). These results suggest that
multiple cellular kinases are capable of phosphorylating I
B
and
regulating different aspects of its function.
To further characterize cellular kinases that phosphorylate
I
B
, we utilized biochemical fractionation and peptide
sequence analysis by ion trap mass spectrometry (MS). This
analysis demonstrated that the catalytic subunit of DNA-dependent
protein kinase, DNA-PKcs, phosphorylated specific residues in the amino
and carboxy termini of I
B
and I
B
. We also investigated the
regulation of I
B
and NF-
B in equine and murine
severe combined immunodeficiency (SCID) cell lines deficient in
DNA-PKcs. SCID cells, which lack the DNA-PKcs gene, exhibited
constitutive nuclear NF-
B DNA binding activity, in
contrast to control cell lines. DNA-PK phosphorylation of
I
B
increased its ability to inhibit NF-
B DNA binding
properties. These studies suggest that I
B is a target for DNA-PK
phosphorylation and that this phosphorylation regulates the DNA binding
properties of NF-
B in the nucleus.
MATERIALS AND METHODS
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Plasmid construction.
Full-length wild-type I
B
cDNA
and I
B
cDNA with mutations at the codons for serine residues 32 and 36 in a pCMV4 vector (gift from Dean Ballard) (16) were
digested with BamHI and HindIII and cloned
into the pGEX-KG vector at BamHI-HindIII
sites. A deletion of the carboxy-terminal 74 amino acids of I
B
,
including the PEST domain, was accomplished by digesting the I
B
cDNA with BamHI and HincII and cloning the
resulting fragment into pGEX-KG cut with BamHI and
SmaI. Another deletion of the C terminus of wild-type
I
B
was accomplished by digesting the I
B
cDNA with BamHI and SacI and cloning the fragment into
pGEX-KG at the BamHI-SacI site to produce an
I
B
cDNA encoding the amino-terminal 138 amino acids of I
B
.
These truncated I
B
cDNAs were then utilized to make
single-stranded M13 DNA templates for mutating either serine residue
36, serine residue 32, or both serine residues to alanine or tyrosine
residue 42 to phenylalanine. The oligonucleotides used for the
site-directed mutagenesis mutations were as follows: for changing
serine residue 32 to alanine, 5'-GAC CGC CAC GAC GCC GGC GGC CTG GAC
TCC-3'; for changing serine residue 36 to alanine, 5'GCG GCC TGG ACG
CCA TGA AAGA-3'; and for changing tyrosine residue 42 to
phenylalanine, 5'-CGA GGA GTT CGA GCA GAT GG-3'. Finally, fragments
extending from amino acids 1 to 53 for both wild-type I
B
and
I
B
with mutations at serine residues 32 and 36 were generated by
BamHI and XhoI digestion of these cDNAs and cloning of the resulting cDNA fragments into pGEX-KG.
B
mutants were constructed by digesting the pCMV4-I
B
cDNA (62) (gift from Dean Ballard) with NcoI and
cloning this full-length I
B
coding sequence into pGEX-KG. This
glutathione S-transferase (GST)-I
B
cDNA was used with
a quick-change site-directed mutagenesis kit (Stratagene) to mutate
serine residues 19 and 23 to alanine by using the oligonucleotide
5'-CGA TGA ATG GTG CGA CGG CGG CCT GGG CGCTC TAGGTCC CGA CG-3 and its
complementary oligonucleotide. The oligonucleotide 5'-ACC TGA CGA CGA
GGA CTA AAA GCT TAG CCC TTG CA-3' and its complementary oligonucleotide were used to delete 54 amino acid residues at the C terminus of I
B
.
I
B
site-directed mutants (with mutations S283A, T291A, and/or
T299A) were prepared by using a quick-change site-directed mutagenesis
kit (Stratagene) to mutate serine residue 283 and threonine
residues 291 and 299 to alanine with oligonucleotides 5'-ATG CTG CCA
GAG GCT GAG GAT GAG GAG-3', 5'-GGA GAG CTA TGA CGC AGA GTC AGA GT-3',
and 5'-TTC ACG GAG TTC GCA GAG GAC GA-3' and the complementary
oligonucleotides, respectively. I
B
proteins with mutations T273A
and T293A were constructed by using I
B
templates containing the
triple mutant (mutations S283A, T291A, and T299A) and oligonucleotides
5'-GCA GCT GGG CCA GCT GGC ACT AGA AAA CCT-3' and 5'-TAT GAC GCA GAG
GCA GAG TTC ACG GAG-3', respectively.
Adenovirus constructs containing wild-type and mutant I
B
were
constructed by inserting the I
B
wild-type cDNA cut with ClaI and SmaI into pBlueScriptSK. An
XbaI-KpnI I
B
cDNA fragment was then
inserted into the pAC/CMVplpa plasmid to generate pAC/CMV-I
B
wt.
The pCMV4-I
B
construct containing mutations at codons
for serine residues 32 and 36 was digested with ClaI
and SmaI and cloned into pAC/CMV plasmid.
Expression of bacterially produced GST-I
B
proteins.
The wild-type and mutant I
B
pGEX-KG constructs were transformed
into Escherichia coli BL21 DE3. Cultures (400 ml) of
E. coli were grown to an optical density at 600 nm of 0.6 to
0.8 and induced with 0.5 mM
isopropyl-
-D-thiogalactopyranoside (IPTG) for 3 h.
Cells were pelleted, resuspended in buffer A (20 mM HEPES [pH 7.9],
400 mM NaCl, 5 mM dithiothreitol [DTT], 50 mM mannitol, 10 mM sodium
ascorbate, 10% glycerol, 0.1 mM EDTA, 0.1% Nonidet P-40 [NP-40], 1 mM phenylmethylsufonyl fluoride [PMSF]), mildly sonicated, and
centrifuged. The supernatant was incubated with 0.5 ml of glutathione
agarose matrix (Sigma) for 1 h at 4°C. The matrix was then
washed four times with buffer A and two times with buffer B (50 mM Tris
[pH 8.0], 120 mM NaCl, 0.5% NP-40, 5 mM DTT, 1 mM PMSF). These GST
fusion proteins were eluted off the matrix by 5 mM glutathione and then
were fractionated through Q-Sepharose columns with a 0.1 to 0.5 M KCl
gradient to remove contaminating DNA.
Cell culture and protein extraction. Wild-type equine fibroblasts (CRL6288) and murine NIH 3T3 cells were purchased from the American Type Culture Collection ATCC. Equine SCID fibroblasts cell line 1863 (82) and murine SCID fibroblast cell line SF19 (39) were described previously. The parental (CB-17), SCID (SCID/St), and SCID-complemented (100E and 50D) cell lines have been previously described (49). HeLa spinner cells were maintained in supplemented Eagle minimal essential media with 5% newborn calf serum at a density of 4 × 105 cells/ml. Tetradecanoyl phorbol acetate (TPA) (Sigma; 50 ng/ml) and/or ionomycin (Calbiochem; 2 µM) was added to either HeLa, 3T3, or SF19 cells as outlined below.
To obtain cytoplasmic proteins, cells were washed with cold phosphate-buffered saline (PBS; pH 7.2), resuspended in buffer C (10 mM HEPES [pH 7.5], 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 50 mM NaF, 1 mM sodium orthovanadate, 5 µM okadaic acid, 0.5 mM PMSF, 5% glycerol, 10 µg of leupeptin and aprotinin per ml), and incubated on ice for 15 min. At the end of incubation, 1/10 volume of 10% NP-40 was added. Cells were vortexed for 30 s and then subjected to Eppendorf centrifugation for 30 s. Supernatants were collected as cytoplasmic proteins. The protein concentrations of the resultant supernatants containing cytoplasmic protein were determined by the Bradford assay using Bio-Rad reagent.Preparation of recombinant adenoviruses.
The production of
recombinant adenovirus utilized previously described methodology
(21, 36). The pAC/CMVplpa plasmids encoding either wild-type
I
B
or I
B
with mutations at serine residues 32 and/or 36 were cotransfected with the pJM17 adenovirus plasmid into 293 cells
(adenovirus E1a-transformed human embryonic kidney cells). Viruses from
293 cell supernatants of cultures showing a complete cytopathic effect
were purified by cesium chloride banding. Virus titers were determined
by plaque assay using serial dilution (38). The pAC/CMVplpa,
pJM17, and control recombinant replication-defective adenoviruses
containing
-galactosidase were obtained from P. Nissen. Adenovirus
infection of murine cell lines was performed by using a multiplicity of
infection of 10 for 24 h.
Gel electrophoresis mobility shift assay.
Nuclei from NIH
3T3, SF19 (murine SCID fibroblasts), CRL6288 (normal equine
fibroblast), and 1863 (equine SCID fibroblasts) cells and the murine
cell lines CB-17, SCID/St, 100E, and 50D were resuspended in buffer
containing 20 mM HEPES (pH 7.5), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mg of PMSF per ml, 10% glycerol, 10 µg of leupeptin, 10 µg of aprotinin per ml and extracted at 4°C on a rocker for 30 min,
followed by Eppendorf centrifugation at 14,000 rpm for 5 min. The
supernatants were collected, and 3 µg of each nuclear extract was
incubated with 0.1 pmol of 32P-labeled double-stranded
B binding site oligonucleotide (5'-GCTGGGGACTTTC-3') or
SP1 binding site oligonucleotide (5'-ATTCGATCGGGGCGGGGCGAGC-3') in buffer containing 1 µg of poly (dI-dC), 1 µg of bovine
serum albumin, 10 mM HEPES (pH 7.9), 0.5 mM DTT, 0.1 mM EDTA, 60 mM KCl, 0.2 mM PMSF, 5 mM MgCl2, and 12% glycerol at room
temperature for 15 min. Samples were analyzed by 5% native
polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.
B
inhibition experiments, bacterially produced I
B
protein was cleaved from the GST moiety with thrombin, followed by the
addition of PMSF. This I
B
protein was then phosphorylated by
purified DNA-PK by using cold ATP. The nonphosphorylated and phosphorylated I
B
proteins were added to the gel retardation reaction mixture 30 min prior to the addition of the
32P-labeled probe. The recombinant p50 and p65 NF-
B
proteins were produced in baculovirus expression vectors and purified
by nickel agarose chromatography.
Immunocytochemistry and confocal microscopy.
Cells,
including HeLa, 50D, and 100E cells, were cultured on coverslips,
washed with PBS, and fixed with methanol at
20°C for 6 min. The
cells were washed twice with PBS, blocked for 30 min in PBS containing
0.5% gelatin and 0.25% bovine serum albumin, and incubated for 1 h at room temperature with one of the following primary antibodies: a
monoclonal antibody directed against DNA-PKcs, a rabbit polyclonal
antibody directed against a carboxy-terminal peptide of I
B
(C-21;
Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), or a rabbit
polyclonal antibody directed against a carboxy-terminal peptide of
I
B
p65 (C-20; Santa Cruz Biotechnology, Inc.). The cells were
washed three times with 0.2% gelatin in PBS and incubated for 1 h
with a goat anti-mouse fluorescein isothiocyanate (FITC) conjugate or a
goat anti-rabbit FITC conjugate (Jackson ImmunoResearch, West Grove,
Pa.). Samples were washed three times and then mounted in a solution of
1 mg of p-phenylenediamine per ml in 90% glycerol. The
preparations were examined on a MRC1024 laser scanning confocal microscope (Bio-Rad, Microscience Division, Cambridge, Mass.) equipped
with a 15-mW air-cooled krypton-argon laser (Ion Laser Technology, Salt
Lake City, Utah) as a light source. The images were constructed from
gray scale confocal fluorescence images with Adobe software.
Immunoprecipitation of DNA-PKcs. Monoclonal antibodies 42-26, 25-4, and 18-2 directed against DNA-PKcs (19, 49) or flag monoclonal antibody (Kodak) was added to column fractions containing DNA-PKcs and incubated at 4°C for 1 h. Protein G-Sepharose was then added for 1 h, and the beads were washed three times with buffer D (20 mM HEPES [pH 7.9], 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 10% glycerol) and one time with buffer containing 12 mM HEPES (pH 7.9), 0.6 mM EDTA, 0.6 mM DTT, 6% glycerol, 60 mM KCl, and 7.5 mM MgCl2. Kinase reactions were then performed.
In vitro kinase assay.
Kinases which bound to GST-I
B
fusion proteins and which were able to phosphorylate I
B
were
assayed by a modification of the previously described methods
(44). Bacterially expressed GST-I
B
proteins were bound
to glutathione agarose matrix (Sigma). HeLa S100 cells or column
fractions were incubated with GST-I
B
-bound matrix in buffer
containing 20 mM HEPES (pH 7.9), 100 mM KCl, 10% glycerol, 0.2 mM
EDTA, 0.5 mM DTT, and 0.5 mM PMSF at 4°C for 1 h. The matrix was
washed twice with buffer containing 50 mM Tris (pH 8.0), 120 mM NaCl,
0.5% NP-40, 5 mM DTT, and 1 mM PMSF and once with the kinase reaction
buffer (50 mM Tris [pH 7.4], 5 mM MnCl2, 5 mM DTT). The
kinase assay was then performed in the kinase reaction buffer with the
addition of 4 µM ATP, 10 µCi of [
-32P]ATP, 5 mM
NaF, 1 mM sodium orthovanadate, 40 µM MgCl2, a protease inhibitor cocktail (Boehringer Mannheim), and 1 mM PMSF at 30°C for
15 min. Similar results were seen when 2.5 µM okadaic acid was added
to the kinase assay mixtures. The matrix was pelleted by
centrifugation, 20 µl of 2× sodium dodecyl sulfate-PAGE (SDS-PAGE) sample buffer was added, and the samples were heated at 95°C for 5 min. The samples were resolved with SDS-12% polyacrylamide gels followed by autoradiography.
Western blot analysis.
Mono S fractions from the
purification scheme for I
B
kinases were mixed with an equal
volume of 2× SDS-PAGE sample buffer, boiled for 5 min, and resolved by
SDS-6% PAGE. Following electrophoresis, the gel was transferred at 60 V to a Hybond-C nitrocellulose membrane (Amersham) for 8 h at
4°C. Affinity-purified goat antibody directed against DNA-PKcs (Santa
Cruz Biotechnology, Inc.; SC-1551) at a 1:2,000 dilution was used as
the primary antibody in the Western blot analysis. Donkey
anti-goat immunoglobulin G horseradish peroxidase (Santa
Cruz Biotechnology, Inc.; SC-2020) was used as the secondary antibody
to detect the DNA-PKcs by enhanced chemiluminiscence reagents (ECL kit;
Amersham).
B
(Santa Cruz Biotechnology, Inc.; SC-371) at a dilution
of 1:3,000 or the p89 subunit of TFIIH (Santa Cruz Biotechnology, Inc.;
SC-230) at a dilution of 1:5,000 before incubation with a second
antibody and development with enhanced chemiluminescence.
Purification of I
B
kinases.
All the purification steps
were conducted at 4°C. HeLa S100 extract (3 g) was prepared from 150 liters of cells (1011 cells) by the method of Dignam
(29) and dialyzed against 0.1 M KCl buffer D (20 mM HEPES
[pH 7.9], 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT). The extract was then
fractionated on a phosphocellulose column which was washed with 150 ml
of 0.1 M KCl buffer D, and the kinase activity was eluted with 100 ml
of buffer D containing 0.3 M or 1.0 M KCl. The I
B
kinase activity
was assayed with GST-I
B
fusion proteins, which extended from
amino acid 1 to 138 and contained either wild-type sequences or
sequences with mutations of serine residues 32 and 36, that were bound
to glutathione agarose beads. The 0.3 M KCl fraction which contained
the majority of the kinase activity was dialyzed against 0.05 M KCl
buffer D and fractionated on a Q-Sepharose column (1 by 2 cm) which was eluted with 1.0 M KCl in buffer D. The active kinase fractions were
pooled, loaded on a Superdex 200 column (26/60; Pharmacia), and eluted
with 0.05 M KCl in buffer D. The active fractions were then pooled and
fractionated with a heparin agarose column (1 by 3 cm). After the
column was washed with 0.05 M KCl in buffer D, it was eluted with a
20-ml linear gradient of 0.05 to 0.8 M KCl in buffer D. The fractions
containing the peak kinase activity were pooled and dialyzed against
0.05 M KCl in buffer D and were fractionated on a Mono Q fast protein
liquid chromatography (FPLC) column (Pharmacia) with a linear gradient
of 0.05 to 1.0 M KCl in buffer D. The fractions active for kinase
activity were then pooled and dialyzed against 0.05 M KCl in buffer D
and fractionated by using a Mono S FPLC column (Pharmacia) with a
linear gradient of 0.05 to 0.6 M KCl. The fractions containing the peak
of kinase activity were dialyzed against 0.1 M KCl in buffer D,
aliquoted, frozen in liquid nitrogen, and stored at
80°C.
Peptide sequencing by ion trap MS of components of the I
B
kinase.
Purified fractions containing the I
B
kinase activity
were pooled, loaded onto an SDS-6% polyacrylamide gel, and
subjected to electrophoresis in Tris-glycine buffer. Coomassie
brilliant blue-stained gel slices containing a 400-kDa protein species
that correlated with the peak of kinase activity for I
B
were
excised and subjected to in gel reduction,
S-carboxyamidomethylation, and tryptic digestion (Promega).
Sequence information on the digestion products was determined by
capillary (Monitor C18 column [0.5 by 150 mm]; Michrom
BioResources) reverse-phase chromatography, coupled to the electrospray
ionization source of a quadruple ion trap mass spectrometer (Finnigan
LCQ, San Jose, Calif.). The instrument was programmed to acquire
successive sets of three-scan modes consisting of full-scan MS over the
m/z range 395 to 1,118 amu, followed by two data-dependent
scans on the most abundant ion in that full scan. These data-dependent
scans allowed the automatic acquisition of high-resolution (zoom scan)
spectra to determine charge state and exact mass and MS/MS spectra for
peptide sequence information. Interpretation of the resulting MS/MS
spectra was facilitated by searching the NCBI nr and dbest databases
with the algorithm Sequest (31) and was then confirmed
manually. The peptide sequences TVGALQVLGTEAQSSLK,
LLLQGEADQSLLTFIDK, and SLGPPQGEEDSVPR had sequence identity with the
DNA-dependent protein kinase catalytic subunit (40).
RESULTS
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Identification of cellular kinases that phosphorylate
I
B
.
To characterize cellular kinases that phosphorylate
I
B
, we prepared S100 extract from both untreated and TPA-treated
HeLa cells (29). The amino-terminal 138 amino acids of
I
B
were fused to GST so that kinases that phosphorylated the
amino terminus of I
B
could be detected. A GST-I
B
fusion
protein containing mutations of serine residues 32 and 36 was also
assayed in an attempt to differentiate kinases with specificity for
these residues (16, 17, 28, 32, 77). Following binding of
cellular proteins to GST-I
B
coupled to glutathione agarose beads,
the beads were extensively washed and kinase reactions were performed, followed by SDS-PAGE and autoradiography.
B
kinase activity which
eluted in the 0.3 M KCl eluate was next fractionated on a Superdex 200 column. Superdex 200 fractionation of proteins isolated from untreated
HeLa cells demonstrated one major peak of activity which migrated
between 600 and 700 kDa (Fig. 1A). The
phorbol ester-treated HeLa extract contained a similar amount of kinase
activity in the 700-kDa fraction from the Superdex 200 column in
addition to a second peak of I
B
kinase activity that migrated
with markers of approximately 40 kDa (Fig. 1B). The specificity of
kinases present in the 700-kDa fraction isolated from either untreated
or TPA-treated HeLa cells was next determined. Kinases present in the
700-kDa fraction from both untreated (Fig. 1C) and TPA-treated (data
not shown) extracts were able to phosphorylate a GST-I
B
fusion
protein with serine residues at positions 32 and 36 (Fig. 1C, lanes 2 and 4) but not a GST-I
B
fusion protein with mutations of these
serine residues (Fig. 1C, lanes 3 and 5).
|
Purification of a cellular kinase that phosphorylates the amino
terminus of I
B
.
To purify kinases present in the 700-kDa
fraction, S100 extract was prepared from 150 liters of untreated
HeLa cells (Fig. 2). The purification
scheme included elution of active kinase fractions from a
phosphocellulose column with 0.3 M KCl, concentration of the sample on
a Q-Sepharose column, and fractionation on a Superdex 200 column. The
kinase activity that eluted in the 600- to 700-kDa Superdex 200 FPLC fraction was then further purified by heparin agarose, mono
Q FPLC, and mono S FPLC chromatography. The fractions from the final
mono S column were subjected to SDS-PAGE and Coomassie staining and
were assayed for their ability to phosphorylate I
B
.
|
B
kinase activity in these
same column fractions (Fig. 3B). The presence of the 400-kDa species
was associated with the peak of I
B
kinase activity in five
different HeLa S100 preparations (data not shown). Following SDS-PAGE
and Coomassie staining, the 400-kDa species was excised from
the gel and subjected to gel tryptic digestion (42). The
resultant mixture was analyzed by capillary high-performance
liquid chromatography (HPLC) ion trap MS which allowed the
acquisition of on-line MS/MS spectra for peptide sequence information.
After manual and computer-assisted interpretation (31),
peptide sequences that corresponded to the amino acid sequences
TVGALQVLGTEAQSSLLK,
LLLQGEADQSLLTFIDK, and
SLGPPQGEEDSUPR in the DNA-dependent protein kinase
catalytic subunit were obtained (40). The 90-kDa protein
seen in the same column fractions as DNA-PK was also analyzed
by peptide microsequence analysis and revealed a novel peptide
sequence of unknown function (50).
|
B
kinase activity was in fact DNA-PKcs (Fig. 3C). Casein kinase II, which
phosphorylates the C terminus of I
B
(10, 57, 61, 81),
was not present in fractions containing DNA-PK, as determined by
Western blot analysis with specific antibodies directed against this
protein (data not shown). Western blot analysis was also used to
analyze whether the Ku antigens (Ku86 and Ku70) copurified with
DNA-PK in our I
B
kinase preparation (37, 74). This
analysis indicated that low levels of the Ku antigens relative to that
of DNA-PKcs were present and that there was increased amounts of Ku70
relative to Ku86 (data not shown). The significance of this
finding remains to be determined.
DNA-PK phosphorylation of I
B
.
DNA-PK is a
serine/threonine kinase which is a member of a family of protein
kinases known as phosphatidylinositol 3 (PI-3) kinases that contain a
conserved kinase domain and include ATM, FRAP, and FRP1 (26, 40,
68). DNA-PK can be detected in cellular extracts prepared from
both the nucleus and the cytoplasm, although the majority of this
activity is found in the nucleus (19, 54). This kinase
comprises two subunits, which include a regulatory subunit that is the
DNA end-binding heterodimer Ku and catalytic subunit DNA-PKcs. The
kinase activity of DNA-PK is stimulated by the addition of
double-stranded DNA and inhibited by the addition of wortmannin
(40). DNA-PK phosphorylates a number of substrates including Jun (9), DNA replication factor A (18),
p53 (33, 54, 56), the RNA polymerase II C-terminal domain
(CTD) (30, 64), simian virus 40 T-antigen (22),
hsp90 (19, 54), SP1 (46), Oct1 (35),
and the serum response factor (59).
B
kinase activity, it was critical to demonstrate that
DNA-PK itself was able to phosphorylate I
B
. A monoclonal antibody
directed against DNA-PKcs was used to immunoprecipitate the kinase from
the mono S column fractions and assay its ability to phosphorylate
I
B
, I
B
, and
-casein (which is a known substrate for
DNA-PK [19, 54]). The unrelated flag monoclonal
antibody was used as a control antibody in these immunoprecipitation
studies. The fractions immunoprecipitated with antibody to
DNA-PKcs were able to efficiently phosphorylate I
B
,
I
B
, and
-casein (Fig. 4A, lanes
1 to 3). In contrast, immunoprecipitation with the control antibody did
not result in phosphorylation of these different substrates (Fig. 4A,
lanes 4 to 6). These data support a role for DNA-PKcs in
phosphorylating both I
B
and I
B
.
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B
kinase activity present in our mono S
column fractions. The addition of wortmannin inhibited the ability of kinases present in the mono S column fraction to phosphorylate I
B
(Fig. 4B, lanes 2 and 3). In contrast, the addition of another kinase
inhibitor, rapamycin, did not prevent the phosphorylation of I
B
(Fig. 4B, lanes 4 and 5). Finally, we demonstrated that the presence of
DNA-PK in cytoplasmic extract was not dependent on the method of
preparation. Immunoprecipitation with a monoclonal antibody directed
against DNA-PKcs demonstrated that cytoplasmic extract prepared by both
the standard S100 preparation procedure (29) and the
rapid-lysis procedure (51) resulted in the presence of
DNA-PK that was capable of phosphorylating I
B
(Fig. 4C, lanes 3 and 6). These results indicate that DNA-PK is able to specifically phosphorylate I
B
.
DNA-PK phosphorylates both the amino and carboxy termini of
I
B
.
We next characterized the ability of a highly purified
DNA-PK prepared by a previously described purification scheme
(19) to phosphorylate GST fusion proteins containing
wild-type and mutant I
B
(Fig. 5A)
and I
B
(Fig. 5B) proteins. Purified DNA-PK and our mono S column
fractions containing DNA-PK gave equivalent specificities for the
I
B
and I
B
substrates (19). Numerous studies have
demonstrated that serine and threonine phosphorylation by
DNA-PK is highly dependent on the presence of free DNA ends (19, 54). Purified DNA-PK phosphorylated
-casein, and
the degree of this phosphorylation was greatly enhanced by the addition of sonicated double-stranded salmon sperm DNA (Fig.
6A, lanes 1 and 2). There was no
phosphorylation of GST by DNA-PK (Fig. 6A, lanes 3 and 4). The GST
fusion protein containing the full-length I
B
protein was
phosphorylated by DNA-PK, and this phosphorylation was stimulated
by the addition of double-stranded DNA (Fig. 6A, lanes 5 and 6).
Mutations of serine residues 32 and 36 in I
B
only slightly
decreased I
B
phosphorylation (Fig. 6A, lanes 7 and 8). An
I
B
protein with its 53 N-terminal amino acids deleted was
strongly phosphorylated by DNA-PK (Fig. 6A, lanes 9 and 10), indicating significant C-terminal phosphorylation of I
B
by
DNA-PK. A deletion of the carboxy-terminal 74 amino acids of
I
B
markedly decreased its phosphorylation (Fig. 6A, lanes 11 and
12). These results were consistent with predominant DNA-PK
phosphorylation of the carboxy terminus of I
B
.
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B
that were
potential sites for DNA-PK phosphorylation. The carboxy terminus of
I
B
is critical for DNA-PK-dependent phosphorylation, as
reflected in the major reduction of I
B
phosphorylation seen as a
result of the deletion of its carboxy-terminal 42 amino acids (Fig. 6B, lanes 1 to 4). Casein kinase II has been demonstrated to phosphorylate the carboxy terminus of I
B
at serine residues 283 and 289 and threonine residues 291 and 299 (57, 61). Mutations of serine residue 283, threonine residue 291, and threonine residue 299 to
alanine eliminated the casein kinase II phosphorylation of I
B
(57) (data not shown). However, DNA-PK was able to
phosphorylate this I
B
mutant in addition to an I
B
mutant
with a mutation at threonine 291, which has been reported to markedly
decrease casein kinase II phosphorylation (57) (Fig. 6B,
lanes 5 to 8). Mutation of additional serine and threonine residues in
the carboxy terminus of I
B
extending between amino acids 251 and
317 revealed that only the mutation of threonine residue 273 markedly
reduced DNA-PK phosphorylation of I
B
(Fig. 6B, lanes 9 and
10). Thus, different residues in the carboxy terminus of I
B
are
critical for phosphorylation by DNA-PK and casein kinase II.
The ability of DNA-PK to phosphorylate residues in the amino
terminus of I
B
was next analyzed. Mutation of serine residues 32 and 36 in wild-type I
B
resulted in a slight decrease in I
B
phosphorylation (Fig. 6D, lanes 1 and 2). Since DNA-PK also
phosphorylates the carboxy terminus of I
B
, a truncated I
B
protein that contained only the amino-terminal 138 amino acids of this
protein was next assayed. DNA-PK was able to phosphorylate this
truncated GST-I
B
protein (Fig. 6C, lane 3), but phosphorylation
of an I
B
protein containing mutations at both serine residues 32 and 36 was significantly reduced (Fig. 6C, lane 5). Next, we determined
whether both of these amino-terminal serine residues were
phosphorylated by DNA-PK. Mutation of serine residue 32 did not
significantly decrease the ability of DNA-PK to phosphorylate
I
B
(Fig. 6C, lane 6), while mutation of serine residue 36 markedly decreased DNA-PK phosphorylation of I
B
(Fig. 6C,
lane 7). Mutation of tyrosine residue 42, which has also been
demonstrated to be a site of I
B
phosphorylation by src-like
kinases, did not alter DNA-PK phosphorylation (45). Finally, an I
B
protein containing mutations at serine residues 32 and 36 and tyrosine residue 42 was not phosphorylated by DNA-PK (Fig. 6C, lane 8). These results indicated that serine 36 was the
predominant site in the amino terminus of I
B
that was
phosphorylated by DNA-PK.
Next we characterized the ability of DNA-PK to phosphorylate
I
B
(62, 76). DNA-PK was able to phosphorylate a
GST fusion protein containing full-length I
B
(Fig. 6D, lane 1).
An I
B
protein with mutations of serine residues 19 and 23 (62) exhibited a slight decrease in phosphorylation by
DNA-PK (Fig. 6D, lane 2). Next, we determined whether DNA-PK
could phosphorylate GST fusion proteins containing the amino-terminal
305 amino acids of I
B
, which lack the PEST domain. DNA-PK was
able to phosphorylate this GST-I
B
protein (Fig. 6D, lane 3),
while an I
B
protein containing mutations of serine residues 19 and 23 exhibited decreased phosphorylation by DNA-PK (Fig. 6D, lane
4). These results are consistent with the ability of DNA-PK to
phosphorylate both the N and C termini of I
B
and I
B
.
Interactions between DNA-PK and I
B
.
Since our
purification scheme was based on the ability of kinases to bind to
I
B
, we next characterized the domains of I
B
that were
involved in interactions between these proteins. S100 extract was
prepared from HeLa cells, and the presence of DNA-PK in this
extract was determined by Western blot analysis (Fig. 7A, lane 1). Thus, we could determine
whether DNA-PK present in the S100 extracts was able to directly
interact with I
B
. The S100 extract was incubated with either GST
alone (Fig. 7A, lane 2) or a variety of GST fusion proteins containing
different portions of I
B
. These included GST fusion proteins with
wild-type I
B
(Fig. 7A, lane 3), an I
B
protein having
mutations of serine residues 32 and 36 (Fig. 7A, lane 4), an I
B
protein with its amino-terminal 53 amino acids deleted (Fig. 7A, lane
5), and a GST-I
B
protein containing either its amino-terminal 138 (Fig. 7A, lane 6) or 53 (Fig. 7A, lane 7) amino acids.
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B
proteins with intact ankyrin repeats and PEST
domains (Fig. 7A, lanes 3 to 5). However, there was a marked decrease
in the interaction of DNA-PKcs with I
B
proteins in which the
majority of the ankyrin repeats were deleted (Fig. 7A, lanes 6 and 7).
Western blot analysis revealed similar quantities of each of the
GST-I
B
fusion proteins used in these assays (Fig. 7B). We noted
that the degree of interaction between DNA-PKcs and I
B
was
greatly diminished with more highly purified preparations of DNA-PK
(data not shown). These results suggested that the interactions between
I
B
and DNA-PKcs likely required the I
B
ankyrin repeats and could be mediated by additional cellular factors that are associated with DNA-PKcs.
SCID cells exhibit constitutive nuclear levels of
NF-
B.
DNA-PK is critical in the process of DNA
double-strand break repair. Cells deficient in DNA-PK are
hypersensitive to ionizing radiation and to other agents which induce
double-strand DNA breaks (14, 55). DNA-PK is also
required in resolving double-strand breaks generated in lymphocyte
precursors during V(D)J rearrangement (15, 49, 72). Germ
line mutations in either chain of the Ku heterodimer or in
DNA-PKcs result in the SCID phenotype (15, 49,
72). Recent data suggest that DNA-PK may play a role in traversing checkpoints after cell cycle arrest (52).
B
protein levels and NF-
B nuclear translocation, we used the murine NIH 3T3 fibroblast cell line and the SCID mouse fibroblast cell
line SF19, which is deficient in DNA-PK activity (39). Western blot analysis with DNA-PKcs antibody confirmed that 3T3 cells contained DNA-PKcs and that this protein was absent from SF19
cells (data not shown). Next we analyzed I
B
protein levels in
cytoplasmic extracts prepared for these cells either prior to treatment
with phorbol esters and ionomycin or following treatment with these
inducers of I
B
degradation. Western blot analysis with rabbit
polyclonal antibody indicated that the I
B
levels were
approximately twofold lower in SF19 cells (Fig.
8A, lane 1) than in 3T3 cells (Fig. 8A,
lane 6). Upon treatment of SF19 cells with TPA and ionomycin,
there was a rapid decrease in I
B
protein levels at 15 min
posttreatment followed by a restoration of I
B
protein levels,
which increased to pretreatment levels between 45 and 60 min
posttreatment (Fig. 8A, lanes 1 to 5). Upon treatment of 3T3 cells with
TPA and ionomycin, a marked decrease in I
B
levels also occurred
at 15 min posttreatment followed by a restoration of I
B
protein
levels, which returned to pretreatment levels between 30 and 60 min posttreatment (Fig. 8A, lanes 6 to 10). As a control, Western blot
analysis was performed with antibody directed against the p89
subunit of TFIIH (Fig. 8B). These results indicated that there were
slight reductions in the levels of I
B
protein in cells lacking
DNA-PKcs but that degradation of I
B
by agents that stimulate
the nuclear translocation of NF-
B was not defective in SCID
cells.
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