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Mol Cell Biol, July 1998, p. 4221-4234, Vol. 18, No. 7
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
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
DNA-Dependent Protein Kinase Phosphorylation of
I
B
and I
B
Regulates NF-
B DNA Binding
Properties
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
.
|
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
.
|
|
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.
|
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.
|
B DNA binding properties differed
in nuclear extract prepared from SF19 and NIH 3T3 cells. Gel
retardation analysis with a 32P-labeled NF-
B DNA
probe was performed with nuclear extract prepared from untreated 3T3
and SF19 cells. There was no binding to the NF-
B probe in 3T3
nuclear extract (Fig. 8C, lane 2), while constitutive DNA binding of
NF-
B was noted with SF19 nuclear extract (Fig. 8C, lane 3). To
confirm that nuclear levels of NF-
B were indeed higher in cells
lacking DNA-PK, we also prepared nuclear extract from control
equine fibroblast cell line CRL6288 and equine SCID fibroblast cell
line 1863 (82). Gel retardation analysis again confirmed
constitutive NF-
B binding activity in nuclear extract prepared
from the equine SCID cells, but not the control equine fibroblast cell
line (Fig. 8C, lanes 4 and 5). Nuclear extracts prepared from untreated
Jurkat cells (Fig. 8C, lane 6) and Jurkat cells treated with TPA and
ionomycin (Fig. 8C, lanes 7 and 8) were also assayed in gel retardation
assays with the NF-
B probe. This analysis demonstrated that the
mobility of the NF-
B gel-retarded complex in Jurkat nuclear
extract was the same as that seen with extracts prepared from the
murine and equine SCID cell lines. Antibodies to either RelA (Fig. 8C,
lane 9) or the p50 subunit of NF-
B (Fig. 8C, lane 10), when
added to the TPA- and ionomycin-treated Jurkat nuclear extract
supershifted the gel-retarded complex, indicating its specificity.
Antibody to the coactivator protein CBP was added as a
control to this gel retardation assay and did not alter the
mobility of the NF-
B complex (Fig. 8C, lane 11). Finally,
to standardize these nuclear extracts, we compared the amounts of SP1
protein prepared from these cell lines by using gel retardation
analysis. These data indicated that the amounts of SP1 in the different
cell lines were similar (Fig. 8D, lanes 1 to 8). These results indicate
that SCID cell lines which lack DNA-PKcs have constitutive nuclear
NF-
B DNA binding activity.
Increases in I
B
levels decrease NF-
B DNA
binding properties in SCID cells.
To determine whether
constitutive NF-
B DNA binding in SCID cells could be modulated
by overexpression of I
B
, we utilized recombinant
adenoviruses containing either flag-tagged wild-type I
B
or a
dominant-negative I
B
protein containing mutations of serine
residues 32 and 36. A recombinant adenovirus expressing the
-galactosidase gene was used as a control. Western blot analysis with a flag monoclonal antibody confirmed the expression of the wild-type and the mutant I
B proteins upon infection of SF19 and 3T3
cells by the recombinant adenoviruses (data not shown). Each of
these adenoviruses was then used to infect 3T3 and SF19 cells, and nuclear extract prepared 24 h later was analyzed by gel
retardation analysis with an NF-
B oligonucleotide probe. Nuclear
extract prepared from SF19 cells but not from 3T3 cells (Fig.
9A, lanes 1 and 5) revealed constitutive
NF-
B DNA binding. A faster-mobility nonspecific gel-retarded
species was present in these nuclear-extract preparations, and its
mobility did not change with the addition of antibodies to p50 or p65,
in contrast to the results seen with the slower-mobility species (data
not shown). Adenoviruses expressing
-galactosidase did not alter the
mobility of the NF-
B-specific gel-retarded species in SF19
extract (Fig. 9A, lanes 2 and 6). In contrast, infection with
adenoviruses containing either wild-type I
B
(Fig. 9A, lanes 3 and
7) or a mutant I
B
with mutations at serine residues 32 and 36 (Fig. 9A, lanes 4 and 8) reduced NF-
B DNA binding in SF19
cells. There was no change in SP1 binding in the 3T3 and SF19 nuclear
extracts infected with these different adenoviruses (Fig. 9B).
The adenovirus construct containing wild-type I
B
was also assayed
in SF19 and NIH 3T3 cells, and similar I
B
protein stability was
found (data not shown).
|
B
protein degradation
(28, 32, 43), could alter the constitutive NF-
B DNA
binding properties in SF19 nuclear extract. Following a 45-min
treatment with 50 µM TPCK, nuclear extract was prepared from 3T3 and
SF19 cells (Fig. 9C, lanes 1 to 4) and subjected to gel retardation
analysis. TPCK was able to reduce NF-
B DNA binding in nuclear
extract prepared from SF19 cells (Fig. 9C, lanes 3 and 4). The addition
of TPCK did not alter SP1 binding in either 3T3 or SF19 cells (data not
shown). Whether the ability of TPCK to prevent I
B degradation is
related to its ability to inhibit protease and/or kinase activity is
unclear (28, 32, 43). These results indicate that increases
in I
B
protein reduce constitutive NF-
B DNA binding
properties in SCID cells.
DNA-PK regulates NF-
B DNA binding properties.
Although we demonstrated that SCID cells exhibited constitutive
nuclear NF-
B DNA binding, it was important to determine whether the lack of DNA-PKcs was responsible for this phenotype. Nuclear extract was prepared from the parental murine cell line, CB-17, a SCID
cell line (SCID-St), and SCID-St cells complemented with portions of
human chromosome 8 either containing the DNA-PKcs gene (100E) or
lacking this gene (50D). Gel retardation analysis indicated that
NF-
B binding was markedly increased in extract prepared from
SCID-St cells (Fig. 10A, lane 3)
compared to that seen in extract from the CB-17 cells (Fig. 10A,
lane 2). Nuclear extract prepared from the
DNA-PKcs-complemented 100E cells had reduced NF-
B
binding activity (Fig. 10A, lane 4) compared to nuclear extract
prepared from 50D cells which lack DNA-PKcs (Fig. 10A, lane 5). SP1
binding levels were similar in nuclear extract prepared from each of
these cells (Fig. 10B). This indicated that the lack of DNA-PKcs
led to constitutive nuclear NF-
B DNA binding activity.
|
B binding, we performed gel retardation analysis with recombinant p50 and p65 NF-
B proteins produced in
baculovirus. Purified I
B
alone and I
B
phosphorylated by
DNA-PK were incubated with the p50 and p65 proteins prior to the
addition of a 32P-labeled NF-
B oligonucleotide
probe. Kinase assays with [
-32P]ATP demonstrated that
the I
B
protein was phosphorylated by DNA-PK under these
conditions (data not shown). Gel retardation analysis indicated that
unphosphorylated I
B
(Fig. 10C, lanes 3 to 5) was a weaker
competitor for p50-p65 binding to the NF-
B probe than the
DNA-PK-phosphorylated I
B
(Fig. 10C, lanes 6 to 8).
Antibody to p65 but not CBP supershifted the p50-p65 complex bound to
the NF-
B oligonucleotide, indicating the specificity of this
binding (Fig. 10C, lanes 9 and 10). These results suggested that
DNA-PK phosphorylation of I
B
increases its interaction with
NF-
B and thus reduces NF-
B DNA binding properties.
Western blot analysis demonstrated that DNA-PKcs was present in
extract prepared from the parental and SCID cells complemented with a
portion of human chromosome 8 containing the DNA-PKcs gene (Fig. 10D, lanes 1, 2, 5, and 6). However, DNA-PKcs was
absent from SCID cells and SCID cells lacking the portion of human
chromosome 8 that contained the DNA-PKcs gene (Fig. 10D, lanes 3, 4, 7, and 8). In addition, Western blot analysis confirmed that the
levels of I
B
in these cell lines were similar although there was
a slight but reproducible reduction of I
B
levels in the SCID cell line (Fig. 10E). These results indicate that DNA-PK phosphorylation of I
B
regulates NF-
B DNA binding properties rather than
significantly altering I
B
levels.
DNA-PK and I
B
distribution in SCID and
DNA-PKcs-complemented SCID cells.
We next determined the
intracellular localization of DNA-PK in HeLa cells, murine SCID
cells lacking the DNA-PKcs gene (50D) and murine SCID cells
complemented with a portion of human chromosome 8 which contains
DNA-PKcs (100E). Immunofluorescence staining with monoclonal
antibody 25-4 directed against human DNA-PKcs followed by
analysis using confocal microscopy indicated that DNA-PKcs was
present predominantly in the nuclei of HeLa (Fig. 11A) and 100E cells (Fig. 11C). As
expected, no DNA-PKcs-specific nuclear staining was observed
in the SCID mouse cell line (50D) which is deficient in both mouse
and human DNA-PKcs (Fig. 11B).
|
B
and the RelA or p65 subunit of NF-
B.
I
B
was present in both the cytoplasm and the nuclei of HeLa and
100E cells (Fig. 11A and C). I
B
staining was slightly increased
in the cytoplasm of 50D SCID cells, but there was reduced I
B
staining in the nuclei of these cells compared to that observed
in the nuclei of 100E and HeLa cells. The RelA or p65 subunit of
NF-
B was present predominantly in the cytoplasm of HeLa and 100E
cells (Fig. 11A and C), whereas it was present in the cytoplasm and in increased amounts in the nuclei of 50D cells (Fig. 11B). Thus, the
absence of DNA-PKcs in the nuclei of SCID mouse cells correlated with decreased nuclear staining of I
B
and with the presence of
increased amounts of RelA in the nuclei of these cells.
DISCUSSION
|
|
|---|
In this study, we characterized cellular kinases that
phosphorylate I
B
. Using a biochemical fractionation scheme, we
isolated a cellular kinase, DNA-PK, that phosphorylated serine
residue 36 and threonine residue 273 in I
B
. Threonine residue 273 in I
B
appeared to be a predominant site of phosphorylation by
DNA-PK. However, the relative effects of phosphorylation of each of
these residues on I
B
function were not addressed in this study.
It is possible that phosphorylation of each of these residues may alter
I
B
function by different mechanisms or that phosphorylation of
both of these sites is required to alter NF-
B binding.
Alternatively, only the predominant carboxy-terminal phosphorylation of
I
B by DNA-PK may be critical for the regulation of NF-
B
DNA binding properties.
The biological relevance of DNA-PK phosphorylation of I
B was
demonstrated by an analysis of SCID cell lines in which DNA-PK activity is absent (15, 49, 72). SCID cell lines from
different species including murine and equine species were found to
have constitutive nuclear NF-
B DNA binding activity when
analyzed in gel retardation assays. This was in contrast to SCID cell
lines complemented with the DNA-PKcs gene (49) in which
there was marked reduction in NF-
B DNA binding activity. These
results and gel retardation assays with unphosphorylated and
DNA-PK-phosphorylated I
B
suggest that DNA-PK
phosphorylation of I
B enhances its ability to associate with
NF-
B and inhibit NF-
B DNA binding properties.
Since DNA-PKcs is predominantly localized to the nucleus (19,
54), it likely phosphorylates I
B in the nucleus. However, we
cannot rule out the possibility that small quantities of DNA-PK may
be present in the cytoplasm and phosphorylate I
B
in the cytoplasm. The ability of double-stranded DNA to markedly stimulate DNA-PK phosphorylation of I
B
would also be consistent with a role for DNA-PK phosphorylation of I
B in the nucleus.
Immunofluorescence studies demonstrate the presence of I
B
in both
the cytoplasm and the nuclei of HeLa and SCID cells complemented with
DNA-PKcs, with reduced levels of I
B
seen in the nuclei of
SCID cells. Previous studies indicate that in some cell lines I
B
is present in both the nucleus and cytoplasm and that its role in the
nucleus may be to inhibit NF-
B-mediated gene expression and
terminate the response to inducers such as TNF-
(2, 3,
60). Increases in I
B phosphorylation by DNA-PK may inhibit
NF-
B DNA binding activity in the nucleus by increasing
associations between I
B
and NF-
B (78, 84).
Decreases in I
B
levels in the nuclei of SCID cells could be due
to the fact that nonphosphorylated I
B is preferentially transported to the cytoplasm compared to phosphorylated I
B
. I
B
can shuttle between the nucleus and the cytoplasm to
promote the efficient nuclear export of NF-
B complexes
(3). This function is mediated by a nuclear export sequence
in the C terminus of I
B
that is homologous to the previously
described export signal found in the human immunodeficiency virus
type 1 Rev protein. It is intriguing that this signal is located
between amino acids 266 and 277 in I
B
, which includes the
threonine residue (residue 273) that is phosphorylated by
DNA-PK (3). Thus, it is possible that the
phosphorylation of I
B
by DNA-PK leads to enhanced
nuclear accumulation of I
B
and more potent inhibition of
NF-
B DNA binding. DNA-PK phosphorylation of serine residue
36 may also lead to higher levels of I
B
in the nucleus by
blocking the ability of inducible kinases such as IKK
and IKK
to
phosphorylate the amino terminus of I
B, which may lead to its
degradation in the nucleus.
I
B
is subject to phosphorylation by a number of different
kinases. These include kinases that are inducible by mitogenic stimuli,
such as IKK
and IKK
, that phosphorylate the amino terminus of
I
B
and lead to its degradation by the proteasome (27, 63, 65, 83, 85). In addition, a phorbol ester-inducible kinase, RSK1,
that specifically phosphorylates I
B
on serine residue 32 has been
identified (71). Whether RSK1 in conjunction with DNA-PK
can phosphorylate both serine residues 32 and 36, similar to the
actions of IKK
and IKK
, and thereby induce I
B
degradation remains to be determined. In addition to amino-terminal phosphorylation of I
B
, carboxy-terminal phosphorylation of I
B
by casein
kinase II has been demonstrated (10, 11, 57, 61, 66). The
four residues in the carboxy terminus of I
B
that are
phosphorylated by casein kinase II are distinct from the predominant
threonine residue that is phosphorylated by DNA-PK. The
phosphorylation of I
B
by casein kinase II is likely involved in
regulating its constitutive stability (11, 57, 61). Our
results suggest that DNA-PK phosphorylation of I
B
is at least
partially distinct from that of casein kinase II and that DNA-PK
phosphorylation does not significantly alter I
B
protein
stability. However, it is intriguing to note that casein kinase II
phosphorylation of the C-terminal PEST domain of I
B
but not of
I
B
markedly increases the ability of I
B
to inhibit
NF-
B DNA binding properties (25, 78). Thus,
DNA-PK and casein kinase II can phosphorylate both I
B
and
I
B
, but these kinases may have differential effects on I
B
and I
B
interactions with NF-
B.
I
B
is a modular protein in which different domains are subject to
regulation by different protein kinases. Inducible kinases, such as
IKK
and IKK
, which are activated by TNF-
and interleukin 1 lead to phosphorylation of the N terminus of I
B
and the
degradation of I
B
(27, 63, 65, 83, 85). Constitutive
phosphorylation of I
B by casein kinase II regulates basal I
B
protein stability (11, 57, 61). In this study, we
demonstrate that DNA-PK phosphorylates both the amino and carboxy
termini of I
B
and I
B
. Further analysis will be required to
determine the relevance of each of these phosphorylation sites. It is
important to note that another member of the PI-3 kinase family, the
product of the ataxia-telangiectasia gene (ATM) (68), has
also been demonstrated to phosphorylate I
B
(47).
Furthermore, an I
B
protein with its amino terminus deleted can
prevent the marked radiosensitivity seen in ataxia-telangiectasia cells
(48). Our results suggest that I
B
is regulated not
only by kinases that lead to its degradation but by kinases that
alter its interactions with NF-
B. This is likely critical in the
control of the expression of genes involved in regulating cellular
growth and proliferation.
ACKNOWLEDGMENTS
|
|
|---|
We thank Sharon Johnson for the preparation of the manuscript;
Shane Fults for the preparation of the figures; and R. Robinson, and K. Pierce for expertise in the digestion and mass spectrometry. We thank
Dean Ballard for providing the I
B
and I
B
cDNAs and Cordula
Kirchgessner for providing SCID and DNA-PKcs-complemented SCID cell
lines.
This work was supported by grants from the NIH, the Veterans Administration, and the Robert Welch Foundation.
Li Liu and Youn-Tae Kwak contributed equally to this work.
FOOTNOTES
* Corresponding author. Mailing address: Division of Hematology-Oncology, Department of Internal Medicine, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, Texas 75235-8594. Phone: (214) 648-7570. Fax: (214) 648-8862. E-mail: Gaynor{at}UTSW.SWMed.edu.
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