Phosphorylation of NF-jB proteins by cyclic GMP-dependent kinase
A noncanonical pathway to NF-jB activation
Bin He
1
and Georg F. Weber
1,2
1
Department of Radiation Oncology, New England Medical Center, Boston, MA, USA;
2
Immunology Program,
Sackler School of Graduate Biomedical Research, Tufts University Medical School, Boston, MA, USA
The transcription factor NF-jB is activated in cellular stress
responses. This requires rapid regulation of its function,
which is accomplished, in part, by various modes of phos-
phorylation. Even though diverse DNA binding subunits of
NF-jB proteins may transactivate from distinct recognition
sequences, the differential regulation of transcription from
the large number of NF-jB responsive sites in various gene
promoters and enhancers has been incompletely understood.
The cyclic GMP-dependent kinase (PKG) is an important
mediator of signal transduction that may induce gene
expression through cAMP response element binding protein
(CREB) and through other, yet undefined, mechanisms. We
have previously characterized a signal transduction pathway
that leads to activation-induced cell death in T-lymphocytes
and involves the activation of PKG. Here we demonstrate
that the NF-jB proteins p65, p49 (also called p52), and p50
are specific substrates for this kinase. PKG dose-dependently
increases the transactivating activity of p65 from the NF-jB
consensus sequence. It also mediates dose-dependently an
increase in transcriptional activity by p49 or p50 from a

of the preformed cytosolic complex, which is typically
accomplished by phosphorylation. NF-jB phosphorylation
is functionally relevant on three levels. Firstly, it targets the
inhibitor I-jB for degradation. Even though phosphoryla-
tion of I-jB is not sufficient to dissociate the complexes of
I-jBandNF-jB in vivo, phosphorylation on serines 32 or
36 is a prerequisite for I-jB degradation in the ubiquitin-
proteasome pathway [6]. Secondly, phosphate residues
contribute to the processing of the p100 and p105 NF-jB
precursor proteins for p50 and p49/p52. A prerequisite for
the proteolytic cleavage of p105 is the phosphorylation of
serines 894 and 908, which are potential recognition sites for
proline-directed serine/threonine kinases, including cyclin-
dependent kinases and Erk2 kinase [7]. The phosphoryla-
tion of the C-terminal region of p105 may be mediated by
cyclic AMP-dependent protein kinase or protein kinase C
[8]. Finally, direct phosphorylation of the DNA-binding
NF-jB subunits can regulate their functions. Phosphoryla-
tion of the DNA-binding NF-jB subunits may modulate
DNA binding affinity, transactivation, or the interaction
with other regulatory proteins. A PKA recognition
sequence within the Rel homology domain, which contains
DNA binding sites and nuclear localization signals, has
been linked to the transformation of avian spleen cells by
v-rel and to the cytoplasmic retention of c-Rel in chicken
embryo fibroblasts [9]. PKA activates NF-jB in a manner
that is independent of I-jB phosphorylation, does not
impair NF-jB interaction with unmodified p105, and
induces transactivation [8,10]. This is accomplished through
the recruitment of CREB-binding protein (CBP)/p300 by

the connection of PKG to gene expression is incompletely
understood. Recently, the localization of the PKG form I in
the cytosol and the nucleus was reported [29,30], suggesting
a broader role for PKG in the regulation of gene transcrip-
tion. Our previous studies [3] suggested that NF-jBmaybe
activated by PKG. Here we demonstrate that p49, p50,
and p65 are substrates for the kinase and we analyze the
mechanisms by which PKG induces NF-jB activation.
Materials and methods
Reagents
Recombinant human NF-jB p49 and p50 were obtained
from Promega. The a-isozyme of cGMP-dependent protein
kinase, purified from bovine lung or recombinant bovine,
was purchased from Promega or Calbiochem.
The following oligonucleotides were utilized in gel shift
reactions after radiolabeling with T4 polynucleotide kinase:
NF-jB consensus (Promega, sense 5¢-AGTTGAGGGGA
CTTTCCCAGGC-3¢), OCT1 (Promega, sense 5¢-TGTCG
AATGCAAATCACTAGAA-3¢), H2K (sense 5¢-GGATC
CCGGTCGGGGGATTCCCCATCTCGG-3¢), j enhan-
cer (sense 5¢-AGCAGAGGGGACTTTCCGAGGC-3¢).
The custom made oligonucleotides were obtained as single
stranded and were annealed to double stranded probes after
phosphorylation with T4 polynucleotide kinase and
[c-
32
P]ATP. Double-stranded poly(dI-dC).poly(dI-dC) was
purchased from Pharmacia. The cGMP-dependent kinase
inhibitor Rp-8-pCPT-cGMPS was purchased from Biolog.
The reporter constructs used in this study included a

kinase A inhibitor peptide) with 1 mgÆmL
)1
Kemptide
substrate (LRRASLG) and 1 m
M
[c-
32
P]ATP (30–40
c.p.m.Æpmol
)1
). The reaction was performed in the presence
or absence of 200 l
M
cyclic GMP at room temperature
for 3 min. The reaction was terminated by spotting 50 lL
onto Whatman P-81 filter paper and immediate immers-
ionin10mL75m
M
H
3
PO
4
for 2 min. This was followed
by five washes in 10 mL 75 m
M
H
3
PO
4
, air drying, and

293T cells (1 · 10
6
per 100 mm diameter Petri dish) were
transiently transfected with 0.3 lg pFLAG-p49 or pRSV-
p65 with CaCl
2
. Twenty-four hours after transfection, the
cells were lyzed in 0.5 mL RIPA buffer (50 m
M
Tris/HCl
pH 7.5, 150 m
M
NaCl, 1% NP-40, 0.5% Na-deoxycholate,
0.1% sodium dodecyl sulfate) and precleared with 30 lL
20% protein A agarose beads overnight. One microgram of
anti-FLAG (mouse IgG) or anti-p65 Ig (rabbit polyclonal
IgG) were added for 2 h at 4 °C followed by pulling-down
with 30 lL 20% protein A agarose beads for an additional
1 h. The agarose beads were pelleted at 14 000 g for 1 min
and washed four times in cold RIPA buffer, then twice in
detergent-free buffer (50 m
M
Tris/HCl pH 7.5, 150 m
M
NaCl). Kinase reaction buffer was directly added to the
pelleted beads with or without PKG and cGMP as indicated
plus 1 lL[c-
32
P]ATP for 15 min at room temperature. The
reaction solutions were then resolved on 8% reducing

of total RNA was used for cDNA synthesis with Super-
script II RNase H
–
reverse transcriptase (Gibco BRL,
USA). The coding sequence of PKG Ia was amplified
with the primers 5¢-AGCATGGGCACCCTGCGGGAT
TTA-3¢ and 5¢-ATTAGAAGTCTATGTCCCAGCCTGA
GTTG-3¢. The amplified product was cloned into the vector
pCR3.1 (Invitrogen Carlsbad, CA) followed by subcloning
into the vector pEF6/His B (Invitrogen, Carlsbad, CA).
Sequence fidelity and accurate reading frame were verified
by DNA sequencing analysis.
Targeted mutations in p65 were generated in positions
276 and 305 by PCR cloning with the Quickchange site
directed mutagenesis kit (Stratagene) according to the
protocol by the manufacturer. The sense mutagenic oligo-
nucleotides used were 5¢-GCGGCGGCCTGCCGACCGG
GAGCTCAGT-3¢ for S276A and 5¢-AAACGTAAAAG
GGCATATGAGACCTTCAAGAGCATC-3¢ for T305A
(mutations in bold). The accuracy of the mutations was
confirmed by DNA sequencing.
p49 (obtained from the NIH AIDS Reagent Repository)
was Flag-tagged at the 5¢-end by PCR using the pri-
mers 5¢-CTGCAGCATGGACTACAAGGACGACGA
TGACAAGGAGAGTTGCTACAACCCAGGTCTG-3¢
and 5¢-GAGAGTTGCTACAACCCAGGTCTG-3¢
with pRSV-p49 as a template. The amplified fragment
was cloned into the vector pCR3.1 and sequence fidelity was
confirmed by DNA sequencing.
Reporter gene assays

for separation on 8% SDS-polyacrylamide gels followed by
Western blotting on PVDF membranes. PKG kinase
activity in the lyzates was confirmed by phosphorylation
of the standard substrate LRRASLG (ÔKemptideÕ)where
indicated.
Western blotting
Cells were lyzed in RIPA (50 m
M
Tris/HCl pH 7.5,
150 m
M
NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1%
sodium dodecyl sulfate) or NTEN buffer (20 m
M
Tris/
HCl, pH 8.0, 120 m
M
NaCl, 0.5% NP40) containing
1m
M
phenylmethanesulfonyl fluoride, 10 lgÆmL
)1
pep-
statin, and 1 m
M
dithiothreitol. The lyzates were centri-
fuged at 15 000 g for 5 min and the protein
concentration was determined in the supernatants.
Twenty micrograms of total protein were resolved on
reducing denaturing SDS/polyacrylamide gels and trans-

b
6 and respond to
conventional antigen (ovalbumin) and to the retroviral
superantigen MTV-7 (Mls-1
a
) [32]. The AF3.G7 hybri-
doma was generated by fusing beef insulin immune
C57BL/6 lymph node cells with the BW5147 thymoma
line. It bears V
b
6andV
a
3.2 and responds to MTV-7
according to interleukin-2 production [33]. After stimula-
tion by conventional antigen or superantigen, the T-cells
were obtained by passage through Cell-ect
TM
columns
(Biotex Laboratories Inc., Edmonton, Alberta, Canada)
for the preparation of nuclear extracts. As control for
activation, measurement of
3
H-thymidine incorporation by
O3 cells after stimulation with mitomycin C-treated LBB
cells as antigen-presenting cells was performed as described
previously [32].
2176 B. He and G. F. Weber (Eur. J. Biochem. 270) Ó FEBS 2003
Results
NF-jB is activated by PKG in T-lymphocytes
T-lymphocytes proliferate in response to engagement of

by conventional antigen. To test whether PKG could
induce the lower NF-jB band, we treated nuclear extracts
from resting O3 T-cell clones or AF3.G7 T-cell hybridoma
cells with PKG and cGMP and found it to induce DNA
binding of the lower band to a NF-jB consensus
Fig. 1. Correlation between PKG activity and NF-jB induction in
T-lymphocyte activation. (A,leftpanel)TimecourseofNF-jB
induction after activation of O3 T-cell clones. O3 cells were stimulated
by the conventional antigen ovalbumin (OVA) or retroviral super-
antigen (MTV-7) for 0, 2, or 4 h. The T-cells were selected for pre-
paration of nuclear extracts, which were then subjected to incubation
with a
32
P-labelled NF-jB consensus probe followed by electropho-
retic mobility shift assay. The relative intensity of the slower and faster
migrating bands after stimulation with MTV or OVA was quantitated
by densitometric measurement. The relative density units for the lower
band are O3 7.0, OVA 2 h 9.8, OVA 4 h 12.8, MTV 2 h 7.9, MTV 4 h
9.7.TherelativedensityunitsfortheupperbandareO36.9,OVA2h
10.5, OVA 4 h 13.3, MTV 2 h 5.5, MTV 4 h 6.7. Comparable levels of
T-cell stimulation were confirmed by tritiated thymidine incorporation
(c.p.m.). (A, right panel) Before stimulation for 4 h, the O3 cells were
pretreated with the inhibitor Rp-8-pCPT-cGMPS. After T-cell selec-
tion, nuclear extracts were prepared and analyzed by gel shift assay for
binding to the H2K probe. (B) Nuclear extracts from the AF3.G7
hybridoma or the O3 clone were phosphorylated in vitro by PKG plus
cGMP followed by analysis of DNA binding to an oligonucleotide
containing the H2K sequence in gel shift assays. The nuclear extract
from O3 cells that had been treated with plate-bound anti-CD3e
antibody served as a positive control for induction of the faster

were transiently cotransfected with pC/EBP-wt-luc reporter (1 lg) and pRSV-p65 (0.3 lg) with or without 3 lg pEF6/HisB-PKG. Transfection of
0.5 lg pRSV-p50 with pC/EBP-wt-luc reporter (1 lg) served as a positive control. Twenty-four hours after transfection, the cells were harvested in
reporter lysis buffer. Forty micrograms of lyzate samples were used for luciferase assays by luminometer and the values obtained for the vector
control group were normalized to 1. The results represent mean ± standard deviation of triplicate samples (top panel). Similar results were
obtained in three independent experiments. Twenty micrograms of lyzates were used for Western blotting to confirm the expression levels of the
transfected proteins (bottom panel). (C) 293T cells were transiently transfected with increasing amounts of PKG in conjunction with either 2 lg
NF-jB consensus reporter or 2 lg pC/EBP-wt-luc reporter. Twenty-four hours after transfection, the cells were harvested in reporter lysis buffer.
Luciferase activity was measured in 40 lg of lyzate samples and the values obtained for the vector control group were normalized to 1. The results
represent mean ± standard deviation of triplicate samples.
2178 B. He and G. F. Weber (Eur. J. Biochem. 270) Ó FEBS 2003
sites. Cotransfection of increasing amounts of PKG dose-
dependently enhanced the transactivation by transfected
p65. The results were consistent with the protein expres-
sion levels and kinase activities in the cell lyzates
(Fig. 2A). Therefore, PKG increases the transactivating
ability of p65.
We also performed cotransfection experiments with a
reporter that contains a nonconsensus NF-jB binding site.
Consistent with earlier reports [35–37], p65 does not
transactivate the nonconsensus motif associated with
C/EBP, which is found in the C-reactive protein promoter.
In this case, the lack of transactivation is not overcome
by cotransfection of PKG (Fig. 2B).
We noted that the transfection of PKG alone was
sufficient to increase the consensus reporter activity by
approximately fourfold (compare Fig. 2A). We therefore
tested whether PKG could stimulate transactivation by
the endogenous NF-jB. 293T cells express substantial
amounts of endogenous p65, but very little p49 and p50.
Consistently, transfected PKG increased the activity of

four consensus sites. 293T cells were transiently cotransfected with
0.5 lgpNF-jB-luc plus 0.3 lgp49or0.3lgpRSV-p50plusincreas-
ing amounts of pEF6/HisB-PKG. 10 ng of the Renilla construct pRL-
SV40 was also cotransfected to normalize the data for transfection
efficiency. Transactivation by p65 (0.3 lgpRSV-p65)inthesame
experiment is shown as a positive control (at the chosen concentrations
of reporter DNA, PKG induces a less than twofold induction of
transactivation by endogenous p65). Expression of the transfected
proteins was confirmed by Western blotting. All panels show the
results of one representative experiment from at least three replicates.
Ó FEBS 2003 Phosphorylation of NF-jB by PKG (Eur. J. Biochem. 270) 2179
approximately fivefold increase in luminescence readout
(Fig. 2C).
The NF-jB protein p50 contains a DNA binding
domain, but no transactivation domain. Nevertheless,
transactivation may be observed after transfection of p50
into cells, presumably due to its binding to endogenous
interaction partners. Those include most prominently p65,
but also Bcl-3 [38]. In addition, p50-dependent transactiva-
tion can occur from a nonconsensus site in conjunction with
C/EBP [35,36]. We used the nonconsensus reporter con-
struct in transient cotransfection assays. No reporter activity
was induced by PKG alone, whereas p50 dose-dependently
increased the luciferase activity (data not shown).
Co-transfection of PKG with low amounts of p50 (0.3 lg
DNA) dose-dependently enhanced its transactivating acti-
vity (Fig. 3A), consistent with an increased affinity of p50 to
this DNA sequence after phosphorylation by PKG. Com-
parable results were obtained with murine p50 (data not
shown). P49 and p50 are related NF-jB subunits [39]. We

inhibitor of PKG, reverses the enzymatic
phosphorylation of p49 or p50. Consistent
with the competition for binding to the kinase
between cGMP and Rp-8-pCPT-cGMPS, the
inhibition is more complete in the absence of
cGMP than in its presence. Autophosphory-
lation of PKG serves as a positive control for
kinase activity. (D) Recombinant His-tagged
p65 was phosphorylated by PKG in vitro in
the absence or in the presence of cGMP (top
panel). 293T cells were transiently transfected
with p65 or FLAG-tagged p49. The cells were
lyzedinRIPAbufferandthetransfected
molecules were pulled down by antibodies to
the p65 or Flag. Kinase reaction buffer plus
[c-
32
P]-ATP was directly added to the pelleted
beads with or without PKG and cGMP for
15 min at room temperature. The reaction
mixtures were analyzed by autoradiography
and Western blotting (bottom panel).
2180 B. He and G. F. Weber (Eur. J. Biochem. 270) Ó FEBS 2003
In summary, the transactivation experiments using
reporter assays indicated that PKG can enhance the
transcriptional activity of the DNA binding NF-jBproteins
p65, p50, and p49 from their cognate recognition sites. In
contrast, PKG does not confer transactivating potential
from the, respectively, noncognate NF-jB sequences.
Because the proteins, p49, p50, and p65 direct transcription

down of p49). The immunoprecipitates and 10% of the input were
resolved on SDS/PAGE and the resulting Western blot was probed
with antibodies to p65, to p52 (recognizes p49), and to PKG.
Detectable levels of endogenous p65 are expressed in 293T cells and are
recognized by the specific antibody, accounting for the band on the p65
blot from untransfected cells. Similar results were obtained in a repeat
experiment. No bands were detected with control immunoglobulin or
in a sample without cell lyzate. (B) 293T cells were transiently trans-
fected by calcium phosphate precipitation with vector control or 3 lg
pEF6-PKG.After24 h,thecellswerelyzedinNTENbuffer.PKGwas
pulled down, the bound proteins were resolved on SDS/PAGE, and
the Western blot was probed with antibodies to PKG and to p65. The
input (2%) is shown in the two left lanes. The two right lanes show the
no cell control and the pull-down with an irrelevant antibody,
respectively. (C) 293T cells were transiently transfected with 3 lg
vector, pRSV-p50, pRSV-p49, or pRSV-p65. After 24 h, the cells were
lyzed for immunoprecipitation. The left panel (Input) shows the
Western blots of whole cell lyzates as a control for transfection effi-
ciency. The right panel (IP) coimmunoprecipitated PKG (top row) and
precipitated or coprecipitated p65 (second row). The immunoglobulin
heavy chain interferes with the detection of pulled-down p50 or p49.
Therefore, the successful immunoprecipitation in these cases was
confirmed by Western blotting for the endogenous precursor proteins
p100, which is recognized by anti-p52 antiserum, and p105, which is
recognized by antibody to p50 (bottom rows). Immunoprecipitation
with an irrelevant antibody and immunoprecipitation without cells
served as negative controls (two right lanes).
Ó FEBS 2003 Phosphorylation of NF-jB by PKG (Eur. J. Biochem. 270) 2181
Similar to p49 and p50, bacterial recombinant His-tagged
p65 was phosphorylated by PKG in vitro and phosphate

p50/p49 (Fig. 5C).
The phosphorylation of p65 occurs on nonconsensus
sites
P65 is a substrate for PKA [11], an enzyme, whose substrate
specificity is similar to PKG. Likely recognition sites for
both enzymes are in positions serine 276 and threonine 305
on p65, and serine 276 has been demonstrated to be
phosphorylated by cyclic AMP-dependent kinase [11]. We
mutated both candidate phosphorylation sites. Phosphory-
lation of the serine in position 276 is known to be essential
for p65 dependent transactivation [11]. In accord with these
previous observations, the transactivating activity was
diminished moderately by the mutation T305A and sub-
stantially by S276A, however, cotransfection of PKG led
to comparable dose-dependent increases in reporter activity
in all cases (Fig. 6A). Consistent with this observation,
synthetic peptides covering the threonine 305 (EKRKRT
YETF)ortheserine276(MQLRRPSDRE) did not
incorporate radioactive phosphate during incubation with
the kinase, whereas the standard substrate peptide
LRRASLG (ÔKemptideÕ) did (Fig. 6B). We also phospho-
rylated bacterial recombinant p65-His [40] with PKG
in vitro and were able to compete the kinase reaction with
the substrate peptide GRTGRRNSI, but not with the
peptides covering threonine 305 or serine 276 (Fig. 6C).
These results suggest the hypothesis that PKG phos-
phorylates p65 in positions distinct from the amino acids
276 and 305.
Fig. 6. The phosphorylation of p65 by PKG does not occur on the
consensus recognition sites. (A) 293T cells were transiently cotrans-

phosphorylation and PKG autophosphorylation were determined by
resolution on reducing denaturing 8% SDS/polyacrylamide gel fol-
lowed by autoradiography.
2182 B. He and G. F. Weber (Eur. J. Biochem. 270) Ó FEBS 2003
Phosphorylation of p50 by PKG directly impacts
its DNA binding characteristics
Changes in transactivating activity may reflect alterations
in DNA binding affinity. We studied the effects of PKG
on oligonucleotide binding by NF-jB proteins in elec-
trophoretic mobility shift assays. Cyclic GMP-dependent
kinase is inactive under standard gel shift assay condi-
tions. Conversely, gel shift assays cannot be performed in
the PKG reaction buffer. We therefore adjusted the kinase
reaction buffer so that we could phosphorylate NF-jB
proteins and then transfer an aliquot to the standard
DNA binding buffer for analysis of phosphorylation-
dependent changes in the DNA binding characteristics.
Phosphorylation by cGMP-dependent kinase did not
affect the binding of p49 or p50 to the H2K probe, to
which these proteins already have high affinity without
being phosphorylated [41]. In contrast, in vitro binding of
recombinant p50 to the NF-jB consensus sequence or to
the nonconsensus NF-jB-C/EBP sequence was increased
by PKG (Fig. 7A,B). The binding affinity of nuclear
extracts from 293T cells transiently transfected with p50
and PKG to the same probes was similarly increased
(Fig. 7C,D). The specificity of the main DNA binding
band from transfectants of p50 or p50 plus PKG was
confirmed by supershift (Fig. 7D).
The subcellular localization of NF-jB subunits

A57034) does not contain any PKG consensus sites, there are
five similar sites with the sequence X(R,K)X(S,T) in p49
(amino acids 76, 195, 201, 231, 430) that may conceivably
serve as candidate recognition motifs. Interestingly, we have
found p49 to be a poor substrate for PKA. The consensus
sites for PKG in p65 are at positions 276 and 305 (GenBank
accession number M62399). The phosphorylation of amino
Fig. 7. PKG dependent phosphorylation of p50 increases its binding affinity to consensus and nonconsensus (jB-C/EBP) sequences. (A and B)
Recombinant p50, 25 ng per sample, were phosphorylated at room temperature for 15 min by 1 U purified PKG with or without 200 l
M
cGMP in
total volume of 10 lL. The reaction mixtures were transferred to DNA binding buffer and incubated for additional 20 min with the indicated
32
P-labeled probes. The reactants were resolved on native 4% polyacrylamide gels and exposed to autoradiography film. (C and D) 293T cells were
transiently transfected with 0.3 lg pRSV-p50 with or without 3 lg pEF6/HisB-PKG. Twenty-four hours after transfection, the cells were har-
vested, washed in NaCl/P
i
, and nuclear extracts were prepared. Ten micrograms of nuclear protein was used for electrophoretic mobility shift assay
with radiolabeled NF-jB consensus or OCT1 oligonucleotides (C), or with radiolabeled jB-C/EBP oligonucleotides (D). The identity of the major
DNA-binding band was confirmed by supershift with 50 ng anti-p50 antiserum, added to the nuclear extracts at room temperature for 10 min
before DNA binding (D). The upper arrow indicates the supershifted band.
Ó FEBS 2003 Phosphorylation of NF-jB by PKG (Eur. J. Biochem. 270) 2183
acid 276 by PKA is of major importance for p65 transcrip-
tional activity. Remarkably, our experiments indicate that
PKG phosphorylates p65 in positions other than the two
consensus sites. The differential phosphorylation of p49 by
PKG but much less by PKA, and the phosphorylation of p65
by PKA and PKG on distinct residues implies multiple
modes of regulation of NF-jB function. It also indicates that
the position, in which phosphorylation occurs, is an import-

system [18]. Cellular NF-jB activation by H
2
O
2
has been
shown to involve protein phosphorylation and therefore to
be mediated indirectly via induction of a kinase [8]. The
cGMP-dependent kinase may thus provide a link for
the NF-jB activation by reactive oxygen species because the
activity of guanylate cyclase, which synthesizes the cofactor
cGMP, can be regulated by redox reactions.
Acknowledgements
This study was supported by National Institutes of Health research
grant CA76176 and Department of Defense breast cancer grant
DAMD17-98-1-8060 to G. F. W. The authors are indebted to Ranjan
Sen for substantial helpful discussions throughout the project. Janet
Stavnezer generously provided the murine p50 DNA. The plasmids
containing human p49, p50, and p65 were obtained from the NIH
AIDS Reagent Repository. The reporter constructs for the noncon-
sensus p50 site were a generous gift from David Samols. Thomas
Maniatis generously provided the purified recombinant p65. We thank
Peter Brodeur for critically reading the manuscript.
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Ó FEBS 2003 Phosphorylation of NF-jB by PKG (Eur. J. Biochem. 270) 2185