It also mediates dose-dependently an increase in transcriptional activity by p49 or p50 from a unique CCAAT/enhance binding protein C/EBP-associ-ated NF-jB site, but not from the consens
Trang 1Phosphorylation of NF-jB proteins by cyclic GMP-dependent kinase
A noncanonical pathway to NF-jB activation
Bin He1and Georg F Weber1,2
1
Department of Radiation Oncology, New England Medical Center, Boston, MA, USA;2Immunology 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 unique CCAAT/enhance binding protein (C/EBP)-associ-ated NF-jB site, but not from the consensus site Phos-phorylation of p65, p50, or p49 does not alter their subcellular distribution Because the release of cytosolic p65/p50 heterodimers into the nucleus is by itself insufficient
to differentiate all the numerous NF-jB promoter sequences, phosphorylation of the DNA-binding subunits reveals a form of differential regulation of NF-jB activity and it implies a novel pathway for PKG-induced gene transcription These observations may bear on mechanisms
of programmed cell death in T-lymphocytes They may also
be relevant to ongoing efforts to induce cancer cell apoptosis through activation of PKG
Keywords: protein kinases; signal transduction; superanti-gens; transcription factors
The transcription factor NF-jB [1,2] mediates a wide range
of cellular stress responses It induces the gene expression
of cytokines, acute phase proteins, and adhesion molecules
In T-lymphocytes, NF-jB regulates activation and
activa-tion-induced cell death [3] and it contributes to T-cell
selection at the double positive stage [4,5] A large number
of gene promoters and enhancers contain NF-jB binding
sites This poses the question how selectivity of
NF-jB-dependent transactivation is accomplished during specific
modes of lymphocyte activation While the release of
p65/p50 into the nucleus is insufficient to differentiate
among diverse binding sites it is clear that there are
mechanisms, which control the interactions of NF-jB with
its various recognition sequences
The biologic roles of NF-jB necessitate a rapid activation
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-jB and NF-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-reland 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
Correspondence to G F Weber, Department of Radiation and
Cancer Biology, New England Medical Center, NEMC #824,
750 Washington Street, Boston, MA 02111, USA.
Fax: 617 636 1766, Tel.: 617 636 9013,
E-mail: gweber@tufts-nemc.org
Abbreviations: PKA, cyclic AMP-dependent kinase; PKG, cyclic
GMP-dependent kinase.
(Received 20 November 2002, revised 19 February 2003,
accepted 14 March 2003)
Trang 2phosphorylated p65 [11–13] Some investigators have not
found p65, p50, or p52 to be phosphorylated by PKA or
PKC [8,14] While p65 and p50 have candidate recognition
sequences for PKA or PKG, p49/p52 does not have such a
consensus site [9,15], however, related sequences are found
in all three molecules
The enzyme cyclic GMP-dependent kinase (cGK, PKG)
is an important mediator of intracellular signal
transduc-tion, involved in such diverse processes as the regulation of
blood vessel tone, platelet aggregation, and long-term
potentiation in memory formation [16–18] Furthermore,
PKG may regulate apoptosis positively [3,19–22] or
negat-ively [23], possibly depending on other modulating
bio-chemical events [24] Efforts have been made to induce
cancer cell apoptosis with sulindac sulfone (exisulind,
Aptosyn), which activates PKG [25–27] The
down-regula-tion of PKG levels in neoplastic ovarian epithelial cells [28]
may contribute to enhanced tumor cell survival Although
PKG, like cyclic AMP-dependent kinase (PKA), can
phosphorylate and activate the transcription factor CREB,
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-jB may be
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-32P]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
commercial NF-jB luciferase reporter (pNF-jB-luc,
Clontech) that contains four consensus NF-jB sites A
luciferase reporter containing the C-reactive protein
promoter (pC/EBP-luc) and a relevant control with a
mutated NF-jB p50 binding site (pC/EBP-mP50-luc)
were generously provided by D Samols (Dept
Biochem-istry, Case Western Reserve University, Cleveland, OH,
USA) The plasmids containing human p49, p50, and
p65 were obtained from the NIH AIDS Reagent
Repository J Stavnezer generously provided the murine
p50 DNA
Kinase reaction The enzymatic activity of cGMP-dependent kinase was analyzed in kinase reaction buffer (250 mM Mes, pH 6.9,
2 mM EGTA, 5 mM magnesium acetate, 50 mM NaCl,
10 mgÆmL)1 BSA, 100 mM dithiothreitol, 2 mM protein kinase A inhibitor peptide) with 1 mgÆmL)1 Kemptide substrate (LRRASLG) and 1 mM [c-32P]ATP (30–40 c.p.m.Æpmol)1) The reaction was performed in the presence
or absence of 200 lM 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-ion in 10 mL 75 mMH3PO4for 2 min This was followed
by five washes in 10 mL 75 mM H3PO4, air drying, and scintillation counting For the analysis of PKG activity in cell lyzates, the PKA inhibitor peptide (Sigma) was present Phosphorylation of PKG substrates (232 ngÆlL)1 for recombinant NF-jB p49 and p50; 500 ngÆlL)1 for p65) was performed in 100 mM Tris/HCl, pH 7.5, 20 mM sodium chloride, 10 mM dithiothreitol, 2 mM magnesium acetate, 200 lM ATP, at room temperature for 15 min The kinase concentration was 3.5 UÆlL)1, while cyclic GMP was present at 200 lM Mops buffer was not used because it is incompatible with the electrophoretic mobi-lity shift assay after transfer There was no loss of PKG enzymatic activity in Tris buffer at the indicated concentration
293T cells (1· 106per 100 mm diameter Petri dish) were transiently transfected with 0.3 lg pFLAG-p49 or pRSV-p65 with CaCl2 Twenty-four hours after transfection, the cells were lyzed in 0.5 mL RIPA buffer (50 mM Tris/HCl
pH 7.5, 150 mMNaCl, 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 4C 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 mM Tris/HCl pH 7.5, 150 mM NaCl) Kinase reaction buffer was directly added to the pelleted beads with or without PKG and cGMP as indicated plus 1 lL [c-32P]ATP for 15 min at room temperature The reaction solutions were then resolved on 8% reducing denaturing SDS-polyacrylamide gel and transferred to poly(vinylidene difluoride) (PVDF) membranes for auto-radiographic exposure and Western blotting
Electrophoretic mobility shift assay DNA binding was assessed by electrophoretic mobility shift according to standard protocols The reaction mixture contained 10 mMTris/HCl, pH 7.5, 1 mMEDTA, pH 8.0,
35 mM NaCl, 50 lgÆmL)1 poly(dI-dC)Æpoly(dI-dC), and 5% glycerol plus labeled probe The DNA binding proteins were transferred from the kinase reaction mixture The reaction was incubated at room temperature for
20 min before separation on a native 4% polyacrylamide gel For supershift, the appropriate antibodies (0.05– 0.1 lg) were incubated with the nuclear extracts for
10 min at room temperature before adding DNA binding buffer
Trang 3Cloning of relevant gene products
To clone mouse PKG Ia cDNA, frozen mouse kidney tissue
was homogenized and total RNA was isolated by using
RNeasy mini kit from Qiagen (Valencia, CA, USA)
following the manufacturer’s protocol One microgram
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
293T cells were plated at 1· 106 cells per 100 mm
diameter Petri dish and were grown for 24 h before
transfection with CaCl2 The commercial pNF-jB-luc
reporter (Clontech, Palo Alto, CA, USA) contains four
NF-jB response elements and was used at 0.5 lg per
transfection (to amplify the signal for the analysis of
transactivation by endogenous p65, we used 2 lg of
reporter DNA) The common internal transfection
standard Renilla in pRL-SV40 (10 ng per transfection)
served as a control for transfection efficiency This
reporter construct was not sensitive to cotransfection of
PKG or NF-jB and provided stable reference values
Renillawas not used in transfection experiments with the
noncommercial reporter constructs because the
lumines-cence intensity is too high compared to the specific
readout Twenty-four hours after transfection, the cells
were harvested in 1 mL reporter lysis buffer (Promega)
and dual luciferase reporter assays were performed
following the protocol provided by the manufacturer
Lyzates were diluted 1 : 40 and 10 lL were used for
measurement in a luminometer (Turner Designs TD-20/20)
In reporter gene experiments without Renilla,
lumine-scene was measured in 40 lg (total protein) of lyzate The
protein concentrations were determined by the BCA
protein assay reagent kit (Pierce) As confirmation of
protein expression, 20 lg of the same lyzates were also used
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 (Kemptide) where indicated
Western blotting Cells were lyzed in RIPA (50 mM Tris/HCl pH 7.5,
150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate) or NTEN buffer (20 mM Tris/ HCl, pH 8.0, 120 mM NaCl, 0.5% NP40) containing
1 mM phenylmethanesulfonyl fluoride, 10 lgÆmL)1 pep-statin, and 1 mM 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-ferred to PVDF membranes The membranes were probed with appropriate antibodies followed by horse-radish peroxidase-conjugated secondary antibodies and development using enhanced chemiluminescence Anti-p65 Ig (C-terminus, rabbit polyclonal), anti-p50 Ig (rabbit antiserum), and anti-p52 Ig (rabbit antiserum, used to detect p49) were obtained from Upstate Biotechnology (Lake Placid, NY, USA) Anti-PKG Ig (C-terminal, amino acids 657–671, rabbit polyclonal) was purchased from Calbiochem (La Jolla, CA, USA) Reprobing of the membranes with anti-tubulin Ig (mouse IgG isotype, Sigma) served as additional loading control
For pull-down assays, transiently transfected 293T cells were lyzed in 0.5 mL NTEN buffer The lyzates were precleared at 4C with 25 lL 20% agarose beads, before addition of 0.5–1 lg of the indicated antibody for 2 h and precipitation with 25 lL 20% agarose beads for 1 h The beads were washed, resuspended in SDS/PAGEsample buffer, and the bound proteins were resolved on 8% SDS/ polyacrylamide gels The proteins of interest were detected
by Western blotting
Cells Reporter assays were performed by transient transfections
of 293T cells O3 is a CD4+ T-helper cell clone derived from BALB/c mice after in vitro selection for proliferation
to ovalbumin in association with BALB/c antigen-present-ing cells [31] O3 cells express Vb6 and respond to conventional antigen (ovalbumin) and to the retroviral superantigen MTV-7 (Mls-1a) [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 Vb6 and Va3.2 and responds to MTV-7 according to interleukin-2 production [33] After stimula-tion by convenstimula-tional antigen or superantigen, the T-cells were obtained by passage through Cell-ectTM columns (Biotex Laboratories Inc., Edmonton, Alberta, Canada) for the preparation of nuclear extracts As control for activation, measurement of3H-thymidine incorporation by O3 cells after stimulation with mitomycin C-treated LBB cells as antigen-presenting cells was performed as described previously [32]
Trang 4NF-jB is activated by PKG in T-lymphocytes
T-lymphocytes proliferate in response to engagement of
their antigen receptor by conventional antigen peptide We
have previously described an alternative signal
transduc-tion pathway, associated with the T-cell antigen receptor
that is induced by superantigen and leads to activation of
PKG and activation-induced cell death [3] Because
T-lymphocyte stimulation is often characterized by NF-jB
translocation to the nucleus and binding to cognate DNA
sequences, we compared the induction of NF-jB following
stimulation by conventional antigen or retroviral
super-antigen in the T-cell clone O3 Consistent with previous
observations [34], the induction of NF-jB by physiologic
T-lymphocyte stimulation is moderate In electrophoretic
mobility shift assays measuring the binding to a NF-jB
consensus probe, the larger and smaller NF-jB complexes
were induced by the conventional antigen In contrast,
predominantly the smaller complex, represented by the
lower band on the gel, was induced by stimulation with
superantigen This was not due to quantitative differences
in stimulation because both modes of T-cell activation
induced comparable levels of tritiated thymidine
incorpor-ation in the same experiment (Fig 1A) To confirm the
dependence of the superantigen mediated induction of
NF-jB, we treated the O3 clones with the cell permeable PKG inhibitor Rp-8-pCPT-cGMPS before stimulation Because superantigen induces predominantly the faster migrating band the gel shift analysis was performed with the H2K probe, which has a higher affinity to p50/p52 than the consensus probe Expectedly, the presence of the PKG inhibitor suppressed the superantigen-dependent induction of NF-jB but had no effect on T-cell stimulation
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, left panel) Time course of NF-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 a32P-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 The relative density units for the upper band are O3 6.9, OVA 2 h
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
migrating NF-jB band Note that the resting levels of NF-jB binding
are relatively high in O3 cells, because the clone depends on the
pre-sence of interleukin-2 in the growth medium, whereas the hybridoma
AF3.G7 does not (C) The induction by PKG plus cGMP of DNA
binding by AF3.G7 cytosol is inhibitable by addition of high
con-centrations of a competing PKG substrate peptide, GRTGRRNSI
(PKI substrate, amounts are l M ) In addition, cGMP and PKG did
not affect Octamer-1 binding in the same experiment (not shown).
Similar results were obtained in at least three additional experiments.
Trang 5oligonucleotide (Fig 1B) A similar induction was seen in
AF3.G7 cytosol and this was inhibitable by increasing
amounts of the competing PKG substrate peptide
GRTGRRNSI (PKI substrate) (Fig 1C) We therefore
set out to investigate the role of PKG in the induction of
NF-jB
PKG increases the transactivating activity of NF-jB proteins from distinct recognition sites
We asked whether PKG can alter the transactivating activity
of p65 as judged by luciferase assays with a commercial
NF-jB luciferase reporter that contains four consensus NF-NF-jB
Fig 2 PKG increases the transactivating activity of p65 from consensus sites but not from a nonconsensus NF-jB rep orter (A) 293T cells were transiently transfected with 300 ng pRSV-p65 and increasing amounts of pEF6/HisB-PKG as indicated Transactivation from the cotransfected pNF-jB-luc reporter (500 ng) was measured by luciferase activity The values are normalized to luminescence induced by 10 ng cotransfected Renilla construct pRL-SV40 and the results (fold induction) are presented as mean ± standard deviation of three samples (top panel) The protein expression of the transfected molecules correlated with the amounts of DNA introduced into the cells, while tubulin (used as a loading control) remained constant (bottom panel) The PKG activity in 20 lg of cell lyzates, as judged by phosphorylation of LRRASLG peptide (kemptide)
in vitro, reflected the amounts of PKG transfected The kinase activity is indicated as mean ± standard deviation (middle panel) (B) 293T cells 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.
Trang 6sites 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
the consensus reporter, but not of the C/EBP-associated
promoter, in a dose-dependent manner, reaching
Fig 3 Transactivation by p50/p49 from a nonconsensus sequence is
enhanced by PKG (A) Transactivation by transfected p50 of a
luci-ferase reporter gene containing a nonconsensus NF-jB site that
overlaps with a C/EBP site (sequence in top panel) 293T cells were
transiently cotransfected with 1 lg pC/EBP-wt-luc reporter (or the
control construct pC/EBP-mp50-luc, in which the p50 binding site is
mutated, represented as pC/EBP (mP50)) with 0.5 lg pRSV-p50 and
the indicated amounts of pEF6/HisB-PKG Twenty-four hours after
transfection, the cells were harvested in reporter lysis buffer and 40 lg
lyzate samples were used for luciferase assays The results, measured as
fold induction, are presented as mean ± standard deviation of three
samples and the values obtained for the vector control group have been
normalized to 1 The transcriptional activity is induced
dose-depend-ently by cotransfection of increasing doses of PKG (second panel from
top) Twenty micrograms of the lyzates were analyzed for PKG kinase
activity (third panel from top), while another 20 lg of lyzates were
used for Western blotting to confirm the protein expression levels
(fourth panel) PKG activity in cell lyzate and Western blotting for the
transfected molecules served as transfection controls (B) PKG
enhances p49-mediated transactivation from the C/EBP-associated
nonconsensus site 293T cells were transiently cotransfected with 1 lg
pC/EBP-wt-luc reporter and 0.5 lg pRSV-p49 plus increasing
amounts of pEF6/HisB-PKG for analysis of luciferase reporter gene
activity The results are represented as fold induction and the values
obtained for the vector control group are normalized to 1 Western
blotting confirmed the expression levels of the transfected molecules
(bottom panel) (C) Neither p49 (top panel) nor p50 (bottom panel)
transactivate from the commercial NF-jB reporter gene containing
four consensus sites 293T cells were transiently cotransfected with
0.5 lg pNF-jB-luc plus 0.3 lg p49 or 0.3 lg pRSV-p50 plus
increas-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 lg pRSV-p65) in the same
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.
Trang 7approximately 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 conjunctransactiva-tion 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 found p49 to also transactivate from the nonconsensus NF-jB site in a manner that could be increased dose-dependently by cotransfected PKG (Fig 3B)
We then tested whether p50 or p49 transactivate the luciferase reporter that contains four NF-jB consensus sites and whether transactivation under these conditions might
be modulated by PKG Luciferase activity was not induced
by transfection of p49 or p50 alone (under the conditions used here, PKG enhances the transactivation by endo-genous p65 less than twofold) Furthermore, cotransfection
of p50 or p49 with increasing amounts of PKG did not lead
to measurable transactivation from the NF-jB consensus luciferase reporter (Fig 3C)
Fig 4 The NF-jB proteins p49, p50, and p65 are substrates for cGMP-dependent kinase (A) Substrate phosphorylation of p49 or p50 depends on the presence of PKG and is enhanced by the addition of cGMP, whereas cGMP in the absence of the kinase does not mediate measurable incorporation of phos-phate Autophosphorylation of PKG is rep-resented as the upper band on all gels and reflects a specificity control for effects on the enzyme (B) Phosphorylation of p49 or p50 by PKG (as well as PKG autophosphorylation) is reversible by titration of a competing substrate peptide for the kinase (GRTGRRNSI), but not a control peptide with a mutated serine (GRTGRRNAI) (C) An analog of cGMP, Rp-8-pCPT-cGMPS, which can act as an 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 lyzed in RIPA buffer and the transfected molecules were pulled down by antibodies to the p65 or Flag Kinase reaction buffer plus [c-32P]-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).
Trang 8In summary, the transactivation experiments using
reporter assays indicated that PKG can enhance the
transcriptional activity of the DNA binding NF-jB proteins
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
from distinct DNA sequences, their activation by PKG
enhances their differential effects
P49, p50, and p65 are substrates for cyclic
GMP-dependent kinase
A possible mechanism to account for PKG-enhanced
transactivation by NF-jB is the phosphorylation of the
DNA binding proteins by the enzyme We therefore tested
whether p49, p50, and p65 are substrates of the kinase
NF-jB p49 and p50 were phosphorylated by cyclic
GMP-dependent kinase and the phosphorylation levels were
enhanced by addition of cyclic GMP (Fig 4A) The
substrate peptide GRTGRRNSI (PKI substrate), but not
the control peptide GRTGRRNAI, inhibited
phosphory-lation of p49 or p50 as well as autophosphoryphosphory-lation of
cGMP-dependent kinase in a dose-dependent manner
(Fig 4B) The kinase reactions on p49 and p50 were also
inhibited by the cGMP-dependent kinase inhibitor
Rp-8-pCPT-cGMPS at a high concentration Consistent with the
competitive function of the compound, the inhibition was complete in the absence of cGMP but partial in the presence
of cGMP (Fig 4C) There is substantial substrate overlap between PKA and PKG Cyclic AMP-dependent kinase also phosphorylated p50 with comparable efficiency, but p49 was phosphorylated strongly by cGMP-dependent kinase and only very weakly by PKA (data not shown)
Fig 5 PKG binds NF-kB proteins (A) 293T cells were transfected
with vector or p65, or were cotransfected with NF-jB p65 plus PKG.
Alternatively, p49 was transfected with or without PKG The cells
were lyzed in NTEN buffer After preclearing, immunoprecipitation
was performed with anti-p65 antibody or anti-Flag antibody (for
pull-down of p49) The immunoprecipitates and 10% of the input were
resolved on SDS/PAGEand 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
pE F6-PKG After 24 h, the cells were lyzed in NTE N buffer PKG was
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).
Trang 9Similar to p49 and p50, bacterial recombinant His-tagged
p65 was phosphorylated by PKG in vitro and phosphate
incorporation was enhanced by the presence of cGMP
(Fig 4D, top panel) We expressed p49 and p65 by transient
transfection in 293T cells P65 was immunoprecipitated with
an anti-p65 Ig and p49 was immunoprecipitated with
an antibody to Flag-tag The pulled-down proteins were
phosphorylated by PKG in vitro Both NF-jB subunits
incorporated radioactive phosphate, although the
phos-phorylation of p65 was substantially weaker than the
phosphorylation of p49 (Fig 4D, bottom panel)
We further confirmed the interaction between p65 or p49
and PKG in vivo by coimmunoprecipitation of the kinase
with an antibody to either p65 or Flag-tag (for p49) after
cotransfection of the kinase with either of the NF-jB
proteins (Fig 5A) Because 293T cells express substantial
levels of NF-jB p65, we tested whether transfected PKG
could pull down endogenous p65 The immunoprecipitation
with anti-PKG antibody efficiently yielded endogenous as
well as transfected PKG In both cases, endogenous NF-jB
p65 was bound, and the band intensity on Western blot
correlated to the amount of PKG present (Fig 5B) To
extend this analysis, we transfected 293T cells with p65, p50,
or p49, immunoprecipitated with antibodies specific to the
transfected gene products, and probed for pulled-down
endogenous PKG In all cases, coimmunoprecipitation of
substantial amounts of PKG was detected Due to the high
expression levels of p65 in 293T cells, the antibody to p65
also pulled-down PKG from untransfected cells Not
unexpectedly, the immunoprecipitations with anti-p50 and
anti-p52 Ig pulled down endogenous p65, suggesting the
possibility of a trimeric complex containing PKG, p65, and
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
YE TF) or the serine 276 (MQLRRPSDRE) did not
incorporate radioactive phosphate during incubation with
the kinase, whereas the standard substrate peptide
LRRASLG (Kemptide) did (Fig 6B) We also
phospho-rylated bacterial recombinant p65-His [40] with PKG
in vitroand 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-fected with 0.5 lg pNF-jB-luc and 0.3 lg pRSV-p65 or its mutants T305A or S276A plus increasing amount of pEF6/HisB-PKG Co-transfected 10 ng of the Renilla construct pRL-SV40 served as a control for transfection efficiency Twenty-four hours after transfec-tion, the cells were harvested in reporter lysis buffer and 10 lL of 1 : 40 diluted lyzates were assayed for luciferase activity The data are pre-sented as fold induction with the values obtained from the vector (pEF6/HisB) transfected cells normalized to 1 The inset shows the PKG-dose dependent increase in transactivation by p65S276 A on an adjusted scale (B) Ten micrograms of the synthetic peptides EK-RKRTYETF (T305p65), MQLRRPSDRE (S276p65), or Kemptide were incubated with or without 1 U PKG plus 200 l M cGMP plus
1 m M [c- 32 P]ATP in total volume of 100 lL for 3 min at room tem-perature One half of the reaction volume was spotted onto filter paper and the reaction was stopped by washes in phosphoric acid The levels
of peptide phosphorylation were determined by scintillation counting and the results are presented as mean values ± standard deviation of three samples (C) Recombinant p65 (10 lgÆmL)1) was phosphoryl-ated by 1 U PKG in kinase reaction buffer at room temperature for
15 min, in the presence or absence of increasing concentrations of the synthetic peptides GRTGRRNSI (PKI substrate), or EKRKRT YETF (T305p65), or MQLRRPSDRE (S276p65) The extent of p65 phosphorylation and PKG autophosphorylation were determined by resolution on reducing denaturing 8% SDS/polyacrylamide gel fol-lowed by autoradiography.
Trang 10Phosphorylation 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
is not affected by phosphorylation with PKG
NF-jB is an inducible transcription factor, which is retained
in the cytosol in resting cells It was therefore possible that
the phosphorylation of DNA binding subunits might affect
their nuclear import as would be reflected in their
subcel-lular distribution Transfection of increasing amounts of PKG did not alter the relative fractions of cotransfected p49, p50, or p65 in the cytosols and nuclei Although not definitive, these observations made phosphorylation-induced changes in the transport and half-lives of these NF-jB subunits unlikely (Fig 8)
Discussion
The incorporation of phosphate into p49 or p50 depends on PKG, is enhanced by cGMP, can be competed by a PKG substrate peptide but not by a control peptide, and is inhibitable by a cGMP analog with inhibition being more efficient in the absence of cGMP than in its presence The sum of these observations indicates that NF-jB p49 and p50 are specific substrates for PKG Similarly, phosphorylation
of p65 is dependent on the kinase, is increased in the presence
of cGMP, and is competed out by a standard substrate peptide Cyclic GMP-dependent kinase, like cyclic AMP-dependent kinase, has a preference for the phosphorylation
of serines or threonines found close to at least two consecutive N-terminal basic residues The standard PKG recognition site is (R,K)(R,K)X(S,T) It is important to note, however, that there are a number of exceptions to this rule The p50 precursor p105 has PKG recognition sites in positions 335 and 940 (GenBank accession numbers M57999
or NM_003998) Although p49 (GenBank accession number 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.