From the Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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The activity of the transcription factor NF- The NF- In most unstimulated cells, NF- Emerging evidence also suggests a second level of controlling NF- As for other members of the atypical protein kinase C family, PKC In this study, we analyzed the role of PKC Cell Culture--
Bovine aortic endothelial cells (BAEC) and
porcine aortic endothelial cells (PAEC) were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum,
L-glutamine (2 mM), penicillin G (100 units/ml), and streptomycin (100 µg/ml). Human umbilical vein
endothelial cells (HUVEC) were grown in M199 medium supplemented with
15% fetal bovine serum, NaH2CO3 (20 mM), HEPES (25 mM), glutamine (5 mM), heparin (100 µg/ml), gentamycin (50 µg/ml), and
endothelial growth factor (50 µg/ml). Primary cultures of PAEC and
HUVEC were used between the fourth and the fifth passage. BAEC were
used between the fifth and the seventh passage. All cells were grown in
culture at 37 °C in a 5% humid CO2 atmosphere. All
media and supplements were from Life Technologies, Inc.
Plasmid Constructs--
The pcDNA3 vector expressing tagged
wild-type Xenopus laevis PKC Transient Transfection and Reporter Assays--
Primary BAEC
were transfected as described (34). Experiments involving RelA
co-transfection were analyzed 20-24 h after transfection. Where
indicated, cells were incubated with human recombinant TNF- PKC p21ras Immunodetection and Activity Assay--
PAEC
and HUVEC were labeled with [35S]Met/Cys for 6 h.
Cells (2-3 × 106) were disrupted (20 min on ice) in
lysis buffer (10 mM Tris·HCl, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 0.2% Triton X-100) supplemented with
phosphatase and protease inhibitors. Lysates were cleared by
centrifugation, and p21ras was immunoprecipitated by incubating
lysates overnight with a rat monoclonal anti-p21ras antibody
coupled to agarose beads (sc-35AC; Santa Cruz Biotechnology). Beads
were washed eight times in wash buffer (50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1%
Triton X-100, 0.005% SDS). Proteins were eluted by boiling in Laemmli
buffer and separated on 15% polyacrylamide gels under denaturing
conditions. Gels were dried and subjected to autoradiography. To
analyze p21ras GTP loading, serum-starved PAEC were labeled
with [32P]orthophosphate for 4 h. Cells were
left untreated or stimulated with TNF- Electrophoretic Mobility Shift Assay--
Whole cell extracts
were prepared from transfected BAEC as described before (38). All
buffers were supplemented with 10 µg/ml aprotinin, 25 µM leupeptin, 1 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Cell extracts were
incubated (30 min at room temperature) with 100,000 cpm of
double-stranded [ RelA Metabolic Labeling and Immunoprecipitation--
PAEC or
BAEC cultured in 10-cm dishes were labeled with
[32P]orthophosphate (500 µCi/ml) in phosphate-free
Dulbecco's modified Eagle's medium for 4 h and stimulated with
TNF- Phosphoamino Acid Analysis--
RelA was immunoprecipitated from
metabolically labeled, TNF- RelA Phosphopeptide Mapping--
BAEC or BAEC transfected with
various RelA constructs as described above were metabolically labeled
with 500 µCi/ml [32P]orthophosphate for 4 h. Cell
extracts were prepared by detergent lysis as described above and
immunoprecipitated with anti-RelA-agarose (Santa Cruz Biotechnology).
Immunoprecipitates were boiled in Laemmli buffer, separated by
polyacrylamide gel electrophoresis, and transferred to a PVDF membrane.
Tryptic digests were obtained as described (40), and equal amounts of
radioactivity were loaded on cellulose plates. The first dimensional
electrophoretic separation was carried out in ammonium carbonate buffer
(pH 8.9). The chromatography was performed in an
n-butanol/pyridine/glacial acetic acid/H2O (37.5:25:7.5:30) buffer. Plates were exposed to x-ray film or analyzed using a PhosphorImager scanning device (Molecular Dynamics).
Activation of PKC
As for PKC Regulation of RelA Transcriptional Activity by PKC
Given that p21ras has been implicated in the PKC
Neither PKC Role of Small GTPases, Raf-1, and PI 3-Kinase--
There are
several potential downstream targets of the p21ras and PKC RelA RHD Is the Target of PKC Regulation of RelA Transcriptional Activity by PKC Phosphorylation of Endogenous RelA--
As previously reported,
RelA is phosphorylated upon stimulation with TNF-
Phosphorylation of RelA has been attributed to phosphorylation of
serines 276 (48) and 529 (50). Both studies could show an exclusive
role for the respective serine in RelA phosphorylation. We investigated
RelA phosphorylation by two-dimensional separation of tryptic
phosphopeptides prepared from nontreated, TNF- Phosphorylation of Overexpressed RelA--
To investigate whether
an exogenous stimulus is necessary to trigger RelA phosphorylation or
alternatively if free, i.e. non-I
Finally, we addressed the topology of RelA phosphorylation. To test to
what degree RelA RHD participates in the overall phosphorylation of
RelA, we transfected BAEC with a RelA mutant that encodes the N-terminal 320 amino acids (RelA/RHD; Ref. 34). Overexpressed RelA/RHD
was readily phosphorylated (Fig. 8A, lane
2), and its phosphorylation was completely inhibited by
co-expressed I
To investigate the contribution of serine 276 to the overall RelA
phosphorylation, RelA was immunoprecipitated from cells transfected
with wild-type RelA (RelA wt) or with a mutated form, where
Ser276 was replaced by Ala (RelA S276A). The
phosphorylation of RelA S276A was significantly lower as compared with
wild type RelA (Fig. 9A). However, the
reduction in phosphorylation was not only caused by a decrease in
specific phosphorylation but also by reduced protein levels of the RelA
S276A mutant. Although EC were transfected with a 3-fold excess of RelA
S276A construct as compared with wild type RelA, the level of
expression of RelA S276A was always lower than wild type RelA (data not
shown). We are currently investigating the causes of this
phenomenon.
Comparison of tryptic peptide maps from wild type RelA and RelA S276A
revealed the specific loss of a phosphopeptide in RelA S276A (Fig.
9B), corresponding to the phosphopeptide a in the endogenous RelA (Fig. 7), which is constitutively phosphorylated in
RelA, and its phosphorylation status is not altered by TNF- Inhibition of RelA Phosphorylation by Blockage of PKC
Having established that PKC It is widely accepted that regulation of NF- Several kinases have been implicated in the regulation of nuclear RelA
transcriptional activity. Protein kinase A is involved in the
regulation of RelA transcriptional activity through phosphorylation of
Ser276 in the consensus site (RRPS) located in the RHD (39,
48). In addition, p38 MAPK has also been implicated in regulating RelA transcriptional activity. However, contrary to protein kinase A, p38
MAPK may not act directly on RelA (51), as suggested by the observation
that inhibition of p38 MAPK does not result in detectable changes in
RelA phosphorylation (16). Casein kinase II has also been shown to
associate with NF- Several downstream effectors may account for the effect of PKC RelA has been shown to be inducibly phosphorylated upon cytokine
stimulation in several cell types, and phosphorylation of the
transactivation domain has been proposed to be a major regulatory mechanism by which the activity of several transcription factors is
controlled (55). Similarly, phosphorylation of the RelA transactivation domain has been reported (56). In particular, inducible phosphorylation of the TA2 (amino acids 428-520) and constitutive
phosphorylation of the TA1 (amino acids 521-551)
activation domains have been suggested to control RelA transcriptional
activity (56). Recently, phosphorylation of the RelA transactivation
domain by RelA-associated casein kinase II has been reported (52), and
the importance of phosphorylation of Ser529 has been
revealed (50). In this study, we present evidence that the RHD domain
contributes substantially to the overall phosphorylation of RelA. We
show that RelA phosphorylation can be inhibited partially by
co-expressing I We further show that RelA is constitutively phosphorylated at multiple
sites and that phosphorylation of some but not all of this site is
increased by TNF- Finally, our data implicate RHD as a central regulator of RelA
transcriptional activity and show that the phosphorylation status of
RHD can modulate the transcriptional activity of the transactivation
domain. This effect seems to be independent of the transactivation
domain itself in that it acts on the RelA as well as on a VP16
transactivation domain (Fig. 3B). It is worthwhile to note
that both RelA and VP16 belong to the same class of acidic transactivators (57). Whether or not this regulatory effect can be
extended to other classes of transactivation domains remains to be
established. The RelA RHD may control the activity of the transactivation domain by several mechanisms. For one, RHD
phosphorylation could induce conformational changes in the
transactivation domain, facilitating interactions with components of
the basal transcriptional machinery, essential for RelA transcriptional
activity (56). Allosteric control of the DNA binding domain over
the transactivation domain has been reported for several transcription
factors (58). Therefore, it would be of interest to obtain crystal
structure data of full-length RelA bound to DNA in its phosphorylated
and nonphosphorylated form.
Second, the phosphorylation status of RHD may regulate interaction of
RelA with nuclear cofactors such as cAMP response element-binding protein-binding protein (CBP/p300) (59, 60). Although not specifically
addressed in this study, it is unlikely that CBP/p300 would be a
cofactor involved in the regulation of RelA transcriptional activity by
PKC Another possible mechanism by which PKC In summary, our data show the existence of a second NF-B
is thought to be regulated mainly through cytoplasmic retention by
I
B molecules. Here we present evidence of a second mechanism of
regulation acting on NF-
B after release from I
B. In endothelial
cells this mechanism involves phosphorylation of the RelA subunit of
NF-
B through a pathway involving activation of protein kinase C
(PKC
) and p21ras. We show that transcriptional activity of
RelA is dependent on phosphorylation of the N-terminal Rel homology
domain but not the C-terminal transactivation domain. Inhibition of
phosphorylation by dominant negative mutants of PKC
or
p21ras results in loss of RelA transcriptional activity without
interfering with DNA binding. Raf/MEK, small GTPases,
phosphatidylinositol 3-kinase, and stress-activated protein kinase
pathways are not involved in this mechanism of regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B/Rel family of dimeric transcription factors is
involved in the immediate early transcription, i.e.
independent of protein synthesis, of a large array of genes induced by
mitogenic or pathogen-associated stimuli. In its active form, NF-
B
is a nuclear homo- or heterodimeric complex of a number of different Rel family members. The canonical and most abundant form of NF-
B is
composed of a 50-kDa (p50, or NF
B1) and a 65-kDa (p65, or RelA)
subunit. Both subunits can form homodimers as well as heterodimers with
other members of the Rel family i.e. c-Rel (Rel), p52
(NF
B2), and RelB (1). All members of the Rel family exhibit
extensive sequence similarity in their N-terminal region referred to as the Rel homology domain
(RHD)1 responsible for DNA
binding and formation of Rel dimers. Only RelA, Rel, and RelB carry a
transcription activating domain, and thus only dimers containing one of
these proteins activate the transcription of
NF-
B-dependent genes efficiently. With respect to
transcription activation, the RelA subunit appears to have the highest activity.
B is constitutively retained in the
cytoplasm by inhibitory proteins of the I
B family, namely I
B
,
I
B
, I
B
, p100, p105, and I
B
(2). Formation of
NF-
B·I
B complexes masks the nuclear localization signal
sequence present in NF-
B molecules and thus prevents their nuclear
translocation. One of the key events in the activation of NF-
B is
the liberation of functional NF-
B dimers from I
B, which results
in the translocation of NF-
B to the nucleus. Cytoplasmic release of
NF-
B dimers involves site-specific phosphorylation of I
B by
kinases of the I
B signalosome (3-6), ubiquitination (7), and
subsequent proteolytic degradation by the 26 S proteasome pathway (8).
Upon nuclear import and binding to specific decameric recognition
motifs, which are reflected by the consensus GGGRNNYYCC (where R
represents A or G and Y represents C or T), NF-
B dimers function as
transcriptional activators. I
B
(9), I
B
, and p105 (10) have
been implicated in the inhibition of DNA binding of NF-
B complexes.
However, there have been several reports showing that NF-
B
transcriptional activity can be blocked without affecting DNA binding.
These include the interactions of NF-
B with the glucocorticoid
receptor (11, 12), the mammalian repressor REP (13), and the
interferon-inducible factor p202 (14).
B
transcriptional activity that acts directly on NF-
B dimers without
influencing the degradation of I
B molecules. For example, ectopic
expression of a dominant negative mutant of the atypical protein kinase
C
(PKC
) or the extracellular signal-regulated kinase 1 inhibit
TNF-
-induced NF-
B activity (15). Similarly, inhibition of p38
mitogen-activated protein kinase (p38 MAPK) has been shown to decrease
TNF-
-induced NF-
B activity and interleukin-6 expression (16).
More recently, tyrosine phosphorylation has been shown to be essential
for NF-
B activity in bacterial lipopolysaccharide (LPS)-induced monocytic THP1 cells (17). Regulation of NF-
B activity
by PKC
, extracellular signal-regulated kinase 1, p38 MAPK, or
tyrosine phosphorylation acts downstream of I
B without interfering with NF-
B nuclear translocation and DNA binding.
is
not activated by Ca2+ or diacylglycerol and is insensitive
to phorbol esters (18). Unresponsiveness of PKC
to Ca2+
and diacylglycerol is consistent with the absence of the
Ca2+ binding C2 domain and the presence of only one
cysteine-rich zinc finger-like motif in the diacylglycerol binding C1
domain of PKC
. PKC
is activated by several lipid mediators
including phosphatidic acid (19) and phosphatidylinositol
3,4,5-trisphosphate (20). PKC
has also been shown to be activated by
TNF-
and interleukin-1 through sphingomyelin hydrolysis and
subsequent generation of ceramide (21-23). Other pathways leading to
PKC
activation include the 21-kDa guanine nucleotide-binding
p21ras (24), which in addition to several growth factors is
also activated by TNF-
as well as LPS (25, 26). Ras GTPases have
been implicated in the signaling of a variety of extracellular stimuli
that control cell proliferation and differentiation. Ras GTPases are
activated by members of the guanine nucleotide exchange factor family,
which increase Ras GTP loading and are negatively regulated by the
GTPase-activating proteins, which enhance the intrinsic rate of
hydrolysis of Ras-bound GTP. Upon binding to GTP, Ras recruits and
activates downstream effectors such as Raf, PI 3-kinase (27) and the
kinase suppressor of Ras (28) by a mechanism that is not well
understood. p21ras has also been implicated in controlling
NF-
B activity in fibroblasts (29, 30).
and p21ras in
regulating NF-
B activity in endothelial cells. We demonstrate that inhibition of either one of these pathways changes the phosphorylation of the RelA subunit and severely impairs NF-
B-mediated transcription without interfering with the ability of NF-
B to bind to DNA.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and rat PKC
dominant
negative mutant were a kind gift of J. Moscat (Universidad
Autónoma, Madrid) and were described elsewhere (15). Expression
vectors encoding wild-type p21ras, a dominant-negative (RasN17)
and a constitutively active mutant (RasV12) were a kind gift from
G. M. Cooper (Harvard Medical School). The inserts were amplified
by polymerase chain reaction with primers carrying appropriate
restriction sites and cloned into pcDNA3HA, which is derived from
pcDNA3 (Invitrogen, Carlsbad, CA) by inserting a DNA fragment
coding for MYPYDVPDYASL, where amino acids 2-12 code for an epitope
derived from the hemagglutinin protein of the human influenza virus.
RhoA, Rac1, and Cdc42 were amplified from HeLa cDNA by polymerase
chain reaction and cloned into pcDNA3HA. Dominant negative mutants
of these small GTPases were generated by overlap extension as described
elsewhere (31). Cdc42N17 was generated by replacing Thr17
with Asn employing the overlapping primers
5'-GTAAAAACTGTCTCCTGATATCCTAC and
5'-GATATCAGGAGACAGTTTTTACCAACAGCACC, Rac1N17 was generated by replacing Thr17 with Asn using the primers
5'-CTGTAGGTAAAAACTGCCTACTGATC and 5'-TGATCAGTAGGCAGTTTTTACCTACAGCTCCG, and RhoAN17 was
generated by replacing Thr19 with Asn using the primers
5'-GTGGAAAGAACTGCTTGCTCATAGTCTTC and 5'-ATGAGCAAGCAGTTCTTTCCACAGGCTCCATC (the underlined base
triplet indicates the mutated amino acid). The Raf-1 dominant negative mutant encompassing the first 259 amino acids encoding the regulatory domain (32) was generated by polymerase chain reaction using full-length human Raf-1 (ATCC 41050) as template. The Src homology 2 (SH2) domain of the 85-kDa regulatory subunit of PI 3-kinase shown to
act as a dominant negative mutant (33) was cloned from BAEC cDNA
and cloned into the pcDNA3 vector. The different fusion proteins
outlined in Fig. 5A were generated by a polymerase chain reaction-based approach and were all expressed from the pcDNA3 vector. The RelA expression plasmid is based on the pcDNA3 vector and comprises the human RelA coding region fused to a N-terminal Myc
tag sequence. The RelA/RHD expression vector has been described elsewhere (34). The RelA DNA binding mutant (RelADNAmut)
that harbors an RF
KA mutation at amino acids 33 and 34, respectively (35), and the RelA mutant harboring a Ser276
Ala substitution were generated by primer overlap extension as
described above. The sequence of all constructs was confirmed by
double-stranded DNA sequencing. The TetO-Luc (pBI5) reporter was a kind
gift of H. Bujard (University of Heidelberg). Other reporter
constructs used in this study were described previously (12,
36).
(R & D,
Systems, Minneapolis, MN; 50 units/ml, 7 h) 40-44 h after
transfection. Cells were washed once in phosphate-buffered saline and
disrupted in lysis buffer (0.1 M
KH2PO4, pH 7.6, 1 mM dithiothreitol
and 0.05% Triton X-100). Luciferase and
-galactosidase activities
were assayed as described previously (12). All experiments were done in
triplicate as indicated, and luciferase activities were normalized to
-galactosidase levels to account for differences in transfection efficiency.
Immunodetection and Activity Assays--
PKC
Western
blot detection was performed on PVDF membranes using a rabbit
polyclonal antibody directed against the C terminus of human PKC
(sc-216; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bands were
visualized using horseradish peroxidase-conjugated donkey anti-rabbit
IgG (Pierce) and the ECL assay (Amersham Pharmacia Biotech) according
to the manufacturer's instructions. Immunoprecipitation of PKC
was
carried out as described previously (37) with the following
modifications. After preclearing with protein G-Sepharose (Amersham
Pharmacia Biotech), extracts were incubated with 3 µg of nonimmune
rabbit IgG or 3 µg of anti-PKC
antibody (sc-216; Santa Cruz
Biotechnology) for 4 h. Antibodies were captured by adding 20 µl
of protein G-Sepharose and washed twice in lysis, twice in Tris/LiCl
and once in 25 mM Tris-HCl buffer. For autophosphorylation experiments, PAEC were serum-starved for 24 h and metabolically labeled with [32P]orthophosphate (200 µCi/ml, 4 h). Immunoprecipitates were obtained as described above, and captured
proteins were eluted by boiling in Laemmli buffer. Proteins were
resolved on 10% polyacrylamide gels under denaturing conditions. The
gels were dried and subjected to autoradiography. For kinase activity
assays, immunoprecipitates obtained from serum-starved PAEC were
incubated in reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM MnCl2,
and 100 µM ATP) supplemented with 3 µg of myelin basic
protein and 3 µCi of [
-32P]ATP. Reactions were
carried out for 20 min at 30 °C and stopped by adding Laemmli
buffer. Proteins were separated on a 12.5% polyacrylamide gel under
denaturing conditions. Gels were dried and quantitated by
Phosphor-Imager analysis (Molecular Dynamics, Inc., Sunnyvale, CA).
(50 units/ml) as indicated.
p21ras immunoprecipitation was carried out as described above.
Bound nucleotides were eluted by incubating immunoprecipitates in
elution buffer (0.2% SDS, 5 mM dithiothreitol, 1 mM GDP, 1 mM GTP 2 mM EDTA) for 15 min at 68 °C. Equal amounts of eluates (500 cpm) were loaded on
polyethylenimine cellulose plates (Merck, Darmstadt, Germany), and
nucleotides were separated by chromatography in 3 M LiCl,
pH 3.4. Plates were dried and quantified by PhosphorImager analysis.
-32P]ATP-radiolabeled NF-
B
oligonucleotide (5'-AGTTGAGGGAATTTCCCAGGC-3'), and the resulting DNA-protein complexes were separated on a 5% polyacrylamide gel in Tris/glycine/EDTA buffer at pH 8.5. The amount of
cell extracts for each binding reaction was adjusted to
-galactosidase activity to compensate for differences in
transfection efficiency.
for 30 min. LPS stimulation was carried out in the presence of
2% dialyzed fetal bovine serum (Sigma) for 60 min. Cells were washed
twice in ice-cold Tris-buffered saline and scraped in 1 ml of lysis
buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 20 mM
-glycerophosphate, 50 mM NaF, 1 mM
orthovanadate, 1 mM EDTA, 1 mM EGTA, 10 µg/ml
aprotinin, 25 µM leupeptin, 1 µM pepstatin,
and 1 mM phenylmethylsulfonyl fluoride). Extracts were homogenized by passing them five times through a 25-gauge needle and
cleared by centrifugation. RelA was immunoprecipitated from precleared
lysates using an agarose-coupled polyclonal antibody directed against
the N terminus of human RelA (sc-109AC; Santa Cruz Biotechnology).
Immunoprecipitates were washed four times in lysis buffer and once in
50 mM Tris-HCl, pH 6.8. Proteins were eluted by boiling in
Laemmli buffer, separated on 10% polyacrylamide gels under denaturing
conditions, and transferred to a PVDF membrane that was subjected to
autoradiography. Sequential immunoprecipitations were carried out using
RelA (sc-109), NF
B1 (sc-114) or I
B
(sc-371; Santa Cruz
Biotechnology) specific antibodies as described elsewhere (39).
-stimulated PAEC and electrophoretically
separated as described above. The band corresponding to RelA was cut
out, and amino acids were prepared by acidic hydrolysis. Phosphoamino
acids were separated by two-dimensional thin layer electrophoresis as
described before (40), and plates were subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and p21ras by TNF-
in
Endothelial Cells--
HUVEC, BAEC, and PAEC expressed similar levels
of PKC
as assayed by Western blotting (Fig.
1A). In the presence of serum, PKC
was constitutively active in these cells (data not shown). However, PKC
activity was significantly reduced when endothelial cells were serum-starved. Under serum starvation, PKC
was activated by both TNF-
and LPS as assayed by PKC
autophosphorylation (Fig. 1B) or kinase activity (Fig. 1C). Maximal PKC
activity was reached 20 min after TNF-
stimulation (Fig.
1C).
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Fig. 1.
Activation of PKC
and p21ras in endothelial cells. A,
expression of PKC
in endothelial cells. Equal amounts of cellular
lysates prepared from HUVEC, PAEC, and BAEC were analyzed by Western
blotting using a PKC
-specific polyclonal antibody. B,
enhanced autophosphorylation of PKC
after TNF-
and LPS
stimulation. Serum-starved PAEC were labeled with
[32P]orthophosphate and stimulated with TNF-
(50 units/ml) or LPS (1 µg/ml) for 15 min. Cell extracts were prepared by
detergent lysis and incubated with nonimmune rabbit IgG or an
anti-PKC
antibody. Immunoprecipitates were separated on 10%
polyacrylamide gels under denaturing conditions. Phosphorylated PKC
was revealed by autoradiography. C, PKC
immune complex kinase assay.
Serum-starved PAEC were stimulated with TNF-
(50 units/ml) as
indicated. PKC
was immunoprecipitated, and kinase reactions were
carried using myelin basic protein as a substrate. D,
p21ras expression in endothelial cells. HUVEC or PAEC were
labeled with [35S]Met/Cys, and Ras was immunoprecipitated
using Y13-259 rat monoclonal antibody in the absence (A) or
presence of a 40-fold molar excess of specific peptide (P).
Proteins were separated on 15% polyacrylamide gels under denaturing
conditions and visualized by autoradiography. E,
serum-starved PAEC were labeled with
[32P]orthophosphate and stimulated with TNF-
(50 units/ml) as indicated. p21ras was immunoprecipitated,
nucleotides were eluted, and equal amounts of radioactive material were
separated by thin layer chromatography. GDP and GTP bands were
quantified by PhosphorImager analysis, and GTP/GDP ratios were
calculated: % GTP = GTP/(GDP × 1.5 + GTP) × 100. Values
present mean ± S.D. (n = 2).
, p21ras activity was constitutively high in
endothelial cells cultured in the presence of serum, which was also
reflected by high MEK1 (mitogen-activated protein and
extracellular signal-regulated kinase kinase 1)
and extracellular signal-regulated kinase/MAPK activity (data not shown
and Ref. 41). Immunoprecipitation of p21ras from PAEC or HUVEC
revealed two closely migrating bands probably corresponding to
processed and nonprocessed form of p21ras (Fig. 1D).
Under serum deprivation, both p21ras and MEK activity were
markedly reduced, and TNF-
induced p21ras activity as
reflected by an increase in Ras GTP loading (Fig. 1E).
and
p21ras--
To test whether PKC
is involved in regulation
of NF-
B activity in endothelial cells, we used a dominant negative
mutant of the rat PKC
in which Lys281 was replaced by
Trp (PKC
mut; Ref. 15). BAEC were transiently
co-transfected with PKC
mut and with a
NF-
B-dependent luciferase reporter (
B-Luc), regulated by three NF-
B consensus sites derived from the porcine E-selectin promoter (12). TNF-
-induced luciferase expression was inhibited in a
dose-dependent manner by increasing amounts of
PKC
mut (Fig.
2A). Furthermore, we
investigated whether PKC
mut would interfere directly
with RelA-mediated transcription. When BAEC were co-transfected with
RelA and increasing amounts PKC
mut together with the
B-Luc reporter, luciferase expression was inhibited in a
dose-dependent manner (Fig. 2B).
PKC
mut was more efficient in repressing RelA activity
than in repressing TNF-
-mediated NF-
B activation, indicating that
TNF-
may generate additional signals that can partially override the
inhibitory effect of PKC
mut.
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Fig. 2.
Inhibition of TNF- -
and RelA-mediated transcription by
PKC
mut and RasN17.
A, BAECs were transfected with the
B-Luc reporter and
increasing amounts of PKC
mut (200, 400, and 600 ng) or
RasN17 (100, 200, 400, and 600 ng). Forty hours after transfection,
cells were stimulated with 50 units/ml TNF-
for 7 h.
B, BAECs were co-transfected with RelA expression plasmid
(30 ng),
B-Luc reporter (700 ng), and 100, 200, 400, and 670 ng of
PKC
mut or RasN17. C, BAECs were
co-transfected with RelA expression plasmid alone (30 ng) or RelA (30 ng) and NF
B1 (20 ng) together with
B-Luc (700 ng) and 500 ng of
PKC
mut or RasN17. D, BAECs were
co-transfected with RelA (30 ng), I
B
-Luc (700 ng), and 500 ng of
PKC
mut or RasN17. The total amount of DNA in all
transfections was kept constant with pcDNA3 plasmid. Luciferase
activities were normalized to
-galactosidase activities to
compensate for differences in transfection efficiency. The
error bars represent mean ± S.D.
(n = 3).
signaling
cascade (24, 42), we tested whether a dominant negative mutant of
p21ras (RasN17) would interfere with NF-
B-mediated
transcription. Overexpression of increasing amounts of RasN17 in BAEC
abolished TNF-
-mediated up-regulation of the
B-Luc reporter in a
dose-dependent manner (Fig. 2A). This inhibitory
effect was more pronounced than the one seen with PKC
mut
(Fig. 2B). We then analyzed whether RasN17 would interfere
directly with RelA activity. Co-transfection of RasN17 with RelA
repressed transcription from the
B-Luc reporter to a similar extent
as PKC
mut (Fig. 2B). Both
PKC
mut and RasN17 also inhibited RelA/NF
B1
transcriptional activity to a similar extent as observed for RelA (Fig.
2C). Comparable results were obtained when RelA was
co-expressed with a reporter construct under the control of the porcine
I
B
promoter (Fig. 2D). The observation that a
constitutive active mutant of p21ras (RasV12) did not
complement the inhibitory effect of PKC
mut suggests that
p21ras does not act downstream of PKC
in controlling RelA
activity. Moreover, we found that a constitutive active form of PKC
,
comprising the catalytic region (amino acids 254-592 of the human
PKC
) did not overcome the inhibitory effect of RasN17 (data not
shown). Taken together, these data suggest that p21ras and
PKC
regulate RelA transcriptional activity by separate pathways.
mut nor RasN17 altered the levels of
overexpressed RelA in BAEC as monitored by Western blotting (data not
shown). Additionally, PKC
mut or RasN17 did not inhibit
DNA binding of RelA as monitored by electrophoretic mobility shift
assay (Fig. 3). We conclude therefore that the inhibitory effect of PKC
mut or RasN17 is not
due to inhibition of RelA DNA binding activity. These data suggest that
regulation of NF-
B activity by PKC
and p21ras acts
downstream of I
B directly on RelA.
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Fig. 3.
PKC mut
and RasN17 do not alter RelA DNA binding activity. Whole cell
extracts were obtained from BAEC transfected with pcDNA3 (control),
RelA (30 ng), or RelA (30 ng) together with wild type PKC
,
PKC
mut, wild type Ras, or RasN17 (all at 500 ng). Cell
extracts were incubated with radiolabeled double-stranded NF-
B
oligonucleotide, and the resulting DNA-protein complexes were separated
on a polyacrylamide gel. Lane I represents the free probe. The
positions of RelA homodimers (
) and RelA/NF
B1 heterodimers (
)
are indicated.
pathway that may account for the regulatory mechanism of p21ras
or PKC
. First, we monitored the effect of p21ras-related
GTPases of the Rho and Rac family, previously shown to regulate NF-
B
activity in fibroblasts (43), on RelA transcriptional activity.
Expression of dominant negative mutants of Cdc42 (Cdc42N17), Rac1
(RacN17), or RhoA (RhoN19) (43) did not inhibit RelA-mediated activation of the
B reporter in BAEC (Fig.
4A). Similar results were
obtained for TNF-
-induced NF-
B activity (data not shown). Raf-1
and PI 3-kinase have been shown to be involved in p21ras (44,
45) and the latter also in PKC
signaling cascades (46) and in
regulating NF-
B activity in fibroblasts (29) and hepatocytes (47),
respectively. To investigate the role of Raf-1 in modulating NF-
B
activity in EC, we used a Raf-1 dominant negative mutant (Raf1-259)
that has been shown to act as a dominant repressor of Ras-Raf-1
signaling (32). This mutant inhibited a RasV12-induced Elk-1- and
c-Jun-dependent reporter system in EC (data not shown). Overexpression of the Raf-1 dominant negative mutant together with RelA
led only to an insignificant reduction of
B-dependent reporter activity (Fig. 4B). Similar results were obtained
expressing a dominant negative mutant of the PI 3-kinase (p85N-SH2;
Ref. 33). Moreover, pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, had no effect on RelA- or TNF-
-induced
B reporter activity. Likewise, wortmannin or LY294002, another inhibitor of PI 3-kinase, did not effect TNF-
-induced I
B
degradation, NF-
B DNA binding, or up-regulation of
NF-
B-dependent endogenous genes, i.e.
I
B
and E-selectin (data not shown).
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Fig. 4.
Role of small GTPases, Raf-1, and PI 3-kinase
on NF- B activation in EC. A,
BAECs were transfected with the
B-Luc reporter (700 ng) and RelA
expression plasmid (30 ng) alone or together with Cdc42N17 (500 ng),
RacN17 (500 ng), or RhoN19 (500 ng). B, BAECs were
transfected with the
B-Luc reporter (700 ng) and RelA expression
plasmid (30 ng) alone or together with increasing amounts of Raf1-259
or p85N-SH2 (100, 250, and 500 ng). C, BAECs were
transfected with the
B-Luc reporter (700 ng) and RelA expression
plasmid (30 ng). Cells were left untreated or were treated with vehicle
(asterisk; Me2SO, 1 µl/ml) or wortmannin
(Wort) at 10, 100, and 1000 nM concentration (16 h, starting 4 h after end of transfection). D, BAECs
were transfected with the
B-Luc reporter (700 ng). Thirty-six hours
after transfection, cells were incubated with vehicle
(asterisk; Me2SO, 1 µl/ml) or wortmannin
(Wort) at 10, 100, and 1000 nM concentration.
TNF-
(50 units/ml) was added 1 h later, and incubation was
continued for 7 h, after which cell extracts were prepared.
Luciferase activities were assayed as described under "Materials and
Methods." The total amount of DNA in all transfections was kept
constant with pcDNA3 plasmid. Luciferase activities were normalized
to
-galactosidase activities to compensate for differences in
transfection efficiency. The error bars represent
mean ± S.D. (n = 3).
and p21ras-mediated
Regulation of Transcriptional Activity--
To monitor which domain of
RelA is targeted by PKC
and p21ras, we constructed different
fusion proteins outlined in Fig.
5A. The first construct was
composed of the DNA binding domain derived from the bacterial
tetracycline repressor (TET) fused to a transactivation domain derived
from the Herpes simplex virus VP16 protein (TET/VP16). The second
construct was composed of the TET DNA binding domain fused to the
C-terminal region of RelA (amino acids 286-551) that includes the
transactivation domain (TET/RelA286-551). In addition, we generated a
construct composed of the RelA RHD fused to the VP16 transactivation
domain (RelA2-320/VP16). Transcriptional activity of constructs
harboring the TET DNA binding domain was analyzed by co-transfection
with a reporter containing seven tetracycline operons (TetO) fused to a
luciferase gene (TetO-Luc). Transcriptional activity of constructs
harboring the RelA DNA binding domain was analyzed by co-transfection
with the
B-Luc reporter. Whereas RelA-mediated transcription was
repressed by both PKC
mut and RasN17 (Fig.
2B), TET/VP16-mediated transcription was not inhibited by
these mutants. TET/RelA286-552-mediated transcription was not
inhibited by PKC
mut or RasN17, while RelA2-320/VP16
transcriptional activity was inhibited by both mutants in a similar
manner to wild type RelA (Fig. 5B). These data suggest that
PKC
and p21ras regulate RelA transcriptional activity by
targeting the RelA RHD.
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Fig. 5.
RelA RHD is the target for
PKC mut- and RasN17-mediated
transcriptional repression. A, schematic representation
of plasmid constructs used for transfections. All constructs were
cloned into the mammalian expression vector pcDNA3. B,
BAEC were transfected with different fusion proteins as outlined in A
(all at 30 ng) and with PKC
mut (500 ng), RasN17 (500 ng),
B-Luc (700 ng), or TETO-Luc (700 ng) reporter as indicated.
C, BAEC were transfected with TET/RelA2-551 (30 ng) along
with 700 ng of
B-Luc or TETO-Luc and protein kinase expression
vectors as indicated (all at 500 ng). 24 h after transfection,
cells were lysed, and luciferase activities were assayed as described
under "Materials and Methods." The total amount of DNA in all
transfections was kept constant with pcDNA3 plasmid. Luciferase
activities were normalized to
-galactosidase activities to
compensate for differences in transfection efficiency. The
error bars represent mean ± S.D.
(n = 3).
and
p21ras Is Dependent on Functional
B Consensus
Sites--
Having established that the regulatory effect of PKC
and
p21ras is dependent on RelA RHD, we analyzed whether DNA
binding through RHD was necessary for inhibition of RelA
transcriptional activity. To test this possibility, we constructed a
fusion protein that contains the TET DNA binding domain and the
full-length RelA (TET/RelA2-551; Fig. 5A) and carries
therefore two DNA binding domains (for TetO and
B consensus binding
sites). This construct allows one to analyze the effect of
PKC
mut and RasN17 on its transcriptional activity
depending on the binding to two different DNA consensus sites. As shown
in Fig. 5C, transcriptional activity of this fusion protein
was repressed by PKC
mut and RasN17 when co-transfected
with the
B-Luc reporter, while it was not affected when the TetO-Luc
reporter was used. Since the
B-Luc reporter harbors the thymidine
kinase minimal promoter, while the TetO-Luc harbors the cytomegalovirus
minimal promoter, we tested whether the use of these two minimal
promoters would account for the differential regulation. To do so, we
constructed a
B reporter containing the same cytomegalovirus minimal
promoter fragment that drives the TetO-Luc construct. When transfected with RelA and PKC
mut or RasN17, this
B reporter
behaved the same way as the reporter construct based on the thymidine
kinase minimal promoter (data not shown). We conclude therefore that
regulation of RelA transcriptional activity by PKC
and
p21ras involves the RelA RHD and is only relevant if RelA binds
DNA through a
B consensus site.
(39, 48, 49). In
endothelial cells, TNF-
induces RelA phosphorylation as analyzed by
immunoprecipitation of RelA from
[32P]orthophosphate-labeled cells (Fig.
6A). Several other
phosphopeptides were co-immunoprecipitated along with RelA. The
identity of the precipitated phosphopeptides was confirmed by Western
blot analysis and by sequential immunoprecipitations and identified as
NF
B1, p105, and I
B
(data not shown). While RelA
phosphorylation was increased upon TNF-
stimulation, NF
B1 was
dephosphorylated (seven independent experiments). The decrease in
intensity of the I
B
corresponding band upon TNF-
stimulation
was due to I
B
degradation as assayed by Western blotting.
Furthermore, we analyzed the identity of phosphoamino acids derived
from RelA isolated from TNF-
-stimulated PAECs by two-dimensional
electrophoresis. As shown in Fig. 6B, these phosphoamino
acids were primarily composed of serine and only to a minor extent
threonine residues, while no tyrosine phosphorylation was observed.
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Fig. 6.
Phosphorylation of RelA. A,
PAEC were metabolically labeled with [32P]orthophosphate
and stimulated with TNF- for 30 min. RelA was immunoprecipitated and
separated on a 10% polyacrylamide gel under denaturing conditions.
B, RelA was immunoprecipitated from TNF-
-stimulated PAEC,
and amino acids were prepared by acidic hydrolysis. Phosphoamino acids
were separated by two-dimensional thin layer electrophoresis and
revealed by autoradiography. The position of the phosphoamino acid
standards is marked by circles.
- (30 min), or LPS-
(60 min) treated BAEC. In quiescent EC, RelA is phosphorylated on
multiple sites, resulting in at least nine distinct phosphopeptides.
Upon TNF-
or LPS stimulation, the pattern of RelA phosphorylation
changes significantly as reflected by an increase in signal intensity
of several phosphopeptides (Fig. 7,
spots b, e, f,
g, h, and i). The increase of
phosphorylation seems to be strongest on peptide b. TNF-
and LPS lead to similar changes in RelA phosphorylation. While peptides
b, e, f, g, and h are phosphorylated to a similar extent in TNF-
- or
LPS-stimulated cells, peptide i seems to be more
phosphorylated in TNF-
-treated cells. These data suggest the
existence of several constitutive and inducible phosphorylation sites
in RelA.
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Fig. 7.
Phosphopeptide map of endogenous RelA.
BAEC were labeled with [32P]orthophosphate and stimulated
with TNF- (100 units/ml; 30 min) or LPS (1 µg/ml; 60 min). Cell
extracts were immunoprecipitated with anti RelA antibody. Equal amounts
(800 cpm) of RelA tryptic digests were analyzed by two-dimensional
separation on thin layer cellulose plates as described under
"Materials and Methods." The sample application point is marked
(+).
B-bound, RelA is
sufficient to trigger phosphorylation, we overexpressed RelA in BAEC
and analyzed the level of phosphorylation. As shown in Fig.
8A (lane
1), overexpressed RelA is readily phosphorylated in
unstimulated cells. TNF-
stimulation did not affect the
phosphorylation status of overexpressed RelA (data not shown),
suggesting that signaling by TNF-
might not be essential for RelA
phosphorylation. We next examined whether phosphorylation of
overexpressed RelA was inhibited by I
B
co-expression under
conditions where all RelA would be complexed to I
B
. I
B
expression resulted in substantial reduction in RelA phosphorylation
(Fig. 8A, lane 5). The decrease in
RelA phosphorylation was not due to a decrease in protein levels as
monitored by immunodetection of RelA on the same membrane used for
phosphorylation analysis (Fig. 8B, compare lane
1 with lane 5). These data indicate
that RelA is not fully phosphorylated when retained by I
B
. This
would suggest three possible scenarios. RelA is phosphorylated (i) in
the cytoplasm upon liberation from I
B, (ii) in the nucleus before
binding to DNA, or (iii) upon binding to DNA. In order to investigate
if DNA binding is necessary for RelA phosphorylation, we expressed a
previously described RelA DNA binding-deficient mutant (35). As shown
in Fig. 8A (lane 3), the
phosphorylation of this DNA binding mutant was strongly reduced as
compared with wild type RelA. The most likely explanation for this
finding is that phosphorylation of RelA occurs after nuclear
translocation and DNA binding.
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Fig. 8.
Regulation of RelA phosphorylation.
A, BAEC were transfected with RelA (100 ng; lane
1), RelA/RHD (100 ng; lane 2),
RelADNAmut (100 ng; lane 3), RelA/RHD
(100 ng) together with I B
(700 ng; lane 4),
or RelA (100 ng) together with I
B
(700 ng; lane
5). Cells were labeled with
[32P]orthophosphate, and cell extracts were
immunoprecipitated with anti-RelA antibody. Immunoprecipitates were
separated on 10% polyacrylamide gels and transferred to a PVDF
membrane. B, to monitor equal protein expression, the
membrane was probed with RelA-specific antibody. The positions of the
endogenous RelA (
) and the immunoglobulin heavy chain (*) are
indicated.
B
(Fig. 8A, lane
4). As for RelA, I
B
co-expression did not change RelA/RHD protein levels as assayed by Western blot analysis (Fig. 8B, lanes 2 and 4).
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Fig. 9.
Phosphorylation of the RelA S276A
mutant. A, BAEC seeded in 10-cm dishes were transfected
with RelA (1800 ng; RelA wt) or with a mutant RelA carrying a
Ser276 Ala substitution (5400 ng; RelA S276A). The
total amount of DNA was kept constant with pcDNA3 plasmid. Cells
were labeled with [32P]orthophosphate, and cell extracts
were immunoprecipitated with anti-RelA antibody. Immunoprecipitates
were separated on 10% polyacrylamide gels. B, tryptic
digests were analyzed by two-dimensional separation on thin layer
cellulose plates as described under "Materials and Methods". The
sample application point is marked (+). The nomenclature of radioactive
spots follows that of Fig. 7 to indicate corresponding spots.
or LPS
stimulation. These data indicate that Ser276 is
constitutively phosphorylated in EC.
and
p21ras Signaling Pathways--
We next investigated
whether inhibition of PKC
or p21ras signaling pathways would
interfere with RelA phosphorylation. Overexpression of RelA together
with PKC
mut or RasN17 significantly decreased RelA
phosphorylation as compared with overexpression of RelA alone (Fig.
10A, top).
PKC
mut or RasN17 did not decrease RelA protein levels as
monitored by immunodetection of RelA (Fig. 10A,
bottom). To monitor which domain of RelA was targeted by
PKC
mut or RasN17, we used different fusion proteins
described above and outlined in Fig. 5A. The phosphorylation
of RelA2-320/VP16 (containing the RelA RHD) was significantly
inhibited by co-expressed PKC
mut or RasN17 (Fig.
10B, top). PKC
mut or RasN17 did
not decrease RelA2-320/VP16 protein Fig. 10B,
bottom). Phosphorylation of the TET/RelA286-551 construct,
which contains the C-terminal RelA transactivation domain, was not
inhibited by PKC
mut or RasN17 (Fig. 10C).
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Fig. 10.
RHD phosphorylation is controlled by
p21ras and PKC . A,
BAEC were transfected with RelA (150 ng) together with pcDNA3
plasmid (control), PKC
mut, or RasN17 (all at 1350 ng).
B, RelA was replaced with 150 ng RelA2-320/VP16.
C, RelA was replaced with 150 ng of TET/RelA286-551. Twenty
hours after transfection, cells were labeled with
[32P]orthophosphate, and proteins were immunoprecipitated
with antibodies directed against the N terminus (A and
B) or the C terminus (C) of RelA. Precipitated
proteins were resolved by SDS-polyacrylamide gel electrophoresis and
transferred to PVDF membranes. Bands were revealed by autoradiography.
Protein bands corresponding to the indicated constructs are marked
(
). The same membrane was subjected to Western blot analysis to
assure equal protein loading and specificity of bands. The bands
corresponding to endogenous RelA (
), Myc-RelA (
), and
RelA2-320/VP16 (
) are indicated.
and p21ras are involved in the
phosphorylation of RelA RHD, we analyzed the phosphorylation pattern of
the RHD by tryptic peptide mapping. Phosphopeptides derived from
RelA/RHD expressed alone or together with PKC
mut or
RasN17 were analyzed by two-dimensional separation on thin layer
cellulose plates. Compared with the phosphopeptide map derived from
endogenous or overexpressed full-length RelA (Fig. 7), the most
striking difference is the disappearance of the most basic peptide
(Figs. 7 and 9, spot a) and the appearance of a
very acidic peptide (Fig. 11,
spot x). The pattern of RHD phosphorylation was substantially modified when RelA/RHD was co-expressed with
PKC
mut or RasN17 as compared with overexpression of
RelA/RHD alone (Fig. 11). These changes were restricted to three
separate peptides and were not equivalent in PKC
mut- and
RasN17-transfected cells. While phosphorylation of peptide b
disappeared in cells transfected with PKC
mut and RasN17
(Fig. 11), phosphorylation of peptides d and g
was only inhibited by PKC
mut and not by RasN17 (Fig.
11). These differences in the phosphorylation pattern again suggest
that PKC
and p21ras feed into at least partially separated
pathways controlling RelA phosphorylation.
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Fig. 11.
Phosphopeptide map of RelA/RHD.
RelA/RHD (150 ng) was expressed alone or together with 1350 ng of
PKC mut or RasN17. Cells were labeled with
[32P]orthophosphate, and cell extracts were
immunoprecipitated with anti-RelA antibody. Equal amounts (2500 cpm) of
RelA/RHD tryptic digests were analyzed by two-dimensional separation on
thin layer cellulose plates. The sample application point is marked
(+).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B transcriptional
activity is controlled mainly by retention of NF-
B in the cytoplasm
by members of the I
B family. In this study, we demonstrate that at
least in endothelial cells there is an additional regulatory system
that controls the transcriptional activity of nuclear NF-
B by
targeting the RelA subunit. This regulatory system involves signaling
through PKC
and p21ras.
B in vivo and to phosphorylate the
C-terminal transcriptional activation domain of RelA in
vitro (52). We now demonstrate that PKC
and p21ras are
two additional components in the regulation of RelA transcriptional activity. We show that inhibition of these signaling cascades results
in decrease of RelA transcriptional activity that correlates with
inhibition of RelA phosphorylation.
or
p21ras over NF-
B. One common feature shared by both PKC
and p21ras is the ability to activate the MEK/extracellular
signal-regulated kinase pathway, which has been suggested to control
NF-
B activity (15, 53). However, we found that at least in
endothelial cells a dominant negative mutant of Raf1 does not interfere
with NF-
B-mediated transcription. Another downstream effector of
p21ras and PKC
is the c-Jun N-terminal kinase signaling
cascade. It is unlikely that this pathway is involved in NF-
B
regulation in endothelial cells, since a dominant negative c-Jun
N-terminal kinase 1 failed to inhibit RelA transcriptional activity
(data not shown). Moreover, a dominant negative mutant of Rac1 (RacN17) efficiently blocked p21ras induced c-Jun N-terminal kinase
activation while it failed to inhibit NF-
B activity (data not shown
and Ref. 54). Inhibition of PI 3-kinase by a dominant negative mutant
or by wortmannin failed to have an effect on RelA-mediated
transcription. Furthermore, inhibition of PI 3-kinase did not impair
TNF-
-induced
B-dependent reporter activity.
B
, which suggests that RelA is phosphorylated upon
liberation from associated I
B molecules. The observation that
phosphorylation of full-length RelA is only partially inhibited by
I
B
overexpression, whereas phosphorylation of RelA RHD is completely inhibited, suggests that the C terminus of RelA is constitutively phosphorylated, while inducible phosphorylation occurs
mainly on the RHD.
or LPS treatment. While the phosphorylation of
some of these sites is regulated by both PKC
and p21ras
signaling cascades, phosphorylation of other sites is not altered by
these pathways. Furthermore, our data suggest that RelA is constitutively phosphorylated at Ser276, and this
phosphorylation is not altered by TNF-
or LPS treatment. In
addition, a RelA S276A mutant retained several phosphorylated sites,
which is different from T cells, where the same mutation completely
abolished RelA phosphorylation (48). Thus, EC show a similar behavior
as fibroblasts, where the RelA S276A mutant can still be phosphorylated
(50).
or p21ras. This hypothesis is supported by the finding
that the VP16 transactivation domain, which is thought not to interact
with CBP/p300, is repressed by PKC
mut or RasN17 when
fused to RelA RHD. Furthermore, the TET/RelA2-551 construct that
harbors the full-length RelA and should be phosphorylated by protein
kinase A and therefore interact with CBP/p300 was only repressed when
bound to a
B-dependent reporter and not when bound to
the TetO reporter. This result favors a model where phosphorylation of
RelA RHD by PKC
and p21ras signaling pathways would modulate
RelA transcriptional activity through conformational changes of
DNA-bound RelA.
and p21ras control
RelA transcriptional activity is through changes in the DNA binding activity of differently phosphorylated RHDs. It has been shown that DNA
binding of RelA can be enhanced by in vitro phosphorylation through protein kinase A and protein kinase C (39). Although our
studies do not show changes of in vitro DNA binding as
monitored by an electrophoretic mobility shift assay (Fig. 3), there
could still be such changes in vivo, since the nuclear
environment is poorly reflected by an in vitro binding assay.
B regulatory
system that controls transcriptional activity after liberation of
NF-
B complexes from their cytoplasmic inhibitors. Although we are
only at the beginning of understanding this regulatory mechanism, we
show evidence that it might include phosphorylation of NF-
B
complexes, and we propose p21ras and PKC
signaling molecules
as being involved in such a control system.
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ACKNOWLEDGEMENTS |
---|
We thank H. Bujard, G. M. Cooper, R. De Martin, and J. Moscat for plasmid constructs; E. Csizmadia for culturing endothelial cells; and F. H. Bach for helpful discussion.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant 1R01HL59476 (to J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Immunobiology Research
Center, Beth Israel Deaconess Medical Center, Harvard Medical School,
99 Brookline Ave., Boston, MA. E-mail:
janrathe{at}caregroup.harvard.edu
§ Present address: Leukosite, Cambridge, MA 02142.
¶ Present address: Zeneca Pharmaceuticals, Macclesfield, Cheshire SK10 4TG3, United Kingdom.
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ABBREVIATIONS |
---|
The abbreviations used are:
RHD, Rel homology
domain;
PKC, protein kinase C
;
MAPK, mitogen-activated protein
kinase;
MEK, mitogen-activated protein and extracellular
signal-regulated kinase kinase;
LPS, bacterial lipopolysaccharide;
TNF-
, tumor necrosis factor-
;
BAEC, bovine aortic endothelial cell(s);
PAEC, porcine aortic endothelial cell(s);
HUVEC, human
umbilical vein endothelial cell(s);
PVDF, polyvinylidene difluoride;
EC, endothelial cell(s);
TET, bacterial tetracycline repressor;
PI, phosphatidylinositol.
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REFERENCES |
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