Regulation of NF-κB RelA Phosphorylation and Transcriptional Activity by p21 and Protein Kinase Cζ in Primary Endothelial Cells

Abstract

The activity of the transcription factor NF-kappaB is thought to be regulated mainly through cytoplasmic retention by IkappaB molecules. Here we present evidence of a second mechanism of regulation acting on NF-kappaB after release from IkappaB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-kappaB through a pathway involving activation of protein kinase Czeta (PKCzeta) and p21(ras). 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 PKCzeta or p21(ras) 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.

Full text

Regulation of NF-
k
B RelA Phosphorylation and
Transcriptional Activity by p21
ras
and Protein Kinase C
z
in
Primary Endothelial Cells*
(Received for publication, August 7, 1998, and in revised form, January 18, 1999)
Josef Anrather‡, Vilmos Csizmadia§, Miguel P. Soares, and Hans Winkler¶
From the Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts 02115
The activity of the transcription factor NF-
k
Bis
thought to be regulated mainly through cytoplasmic re-
tention by I
k
B molecules. Here we present evidence of a
second mechanism of regulation acting on NF-
k
B after
release from I
k
B. In endothelial cells this mechanism
involves phosphorylation of the RelA subunit of NF-
k
B
through a pathway involving activation of protein ki-
nase C
z
(PKC
z
) and p21
ras
. 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
z
or p21
ras
results
in loss of RelA transcriptional activity without interfer-
ing with DNA binding. Raf/MEK, small GTPases, phos-
phatidylinositol 3-kinase, and stress-activated protein
kinase pathways are not involved in this mechanism of
regulation.
The NF-
k
B/Rel family of dimeric transcription factors is in-
volved in the immediate early transcription, i.e. independent of
protein synthesis, of a large array of genes induced by mito-
genic or pathogen-associated stimuli. In its active form, NF-
k
B
is a nuclear homo- or heterodimeric complex of a number of
different Rel family members. The canonical and most abun-
dant form of NF-
k
B is composed of a 50-kDa (p50, or NF
k
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
k
B2), and RelB (1). All
members of the Rel family exhibit extensive sequence similar-
ity 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-
k
B-dependent
genes efficiently. With respect to transcription activation, the
RelA subunit appears to have the highest activity.
In most unstimulated cells, NF-
k
B is constitutively retained
in the cytoplasm by inhibitory proteins of the I
k
B family,
namely I
k
B
a
,I
k
B
b
,I
k
B
g
, p100, p105, and I
k
B
e
(2). Formation
of NF-
k
BzI
k
B complexes masks the nuclear localization signal
sequence present in NF-
k
B molecules and thus prevents their
nuclear translocation. One of the key events in the activation of
NF-
k
B is the liberation of functional NF-
k
B dimers from I
k
B,
which results in the translocation of NF-
k
B to the nucleus.
Cytoplasmic release of NF-
k
B dimers involves site-specific
phosphorylation of I
k
B by kinases of the I
k
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 repre-
sents A or G and Y represents C or T), NF-
k
B dimers function
as transcriptional activators. I
k
B
a
(9), I
k
B
b
, and p105 (10)
have been implicated in the inhibition of DNA binding of
NF-
k
B complexes. However, there have been several reports
showing that NF-
k
B transcriptional activity can be blocked
without affecting DNA binding. These include the interactions
of NF-
k
B with the glucocorticoid receptor (11, 12), the mam-
malian repressor REP (13), and the interferon-inducible factor
p202 (14).
Emerging evidence also suggests a second level of controlling
NF-
k
B transcriptional activity that acts directly on NF-
k
B
dimers without influencing the degradation of I
k
B molecules.
For example, ectopic expression of a dominant negative mutant
of the atypical protein kinase C
z
(PKC
z
) or the extracellular
signal-regulated kinase 1 inhibit TNF-
a
-induced NF-
k
B activ-
ity (15). Similarly, inhibition of p38 mitogen-activated protein
kinase (p38 MAPK) has been shown to decrease TNF-
a
-induced
NF-
k
B activity and interleukin-6 expression (16). More re-
cently, tyrosine phosphorylation has been shown to be essential
for NF-
k
B activity in bacterial lipopolysaccharide (LPS)-in-
duced monocytic THP1 cells (17). Regulation of NF-
k
B activ-
ity by PKC
z
, extracellular signal-regulated kinase 1, p38
MAPK, or tyrosine phosphorylation acts downstream of I
k
B
without interfering with NF-
k
B nuclear translocation and DNA
binding.
As for other members of the atypical protein kinase C family,
PKC
z
is not activated by Ca
21
or diacylglycerol and is insensi-
tive to phorbol esters (18). Unresponsiveness of PKC
z
to Ca
21
and diacylglycerol is consistent with the absence of the Ca
21
binding C2 domain and the presence of only one cysteine-rich
zinc finger-like motif in the diacylglycerol binding C1 domain of
PKC
z
. PKC
z
is activated by several lipid mediators including
phosphatidic acid (19) and phosphatidylinositol 3,4,5-trisphos-
phate (20). PKC
z
has also been shown to be activated by TNF-
a
* 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. This 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 Re-
search Center, Beth Israel Deaconess Medical Center, Harvard Medical
School, 99 Brookline Ave., Boston, MA. E-mail: janrathe@caregroup.
harvard.edu
§ Present address: Leukosite, Cambridge, MA 02142.
¶Present address: Zeneca Pharmaceuticals, Macclesfield, Cheshire
SK10 4TG3, United Kingdom.
1
The abbreviations used are: RHD, Rel homology domain; PKC
z
,
protein kinase C
z
; MAPK, mitogen-activated protein kinase; MEK,
mitogen-activated protein and extracellular signal-regulated kinase ki-
nase; LPS, bacterial lipopolysaccharide; TNF-
a
, tumor necrosis fac-
tor-
a
; 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.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 19, Issue of May 7, pp. 13594–13603, 1999
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org13594
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and interleukin-1 through sphingomyelin hydrolysis and sub-
sequent generation of ceramide (21–23). Other pathways lead-
ing to PKC
z
activation include the 21-kDa guanine nucleotide-
binding p21
ras
(24), which in addition to several growth factors
is also activated by TNF-
a
as well as LPS (25, 26). Ras GTPases
have been implicated in the signaling of a variety of extracel-
lular stimuli that control cell proliferation and differentiation.
Ras GTPases are activated by members of the guanine nucle-
otide exchange factor family, which increase Ras GTP loading
and are negatively regulated by the GTPase-activating pro-
teins, 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. p21
ras
has also been implicated in controlling
NF-
k
B activity in fibroblasts (29, 30).
In this study, we analyzed the role of PKC
z
and p21
ras
in
regulating NF-
k
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-
k
B-mediated transcription without interfering with the ability
of NF-
k
B to bind to DNA.
MATERIALS AND METHODS
Cell Culture—Bovine aortic endothelial cells (BAEC) and porcine
aortic endothelial cells (PAEC) were grown in Dulbecco’s modified Ea-
gle’s medium supplemented with 10% fetal bovine serum, L-glutamine
(2 mM), penicillin G (100 units/ml), and streptomycin (100
m
g/ml).
Human umbilical vein endothelial cells (HUVEC) were grown in M199
medium supplemented with 15% fetal bovine serum, NaH
2
CO
3
(20 mM),
HEPES (25 mM), glutamine (5 mM), heparin (100
m
g/ml), gentamycin
(50
m
g/ml), and endothelial growth factor (50
m
g/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 CO
2
atmosphere.
All media and supplements were from Life Technologies, Inc.
Plasmid Constructs—The pcDNA3 vector expressing tagged wild-
type Xenopus laevis PKC
z
and rat PKC
z
dominant negative mutant
were a kind gift of J. Moscat (Universidad Auto´noma, Madrid) and were
described elsewhere (15). Expression vectors encoding wild-type p21
ras
,
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 gener-
ated by replacing Thr
17
with Asn employing the overlapping primers
59-GTAAAAACTGTCTCCTGATATCCTAC and 59-GATATCAGGAGA-
CAGTTTTTACCAACAGCACC, Rac1N17 was generated by replacing
Thr
17
with Asn using the primers 59-CTGTAGGTAAAAACTGCCTACT-
GATC and 59-TGATCAGTAGGCAGTTTTTACCTACAGCTCCG, and
RhoAN17 was generated by replacing Thr
19
with Asn using the primers
59-GTGGAAAGAACTGCTTGCTCATAGTCTTC and 59-ATGAGCAAG-
CAGTTCTTTCCACAGGCTCCATC (the underlined base triplet indi-
cates 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 hu-
man 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. 5Awere 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 (RelA
DNAmut
) that harbors an RF 3KA mutation at
amino acids 33 and 34, respectively (35), and the RelA mutant harbor-
ing a Ser
276
3Ala substitution were generated by primer overlap
extension as described above. The sequence of all constructs was con-
firmed by double-stranded DNA sequencing. The TetO-Luc (pBI5) re-
porter was a kind gift of H. Bujard (University of Heidelberg). Other
reporter constructs used in this study were described previously
(12, 36).
Transient Transfection and Reporter Assays—Primary BAEC were
transfected as described (34). Experiments involving RelA co-transfec-
tion were analyzed 20–24 h after transfection. Where indicated, cells
were incubated with human recombinant TNF-
a
(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 MKH
2
PO
4
,pH7.6,1mMdithiothreitol and 0.05% Triton
X-100). Luciferase and
b
-galactosidase activities were assayed as de-
scribed previously (12). All experiments were done in triplicate as
indicated, and luciferase activities were normalized to
b
-galactosidase
levels to account for differences in transfection efficiency.
PKC
z
Immunodetection and Activity Assays—PKC
z
Western blot
detection was performed on PVDF membranes using a rabbit polyclonal
antibody directed against the C terminus of human PKC
z
(sc-216;
Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Bands were visual-
ized 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
z
was
carried out as described previously (37) with the following modifica-
tions. After preclearing with protein G-Sepharose (Amersham Pharma-
cia Biotech), extracts were incubated with 3
m
g of nonimmune rabbit
IgG or 3
m
g of anti-PKC
z
antibody (sc-216; Santa Cruz Biotechnology)
for 4 h. Antibodies were captured by adding 20
m
l of protein G-Sepha-
rose and washed twice in lysis, twice in Tris/LiCl and once in 25 mM
Tris-HCl buffer. For autophosphorylation experiments, PAEC were se-
rum-starved for 24 h and metabolically labeled with [
32
P]orthophos-
phate (200
m
Ci/ml, 4 h). Immunoprecipitates were obtained as de-
scribed above, and captured proteins were eluted by boiling in Laemmli
buffer. Proteins were resolved on 10% polyacrylamide gels under dena-
turing conditions. The gels were dried and subjected to autoradiogra-
phy. For kinase activity assays, immunoprecipitates obtained from
serum-starved PAEC were incubated in reaction buffer (50 mMTris-
HCl, pH 7.5, 5 mMMgCl
2
,1mMMnCl
2
, and 100
m
MATP) supplemented
with 3
m
g of myelin basic protein and 3
m
Ci of [
g
-
32
P]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).
p21
ras
Immunodetection and Activity Assay—PAEC and HUVEC
were labeled with [
35
S]Met/Cys for 6 h. Cells (2–3 310
6
) were disrupted
(20 min on ice) in lysis buffer (10 mMTriszHCl, pH 7.5, 150 mMNaCl, 1
mMEDTA, 0.2% Triton X-100) supplemented with phosphatase and
protease inhibitors. Lysates were cleared by centrifugation, and p21
ras
was immunoprecipitated by incubating lysates overnight with a rat
monoclonal anti-p21
ras
antibody coupled to agarose beads (sc-35AC;
Santa Cruz Biotechnology). Beads were washed eight times in wash
buffer (50 mMHEPES, pH 7.4, 500 mMNaCl, 5 mMMgCl
2
, 0.1% Triton
X-100, 0.005% SDS). Proteins were eluted by boiling in Laemmli buffer
and separated on 15% polyacrylamide gels under denaturing condi-
tions. Gels were dried and subjected to autoradiography. To analyze
p21
ras
GTP loading, serum-starved PAEC were labeled with [
32
P]ortho-
phosphate for 4 h. Cells were left untreated or stimulated with TNF-
a
(50 units/ml) as indicated. p21
ras
immunoprecipitation was carried out
as described above. Bound nucleotides were eluted by incubating im-
munoprecipitates in elution buffer (0.2% SDS, 5 mMdithiothreitol, 1 mM
GDP, 1 mMGTP2mMEDTA) 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 chro-
matography in 3 MLiCl, pH 3.4. Plates were dried and quantified by
PhosphorImager analysis.
Electrophoretic Mobility Shift Assay—Whole cell extracts were pre-
pared from transfected BAEC as described before (38). All buffers were
supplemented with 10
m
g/ml aprotinin, 25
m
Mleupeptin, 1
m
Mpepsta-
tin, and 1 mMphenylmethylsulfonyl fluoride. Cell extracts were incu-
bated (30 min at room temperature) with 100,000 cpm of double-
stranded [
g
-
32
P]ATP-radiolabeled NF-
k
B oligonucleotide (59-AGTTGA-
GGGAATTTCCCAGGC-39), 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
b
-galactosidase activity to compensate for differences in
transfection efficiency.
RelA Metabolic Labeling and Immunoprecipitation—PAEC or BAEC
cultured in 10-cm dishes were labeled with [
32
P]orthophosphate (500
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m
Ci/ml) in phosphate-free Dulbecco’s modified Eagle’s medium for 4 h
and stimulated with TNF-
a
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 mMTris, pH 7.5, 150 mMNaCl, 1% Nonidet P-40,
0.5% deoxycholate, 0.1% SDS, 20 mM
b
-glycerophosphate, 50 mMNaF,
1mMorthovanadate, 1 mMEDTA, 1 mMEGTA, 10
m
g/ml aprotinin, 25
m
Mleupeptin, 1
m
Mpepstatin, and 1 mMphenylmethylsulfonyl fluoride).
Extracts were homogenized by passing them five times through a 25-
gauge needle and cleared by centrifugation. RelA was immunoprecipi-
tated from precleared lysates using an agarose-coupled polyclonal an-
tibody 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 mMTris-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 mem-
brane that was subjected to autoradiography. Sequential immunopre-
cipitations were carried out using RelA (sc-109), NF
k
B1 (sc-114) or
I
k
B
a
(sc-371; Santa Cruz Biotechnology) specific antibodies as de-
scribed elsewhere (39).
Phosphoamino Acid Analysis—RelA was immunoprecipitated from
metabolically labeled, TNF-
a
-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.
RelA Phosphopeptide Mapping—BAEC or BAEC transfected with
various RelA constructs as described above were metabolically labeled
with 500
m
Ci/ml [
32
P]orthophosphate for 4 h. Cell extracts were pre-
pared by detergent lysis as described above and immunoprecipitated
with anti-RelA-agarose (Santa Cruz Biotechnology). Immunoprecipi-
tates 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/H
2
O (37.5:25:7.5:30) buffer. Plates were exposed to x-ray film or
analyzed using a PhosphorImager scanning device (Molecular Dynamics).
RESULTS
Activation of PKC
z
and p21
ras
by TNF-
a
in Endothelial
Cells—HUVEC, BAEC, and PAEC expressed similar levels of
PKC
z
as assayed by Western blotting (Fig. 1A). In the presence
of serum, PKC
z
was constitutively active in these cells (data
not shown). However, PKC
z
activity was significantly reduced
when endothelial cells were serum-starved. Under serum star-
vation, PKC
z
was activated by both TNF-
a
and LPS as assayed
by PKC
z
autophosphorylation (Fig. 1B) or kinase activity (Fig.
1C). Maximal PKC
z
activity was reached 20 min after TNF-
a
stimulation (Fig. 1C).
As for PKC
z
, p21
ras
activity was constitutively high in endo-
thelial cells cultured in the presence of serum, which was also
reflected by high MEK1 (mitogen-activated protein and extra-
cellular signal-regulated kinase kinase 1) and extracellular
signal-regulated kinase/MAPK activity (data not shown and
Ref. 41). Immunoprecipitation of p21
ras
from PAEC or HUVEC
revealed two closely migrating bands probably corresponding
to processed and nonprocessed form of p21
ras
(Fig. 1D). Under
serum deprivation, both p21
ras
and MEK activity were mark-
edly reduced, and TNF-
a
induced p21
ras
activity as reflected by
an increase in Ras GTP loading (Fig. 1E).
Regulation of RelA Transcriptional Activity by PKC
z
and
p21
ras
—To test whether PKC
z
is involved in regulation of
NF-
k
B activity in endothelial cells, we used a dominant nega-
tive mutant of the rat PKC
z
in which Lys
281
was replaced by
Trp (PKC
z
mut
; Ref. 15). BAEC were transiently co-transfected
with PKC
z
mut
and with a NF-
k
B-dependent luciferase reporter
(
k
B-Luc), regulated by three NF-
k
B consensus sites derived
from the porcine E-selectin promoter (12). TNF-
a
-induced lu-
ciferase expression was inhibited in a dose-dependent manner
by increasing amounts of PKC
z
mut
(Fig. 2A). Furthermore, we
investigated whether PKC
z
mut
would interfere directly with
RelA-mediated transcription. When BAEC were co-transfected
with RelA and increasing amounts PKC
z
mut
together with the
k
B-Luc reporter, luciferase expression was inhibited in a dose-
dependent manner (Fig. 2B). PKC
z
mut
was more efficient in
repressing RelA activity than in repressing TNF-
a
-mediated
NF-
k
B activation, indicating that TNF-
a
may generate addi-
tional signals that can partially override the inhibitory effect of
PKC
z
mut
.
Given that p21
ras
has been implicated in the PKC
z
signaling
cascade (24, 42), we tested whether a dominant negative mu-
tant of p21
ras
(RasN17) would interfere with NF-
k
B-mediated
transcription. Overexpression of increasing amounts of
RasN17 in BAEC abolished TNF-
a
-mediated up-regulation of
the
k
B-Luc reporter in a dose-dependent manner (Fig. 2A).
This inhibitory effect was more pronounced than the one seen
with PKC
z
mut
(Fig. 2B). We then analyzed whether RasN17
would interfere directly with RelA activity. Co-transfection of
RasN17 with RelA repressed transcription from the
k
B-Luc
reporter to a similar extent as PKC
z
mut
(Fig. 2B). Both
PKC
z
mut
and RasN17 also inhibited RelA/NF
k
B1 transcrip-
tional 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
k
B
a
FIG.1. Activation of PKC
z
and p21
ras
in endothelial cells. A,
expression of PKC
z
in endothelial cells. Equal amounts of cellular
lysates prepared from HUVEC, PAEC, and BAEC were analyzed by
Western blotting using a PKC
z
-specific polyclonal antibody. B, en-
hanced autophosphorylation of PKC
z
after TNF-
a
and LPS stimulation.
Serum-starved PAEC were labeled with [
32
P]orthophosphate and stim-
ulated with TNF-
a
(50 units/ml) or LPS (1
m
g/ml) for 15 min. Cell
extracts were prepared by detergent lysis and incubated with nonim-
mune rabbit IgG or an anti-PKC
z
antibody. Immunoprecipitates were
separated on 10% polyacrylamide gels under denaturing conditions.
Phosphorylated PKC
z
was revealed by autoradiography. C, PKC
z
im-
mune complex kinase assay. Serum-starved PAEC were stimulated
with TNF-
a
(50 units/ml) as indicated. PKC
z
was immunoprecipitated,
and kinase reactions were carried using myelin basic protein as a
substrate. D, p21
ras
expression in endothelial cells. HUVEC or PAEC
were labeled with [
35
S]Met/Cys, and Ras was immunoprecipitated us-
ing 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 [
32
P]ortho-
phosphate and stimulated with TNF-
a
(50 units/ml) as indicated. p21
ras
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 5GTP/(GDP 31.5 1GTP) 3
100. Values present mean 6S.D. (n52).
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promoter (Fig. 2D). The observation that a constitutive active
mutant of p21
ras
(RasV12) did not complement the inhibitory
effect of PKC
z
mut
suggests that p21
ras
does not act downstream
of PKC
z
in controlling RelA activity. Moreover, we found that a
constitutive active form of PKC
z
, comprising the catalytic re-
gion (amino acids 254–592 of the human PKC
z
) did not over-
come the inhibitory effect of RasN17 (data not shown). Taken
together, these data suggest that p21
ras
and PKC
z
regulate
RelA transcriptional activity by separate pathways.
Neither PKC
z
mut
nor RasN17 altered the levels of overex-
pressed RelA in BAEC as monitored by Western blotting (data
not shown). Additionally, PKC
z
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
z
mut
or RasN17 is not due to inhibition of RelA
DNA binding activity. These data suggest that regulation of
NF-
k
B activity by PKC
z
and p21
ras
acts downstream of I
k
B
directly on RelA.
Role of Small GTPases, Raf-1, and PI 3-Kinase—There are
several potential downstream targets of the p21
ras
and PKC
z
pathway that may account for the regulatory mechanism of
p21
ras
or PKC
z
. First, we monitored the effect of p21
ras
-related
GTPases of the Rho and Rac family, previously shown to reg-
ulate NF-
k
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
k
B reporter in BAEC
(Fig. 4A). Similar results were obtained for TNF-
a
-induced
NF-
k
B activity (data not shown). Raf-1 and PI 3-kinase have
been shown to be involved in p21
ras
(44, 45) and the latter also
in PKC
z
signaling cascades (46) and in regulating NF-
k
B ac-
tivity in fibroblasts (29) and hepatocytes (47), respectively. To
investigate the role of Raf-1 in modulating NF-
k
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
k
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-
a
-induced
k
B reporter activity. Like-
wise, wortmannin or LY294002, another inhibitor of PI 3-ki-
nase, did not effect TNF-
a
-induced I
k
B
a
degradation, NF-
k
B
DNA binding, or up-regulation of NF-
k
B-dependent endoge-
nous genes, i.e. I
k
B
a
and E-selectin (data not shown).
RelA RHD Is the Target of PKC
z
and p21
ras
-mediated Regu-
lation of Transcriptional Activity—To monitor which domain of
RelA is targeted by PKC
z
and p21
ras
, we constructed different
fusion proteins outlined in Fig. 5A. The first construct was
composed of the DNA binding domain derived from the bacte-
rial 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 do-
main (TET/RelA286–551). In addition, we generated a con-
struct composed of the RelA RHD fused to the VP16 transac-
tivation domain (RelA2–320/VP16). Transcriptional activity of
constructs harboring the TET DNA binding domain was ana-
lyzed by co-transfection with a reporter containing seven tet-
racycline operons (TetO) fused to a luciferase gene (TetO-Luc).
Transcriptional activity of constructs harboring the RelA DNA
FIG.2.Inhibition of TNF-
a
- and RelA-mediated transcription
by PKC
z
mut
and RasN17. A, BAECs were transfected with the
k
B-Luc
reporter and increasing amounts of PKC
z
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-
a
for7h.B, BAECs were
co-transfected with RelA expression plasmid (30 ng),
k
B-Luc reporter
(700 ng), and 100, 200, 400, and 670 ng of PKC
z
mut
or RasN17. C,
BAECs were co-transfected with RelA expression plasmid alone (30 ng)
or RelA (30 ng) and NF
k
B1 (20 ng) together with
k
B-Luc (700 ng) and
500 ng of PKC
z
mut
or RasN17. D, BAECs were co-transfected with RelA
(30 ng), I
k
B
a
-Luc (700 ng), and 500 ng of PKC
z
mut
or RasN17. The total
amount of DNA in all transfections was kept constant with pcDNA3
plasmid. Luciferase activities were normalized to
b
-galactosidase activ-
ities to compensate for differences in transfection efficiency. The error
bars represent mean 6S.D. (n53).
FIG.3. PKC
z
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
z
, PKC
z
mut
, wild type Ras, or RasN17 (all at 500 ng). Cell
extracts were incubated with radiolabeled double-stranded NF-
k
B oli-
gonucleotide, and the resulting DNA-protein complexes were separated
on a polyacrylamide gel. Lane I represents the free probe. The positions
of RelA homodimers (Œ) and RelA/NF
k
B1 heterodimers (L) are
indicated.
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binding domain was analyzed by co-transfection with the
k
B-
Luc reporter. Whereas RelA-mediated transcription was re-
pressed by both PKC
z
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
z
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
z
and
p21
ras
regulate RelA transcriptional activity by targeting the
RelA RHD.
Regulation of RelA Transcriptional Activity by PKC
z
and
p21
ras
Is Dependent on Functional
k
B Consensus Sites—Having
established that the regulatory effect of PKC
z
and p21
ras
is
dependent on RelA RHD, we analyzed whether DNA binding
through RHD was necessary for inhibition of RelA transcrip-
tional 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 there-
fore two DNA binding domains (for TetO and
k
B consensus
binding sites). This construct allows one to analyze the effect of
PKC
z
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
z
mut
and RasN17 when co-transfected with
the
k
B-Luc reporter, while it was not affected when the TetO-
Luc reporter was used. Since the
k
B-Luc reporter harbors the
thymidine kinase minimal promoter, while the TetO-Luc har-
bors 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
k
B reporter
containing the same cytomegalovirus minimal promoter frag-
ment that drives the TetO-Luc construct. When transfected
with RelA and PKC
z
mut
or RasN17, this
k
B reporter behaved
the same way as the reporter construct based on the thymidine
kinase minimal promoter (data not shown). We conclude there-
fore that regulation of RelA transcriptional activity by PKC
z
and p21
ras
involves the RelA RHD and is only relevant if RelA
binds DNA through a
k
B consensus site.
Phosphorylation of Endogenous RelA—As previously re-
ported, RelA is phosphorylated upon stimulation with TNF-
a
FIG.4.Role of small GTPases, Raf-1, and PI 3-kinase on NF-
k
B
activation in EC. A, BAECs were transfected with the
k
B-Luc re-
porter (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
k
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 trans-
fected with the
k
B-Luc reporter (700 ng) and RelA expression plasmid
(30 ng). Cells were left untreated or were treated with vehicle (asterisk;
Me
2
SO, 1
m
l/ml) or wortmannin (Wort) at 10, 100, and 1000 nMconcen-
tration (16 h, starting 4 h after end of transfection). D, BAECs were
transfected with the
k
B-Luc reporter (700 ng). Thirty-six hours after
transfection, cells were incubated with vehicle (asterisk;Me
2
SO, 1
m
l/
ml) or wortmannin (Wort) at 10, 100, and 1000 nMconcentration. TNF-
a
(50 units/ml) was added 1 h later, and incubation was continued for 7 h,
after which cell extracts were prepared. Luciferase activities were as-
sayed 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
b
-galactosidase activities to
compensate for differences in transfection efficiency. The error bars
represent mean 6S.D. (n53).
FIG.5.RelA RHD is the target for PKC
z
mut
- and RasN17-medi-
ated transcriptional repression. A, schematic representation of
plasmid constructs used for transfections. All constructs were cloned
into the mammalian expression vector pcDNA3. B, BAEC were trans-
fected with different fusion proteins as outlined in A (all at 30 ng) and
with PKC
z
mut
(500 ng), RasN17 (500 ng),
k
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
k
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
b
-galactosidase activities to compensate
for differences in transfection efficiency. The error bars represent
mean 6S.D. (n53).
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(39, 48, 49). In endothelial cells, TNF-
a
induces RelA phospho-
rylation as analyzed by immunoprecipitation of RelA from
[
32
P]orthophosphate-labeled cells (Fig. 6A). Several other phos-
phopeptides 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
k
B1, p105, and I
k
B
a
(data not shown).
While RelA phosphorylation was increased upon TNF-
a
stim-
ulation, NF
k
B1 was dephosphorylated (seven independent ex-
periments). The decrease in intensity of the I
k
B
a
correspond-
ing band upon TNF-
a
stimulation was due to I
k
B
a
degradation
as assayed by Western blotting. Furthermore, we analyzed the
identity of phosphoamino acids derived from RelA isolated from
TNF-
a
-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 resi-
dues, while no tyrosine phosphorylation was observed.
Phosphorylation of RelA has been attributed to phosphoryl-
ation of serines 276 (48) and 529 (50). Both studies could show
an exclusive role for the respective serine in RelA phosphoryl-
ation. We investigated RelA phosphorylation by two-dimen-
sional separation of tryptic phosphopeptides prepared from
nontreated, TNF-
a
- (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-
a
or LPS stimulation, the pattern of RelA phosphorylation
changes significantly as reflected by an increase in signal in-
tensity 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-
a
and LPS lead to similar changes in RelA
phosphorylation. While peptides b,e,f,g, and hare phospho-
rylated to a similar extent in TNF-
a
- or LPS-stimulated cells,
peptide iseems to be more phosphorylated in TNF-
a
-treated
cells. These data suggest the existence of several constitutive
and inducible phosphorylation sites in RelA.
Phosphorylation of Overexpressed RelA—To investigate
whether an exogenous stimulus is necessary to trigger RelA
phosphorylation or alternatively if free, i.e. non-I
k
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 phos-
phorylated in unstimulated cells. TNF-
a
stimulation did not
affect the phosphorylation status of overexpressed RelA (data
not shown), suggesting that signaling by TNF-
a
might not be
essential for RelA phosphorylation. We next examined whether
phosphorylation of overexpressed RelA was inhibited by I
k
B
a
co-expression under conditions where all RelA would be com-
plexed to I
k
B
a
.I
k
B
a
expression resulted in substantial reduc-
tion 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
k
B
a
. This would suggest
three possible scenarios. RelA is phosphorylated (i) in the cy-
toplasm upon liberation from I
k
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 phosphoryla-
tion, we expressed a previously described RelA DNA binding-
deficient mutant (35). As shown in Fig. 8A(lane 3), the phos-
phorylation 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.
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 phospho-
rylated (Fig. 8A,lane 2), and its phosphorylation was com-
pletely inhibited by co-expressed I
k
B
a
(Fig. 8A,lane 4). As for
RelA, I
k
B
a
co-expression did not change RelA/RHD protein
levels as assayed by Western blot analysis (Fig. 8B,lanes 2
and 4).
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 mu-
tated form, where Ser
276
was replaced by Ala (RelA S276A).
The phosphorylation of RelA S276A was significantly lower as
compared with wild type RelA (Fig. 9A). However, the reduc-
tion 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 ain
the endogenous RelA (Fig. 7), which is constitutively phospho-
rylated in RelA, and its phosphorylation status is not altered by
TNF-
a
or LPS stimulation. These data indicate that Ser
276
is
constitutively phosphorylated in EC.
FIG.6.Phosphorylation of RelA. A,
PAEC were metabolically labeled with
[
32
P]orthophosphate and stimulated with
TNF-
a
for 30 min. RelA was immunopre-
cipitated and separated on a 10% poly-
acrylamide gel under denaturing condi-
tions. B, RelA was immunoprecipitated
from TNF-
a
-stimulated PAEC, and amino
acids were prepared by acidic hydrolysis.
Phosphoamino acids were separated by
two-dimensional thin layer electrophore-
sis and revealed by autoradiography. The
position of the phosphoamino acid stand-
ards is marked by circles.
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Inhibition of RelA Phosphorylation by Blockage of PKC
z
and
p21
ras
Signaling Pathways—We next investigated whether in-
hibition of PKC
z
or p21
ras
signaling pathways would interfere
with RelA phosphorylation. Overexpression of RelA together
with PKC
z
mut
or RasN17 significantly decreased RelA phos-
phorylation as compared with overexpression of RelA alone
(Fig. 10A,top). PKC
z
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
z
mut
or RasN17, we used different fusion proteins de-
scribed above and outlined in Fig. 5A. The phosphorylation of
RelA2–320/VP16 (containing the RelA RHD) was significantly
inhibited by co-expressed PKC
z
mut
or RasN17 (Fig. 10B,top).
PKC
z
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
z
mut
or RasN17 (Fig. 10C).
Having established that PKC
z
and p21
ras
are involved in the
phosphorylation of RelA RHD, we analyzed the phosphoryla-
tion pattern of the RHD by tryptic peptide mapping. Phos-
phopeptides derived from RelA/RHD expressed alone or to-
gether with PKC
z
mut
or RasN17 were analyzed by two-
dimensional separation on thin layer cellulose plates.
Compared with the phosphopeptide map derived from endoge-
nous or overexpressed full-length RelA (Fig. 7), the most strik-
ing 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
FIG.7.Phosphopeptide map of endogenous RelA. BAEC were labeled with [
32
P]orthophosphate and stimulated with TNF-
a
(100 units/ml;
30 min) or LPS (1
m
g/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 (1).
FIG.8.Regulation of RelA phosphorylation. A, BAEC were transfected with RelA (100 ng; lane 1), RelA/RHD (100 ng; lane 2), RelA
DNAmut
(100 ng; lane 3), RelA/RHD (100 ng) together with I
k
B
a
(700 ng; lane 4), or RelA (100 ng) together with I
k
B
a
(700 ng; lane 5). Cells were labeled
with [
32
P]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 (Œ) and the immunoglobulin heavy chain (*) are indicated.
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 Ser
276
3Ala substitution (5400 ng; RelA S276A). The total amount of DNA was kept constant with pcDNA3 plasmid.
Cells were labeled with [
32
P]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 (1). The nomenclature of radioactive spots follows that of Fig. 7 to indicate
corresponding spots.
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was substantially modified when RelA/RHD was co-expressed
with PKC
z
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
z
mut
-
and RasN17-transfected cells. While phosphorylation of pep-
tide bdisappeared in cells transfected with PKC
z
mut
and
RasN17 (Fig. 11), phosphorylation of peptides dand gwas only
inhibited by PKC
z
mut
and not by RasN17 (Fig. 11). These
differences in the phosphorylation pattern again suggest that
PKC
z
and p21
ras
feed into at least partially separated path-
ways controlling RelA phosphorylation.
DISCUSSION
It is widely accepted that regulation of NF-
k
B transcrip-
tional activity is controlled mainly by retention of NF-
k
Binthe
cytoplasm by members of the I
k
B family. In this study, we
demonstrate that at least in endothelial cells there is an addi-
tional regulatory system that controls the transcriptional ac-
tivity of nuclear NF-
k
B by targeting the RelA subunit. This
regulatory system involves signaling through PKC
z
and p21
ras
.
Several kinases have been implicated in the regulation of
nuclear RelA transcriptional activity. Protein kinase A is in-
volved in the regulation of RelA transcriptional activity
through phosphorylation of Ser
276
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-
k
Bin vivo and to phosphorylate the
C-terminal transcriptional activation domain of RelA in vitro
(52). We now demonstrate that PKC
z
and p21
ras
are two addi-
tional components in the regulation of RelA transcriptional
activity. We show that inhibition of these signaling cascades
results in decrease of RelA transcriptional activity that corre-
lates with inhibition of RelA phosphorylation.
Several downstream effectors may account for the effect of
PKC
z
or p21
ras
over NF-
k
B. One common feature shared by
both PKC
z
and p21
ras
is the ability to activate the MEK/extra-
cellular signal-regulated kinase pathway, which has been sug-
gested to control NF-
k
B activity (15, 53). However, we found
that at least in endothelial cells a dominant negative mutant of
Raf1 does not interfere with NF-
k
B-mediated transcription.
Another downstream effector of p21
ras
and PKC
z
is the c-Jun
N-terminal kinase signaling cascade. It is unlikely that this
pathway is involved in NF-
k
B regulation in endothelial cells,
since a dominant negative c-Jun N-terminal kinase 1 failed to
inhibit RelA transcriptional activity (data not shown). More-
over, a dominant negative mutant of Rac1 (RacN17) efficiently
blocked p21
ras
induced c-Jun N-terminal kinase activation
while it failed to inhibit NF-
k
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
FIG. 10. RHD phosphorylation is controlled by p21
ras
and PKC
z
.A, BAEC were transfected with RelA (150 ng) together with pcDNA3
plasmid (control), PKC
z
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 [
32
P]orthophosphate, and proteins were immunoprecipitated with
antibodies directed against the N terminus (Aand 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 (3). 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 (Œ) are indicated.
FIG. 11. Phosphopeptide map of RelA/RHD. RelA/RHD (150 ng) was expressed alone or together with 1350 ng of PKC
z
mut
or RasN17. Cells
were labeled with [
32
P]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 (1).
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did not impair TNF-
a
-induced
k
B-dependent reporter activity.
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 tran-
scription factors is controlled (55). Similarly, phosphorylation
of the RelA transactivation domain has been reported (56). In
particular, inducible phosphorylation of the TA
2
(amino acids
428–520) and constitutive phosphorylation of the TA
1
(amino
acids 521–551) activation domains have been suggested to con-
trol RelA transcriptional activity (56). Recently, phosphoryla-
tion of the RelA transactivation domain by RelA-associated
casein kinase II has been reported (52), and the importance of
phosphorylation of Ser
529
has been revealed (50). In this study,
we present evidence that the RHD domain contributes substan-
tially to the overall phosphorylation of RelA. We show that
RelA phosphorylation can be inhibited partially by co-express-
ing I
k
B
a
, which suggests that RelA is phosphorylated upon
liberation from associated I
k
B molecules. The observation that
phosphorylation of full-length RelA is only partially inhibited
by I
k
B
a
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.
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-
a
or LPS treatment. While the
phosphorylation of some of these sites is regulated by both
PKC
z
and p21
ras
signaling cascades, phosphorylation of other
sites is not altered by these pathways. Furthermore, our data
suggest that RelA is constitutively phosphorylated at Ser
276
,
and this phosphorylation is not altered by TNF-
a
or LPS treat-
ment. In addition, a RelA S276A mutant retained several phos-
phorylated 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).
Finally, our data implicate RHD as a central regulator of
RelA transcriptional activity and show that the phosphoryla-
tion status of RHD can modulate the transcriptional activity of
the transactivation domain. This effect seems to be independ-
ent 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 transacti-
vation 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 con-
formational changes in the transactivation domain, facilitating
interactions with components of the basal transcriptional ma-
chinery, essential for RelA transcriptional activity (56). Allo-
steric control of the DNA binding domain over the transactiva-
tion domain has been reported for several transcription factors
(58). Therefore, it would be of interest to obtain crystal struc-
ture data of full-length RelA bound to DNA in its phosphoryl-
ated and nonphosphorylated form.
Second, the phosphorylation status of RHD may regulate
interaction of RelA with nuclear cofactors such as cAMP re-
sponse 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
z
or p21
ras
.
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
z
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
k
B-dependent reporter and not
when bound to the TetO reporter. This result favors a model
where phosphorylation of RelA RHD by PKC
z
and p21
ras
sig-
naling pathways would modulate RelA transcriptional activity
through conformational changes of DNA-bound RelA.
Another possible mechanism by which PKC
z
and p21
ras
con-
trol 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.
In summary, our data show the existence of a second NF-
k
B
regulatory system that controls transcriptional activity after
liberation of NF-
k
B complexes from their cytoplasmic inhibi-
tors. Although we are only at the beginning of understanding
this regulatory mechanism, we show evidence that it might
include phosphorylation of NF-
k
B complexes, and we propose
p21
ras
and PKC
z
signaling molecules as being involved in such
a control system.
Acknowledgments—We thank H. Bujard, G. M. Cooper, R. De
Martin, and J. Moscat for plasmid constructs; E. Csizmadia for cultur-
ing endothelial cells; and F. H. Bach for helpful discussion.
REFERENCES
1. Siebenlist, U., Franzoso, G., and Brown, K. (1994) Annu. Rev. Cell Biol. 10,
405–455
2. Whiteside, S. T., and Israel, A. (1997) Semin. Cancer Biol. 8, 75–82
3. Didonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M.
(1997) Nature 388, 548–554
4. Mercurio, F., Zhu, H. Y., Murray, B. W., Shevchenko, A., Bennett, B. L., Li,
J. W., Young, D. B., Barbosa, M., and Mann, M. (1997) Science 278,
860–866
5. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science
278, 866–869
6. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997)
Cell 91, 243–252
7. Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell
78, 773–785
8. Traenckner, E. B., Wilk, S., and Baeuerle, P. A. (1994) EMBO J. 13, 5433–5441
9. Read, M. A., Neish, A. S., Luscinskas, F. W., Palombella, V. J., Maniatis, T.,
and Collins, T. (1995) Immunity 2, 493–506
10. Baldassarre, F., Mallardo, M., Mezza, E., Scala, G., and Quinto, I. (1995)
J. Biol. Chem. 270, 31244–31248
11. Caldenhoven, E., Liden, J., Wissink, S., Van de Stolpe, A., Raaijmakers, J.,
Koenderman, L., Okret, S., Gustafsson, J. A., and Van der Saag, P. T. (1995)
Mol. Endocrinol. 9, 401–412
12. Brostjan, C., Anrather, J., Csizmadia, V., Natarajan, G., and Winkler, H.
(1997) J. Immunol. 158, 3836–3844
13. Kannabiran, C., Zeng, X. Y., and Vales, L. D. (1997) Mol. Cell. Biol. 17, 1–9
14. Min, W., Ghosh, S., and Lengyel, P. (1996) Mol. Cell. Biol. 16, 359–68
15. Berra, E., Diaz Meco, M. T., Lozano, J., Frutos, S., Municio, M. M., Sanchez, P.,
Sanz, L., and Moscat, J. (1995) EMBO J. 14, 6157–6163
16. Beyaert, R., Cuenda, A., Vanden, B. W., Plaisance, S., Lee, J. C., Haegeman,
G., Cohen, P., and Fiers, W. (1996) EMBO J. 15, 1914–1923
17. Yoza, B. K., Hu, J., and Mccall, C. E. (1996) J. Biol. Chem. 271, 18306–18309
18. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y.
(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3099–3103
19. Nakanishi, H., and Exton, J. H. (1992) J. Biol. Chem. 267, 16347–16354
20. Nakanishi, H., Brewer, K. A., and Exton, J. H. (1993) J. Biol. Chem. 268, 13–16
21. Rzymkiewicz, D. M., Tetsuka, T., Daphna-Iken, D., Srivastava, S., and
Morrison, A. R. (1996) J. Biol. Chem. 271, 17241–17246
22. Mu¨ller, G., Ayoub, M., Storz, P., Rennecke, J., Fabbro, D., and Pfizenmaier, K.
(1995) EMBO J. 14, 1961–1969
23. Lozano, J., Berra, E., Municio, M. M., Diaz-Meco, M. T., Dominguez, I., Sanz,
L., and Moscat, J. (1994) J. Biol. Chem. 269, 19200–19202
24. Diaz-Meco, M. T., Lozano, J., Municio, M. M., Berra, E., Frutos, S., Sanz, L.,
and Moscat, J. (1994) J. Biol. Chem. 269, 31706–31710
25. Trent, J. C. N., McConkey, D. J., Loughlin, S. M., Harbison, M. T., Fernandez,
A., and Ananthaswamy, H. N. (1996) EMBO J. 15, 4497–4505
26. Geppert, T. D., Whitehurst, C. E., Thompson, P., and Beutler, B. (1994) Mol.
Med. 1, 93–103
27. Marshall, M. (1995) Mol. Reprod. Dev. 42, 493–499
28. Therrien, M., Chang, H. C., Solomon, N. M., Karim, F. D., Wassarman, D. A.,
and Rubin, G. M. (1995) Cell 83, 879–888
Regulation of RelA Transcriptional Activity13602
by guest on October 18, 2015http://www.jbc.org/Downloaded from

29. Finco, T. S., and Baldwin, A. S., Jr. (1993) J. Biol. Chem. 268, 17676–17679
30. Finco, T. S., Westwick, J. K., Norris, J. L., Beg, A. A., Der, C. J., and Baldwin,
A. S., Jr. (1997) J. Biol. Chem. 272, 24113–24116
31. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989)
Gene (Amst.)77, 51–59
32. Bruder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes Dev. 6, 545–56
33. Sharma, P. M., Egawa, K., Huang, Y., Martin, J. L., Huvar, I., Boss, G. R., and
Olefsky, J. M. (1998) J. Biol. Chem. 273, 18528–18537
34. Anrather, J., Csizmadia, V., Brostjan, C., Soares, M. P., Bach, F. H., and
Winkler, H. (1997) J. Clin. Invest. 99, 763–772
35. Franzoso, G., Carlson, L., Brown, K., Daucher, M. B., Bressler, P., and
Siebenlist, U. (1996) EMBO J. 15, 3403–3412
36. Cheng, Q., Cant, C. A., Moll, T., Hofer-Warbinek, R., Wagner, E., Birnstiel,
M. L., Bach, F. H., and De Martin, R. (1994) J. Biol. Chem. 269,
13551–13557
37. Gomez, J., Garcia, A., Borlado, L. R., Bonay, P., Martineza, C., Silva, A.,
Fresno, M., Carrera, A. C., Eicherstreiber, C., and Rebollo, A. (1997)
J. Immunol. 158, 1516–1522
38. Krappmann, D., Wulczyn, F. G., and Scheidereit, C. (1996) EMBO J. 15,
6716–6726
39. Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4597–4607
40. Boyle, W., Van der Geer, P., and Hunter, T. (1991) in Methods Enzymol. 201,
110–149
41. Read, M. A., Whitley, M. Z., Gupta, S., Pierce, J. W., Best, J., Davis, R. J., and
Collins, T. (1997) J. Biol. Chem. 272, 2753–2761
42. Liao, D. F., Monia, B., Dean, N., and Berk, B. C. (1997) J. Biol. Chem. 272,
6146–6150
43. Perona, R., Montaner, S., Saniger, L., Sanchez-Perez, I., Bravo, R., and Lacal,
J. C. (1997) Genes Dev. 11, 463–475
44. Van Aelst, L., Barr, M., Marcus, S., Polverino, A., and Wigler, M. (1993) Proc.
Natl. Acad. Sci. U. S. A. 90, 6213–6217
45. Yan, J., Roy, S., Apolloni, A., Lane, A., and Hancock, J. F. (1998) J. Biol. Chem.
273, 24052–24056
46. Standaert, M. L., Galloway, L., Karnam, P., Bandyopadhyay, G., Moscat, J.,
and Farese, R. V. (1997) J. Biol. Chem. 272, 30075–30082
47. Reddy, S. A. G., Huang, J. H., and Liao, W. S. L. (1997) J. Biol. Chem. 272,
29167–29173
48. Zhong, H. H., Suyang, H., Erdjumentbromage, H., Tempst, P., and Ghosh, S.
(1997) Cell 89, 413–424
49. Ollivier, V., Parry, G., Cobb, R. R., de Prost, D., and Mackman, N. (1996)
J. Biol. Chem. 271, 20828–20835
50. Wang, D., and Baldwin, A. S., Jr. (1998) J. Biol. Chem. 273, 29411–29416
51. Wesselborg, S., Bauer, M., Vogt, M., Schmitz, M. L., and Schulze-Osthoff, K.
(1997) J. Biol. Chem. 272, 12422–12429
52. Bird, T. A., Schooley, K., Dower, S. K., Hagen, H., and Virca, G. D. (1997)
J. Biol. Chem. 272, 32606–32612
53. Marshall, M. S. (1995) FASEB J. 9, 1311–1318
54. Min, W., and Pober, J. S. (1997) J. Immunol. 159, 3508–3518
55. Boulikas, T. (1995) Crit. Rev. Eukaryot. Gene Expr. 5, 1–77
56. Schmitz, M. L., dos Santos Silva, M. A., and Baeuerle, P. A. (1995) J. Biol.
Chem. 270, 15576–15584
57. Schmitz, M. L., dos Santos Silva, M. A., Altmann, H., Czisch, M., Holak, T. A.,
and Baeuerle, P. A. (1994) J. Biol. Chem. 269, 25613–25620
58. Lefstin, J. A., and Yamamoto, K. R. (1998) Nature 392, 885–888
59. Perkins, N. D., Felzien, L. K., Betts, J. C., Leung, K. Y., Beach, D. H., and
Nabel, G. J. (1997) Science 275, 523–527
60. Gerritsen, M. E., Williams, A. J., Neish, A. S., Moore, S., Shi, Y., and Collins,
T. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2927–2932
Regulation of RelA Transcriptional Activity 13603
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Soares and Hans Winkler
Josef Anrather, Vilmos Csizmadia, Miguel P.
Endothelial Cells in Primaryζand Protein Kinase C ras
and Transcriptional Activity by p21
B RelA PhosphorylationκRegulation of NF-
CELL BIOLOGY AND METABOLISM:
doi: 10.1074/jbc.274.19.13594
1999, 274:13594-13603.J. Biol. Chem.
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