TRAF6 and C-SRC induce synergistic AP-1 activation viaPI3-kinase–AKT–JNK pathway Megumi Funakoshi-Tago1, Kenji Tago2, Yoshiko Sonoda1, Shin-ichi Tominaga2and Tadashi Kasahara1 1 Departme
Trang 1TRAF6 and C-SRC induce synergistic AP-1 activation via
PI3-kinase–AKT–JNK pathway
Megumi Funakoshi-Tago1, Kenji Tago2, Yoshiko Sonoda1, Shin-ichi Tominaga2and Tadashi Kasahara1 1
Department of Biochemistry, Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105–8512, Japan;
2
Department of Biochemistry, Jichi Medical School, 3311–1 Minamikawachi-machi, Tochigi-ken, 329–0433, Japan
Interleukin-1 (IL-1) induces multiple genes via activation of
transcription factors that include NF-jB and activator
protein-1 (AP-1).We found that IL-1-mediated c-Src
acti-vation was required for AP-1 actiacti-vation, but not for NF-jB
activation and also revealed that c-Src-induced AP-1
acti-vation was enhanced synergistically by the coexpression of
TNF receptor associated factor 6 (TRAF6).In addition,
c-Src interacts with TRAF6 in response to IL-1 and this
interaction is required for c-Src activity.However, neither
dominant negative mutants of TRAF6 (TRAF6 DN) nor
kinase-dead mutant of c-Src (c-Src KD) counteracted
each-induced AP-1 activation, suggesting no hierarchy between
these two molecules.During the TRAF6 and c-Src-induced
AP-1 activation, phosphatidylinositol 3 (PI3)-kinase, its
downstream signaling molecule, Akt and c-Jun N-terminal kinase (JNK) were significantly activated and inhibition of these kinase activities down-regulated AP-1 activation through the suppression of c-fos expression.Furthermore, TRAF6 and c-Src-induced JNK activation was significantly inhibited by PI3-kinase inhibitor or a dominant negative mutant of Akt (Akt DN).Taken together, our results demonstrate that c-Src and TRAF6 are key mediators of IL-1-induced AP-1 activation and provide evidence of cross talk between c-Src and TRAF6 molecules through PI3 kinase–Akt–JNK pathways
Keywords: NF-jB activation; activator protein-1 (AP-1); Src kinase; TRAF6; c-Jun N-terminal kinase (JNK)
Interleukin-1 (IL-1) is a potent activator of immune and
inflammatory responses that exert various biological
acti-vities mostly through rapid and marked activation of
transcription factors, nuclear factor-jB (NF-jB) and
acti-vator protein-1 (AP-1).Both NF-jB and AP-1 are
import-ant regulators of numerous cytokine genes including IL-6,
IL-8, monocyte chemotactic protein-1 (MCP-1) and the
induction of adhesion molecules [1–3].Recently,
intracel-lular events mediated by IL-1 receptor type I (IL-1RI) have
been extensively explored.That is, binding of IL-1 to the
IL)1RI receptor allows association with the IL-1R
acces-sory protein (IL-1R AcP), that causes the recruitment of
MyD88, an adapter protein, to the IL-1RI complex [4–6]
This in turn leads to the recruitment of a serine/threonine
kinase, IL-1 receptor-associated kinases (IRAK) to the
receptor complex.Then, IRAK interacts with tumor
necrosis factor receptor-associated factor 6 (TRAF6)
[5–9].Kinase, TAK1 and two interacting proteins, TAB1
and TAB2, are also implicated in IL-1 signaling [10,11], in
which TAK1 activates the kinase, NIK, directly, leading to
the activation of NF-jB.IL-1-dependent signaling also lead
to the activation of c-Jun N-terminal kinase (JNK) and other mitogen-activated protein kinase [6,9,12–14], that results in the phosphorylation and activation of AP-1 The above model is based mainly on results obtained through the isolation of specific protein complexes, analyses
of protein–protein interactions (yeast two-hybrid screens) and data from gene knockout studies.However, the detailed mechanisms of IL-1-dependent signaling pathways are still not well defined.While some investigators have reported the involvement of tyrosine kinases in IL-1 signaling, the involvement of Src family kinases has received little attention [15,16]
c-Src tyrosine kinase is a member of a family of nine closely related tyrosine kinases defined by a common structure that includes SH2 and SH3 protein interaction domains, a catalytic region and a negative-regulatory tyrosine located near the carboxyl terminus [18–20].Src kinases have been implicated in multiple signaling pathways that regulate cellular growth, migration and cell survival [19,20].Functional roles of Src family kinases have been well documented in leukocyte signaling processes such as cell migration, adhesion, phagocytosis, cell survival [21–23] or immune response signalings through T cell and B cell receptors [24,25].It has been demonstrated recently that TRANCE, a TNF family member, activates Akt/PKB in osteoclasts and dendritic cells through a signaling complex involving TRAF6 and c-Src [26].Src family kinases are presumed to be a prerequisite for survival signaling using the TNF-family receptors, RANKL/TRANCE [27].In spite of the widespread expression of c-Src, targeted disrup-tion of the c-src gene in mice leads to one predominant
Correspondence to T.Kasahara, Department of Biochemistry,
Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minato-ku,
Tokyo 105-8512, Japan.Fax/Tel: +81 3 5400 2697,
E-mail: kasahara-td@kyoritsu-ph.ac.jp
Abbreviations: NF-jB, nuclear factor-kappaB; AP-1, activator
protein-1; TRAF6, TNF receptor associated factor 6; PI3-kinase,
phosphatidyl-inositol 3-kinase; JNK, c-Jun N-terminal kinase;
DN, dominant negative mutant; IL-1, interleukin-1.
(Received 29 September 2002, revised 15 December 2002,
accepted 27 January 2003)
Trang 2phenotype, osteopetrosis – a failure to break down bone –
resulting from an intrinsic defect in osteoclasts, a cell type
that expresses high levels of Src [27,28].IL-1 also regulates
cytoskeletal organization in osteoclasts via a TRAF6/c-Scr
complex [29].These findings strongly support the fact that
IL-1 signaling is closely related to that by TRAF6 and c-Src
However, downstream targets of c-Src kinase, particularly
in IL-1-mediated signaling processes, that lead to NF-jB or
AP-1 activation, remain to be defined
We describe here that IL-1 induces marked c-Src kinase
activation and that c-Src activity is involved in AP-1 but
NF-jB activation.In addition, c-Src-induced AP-1
activa-tion is synergistically augmented by the coexpression of
TRAF6.This synergistic AP-1 activation is mediated mostly
by the PI3-kinase/Akt/c-Jun N-terminal kinase (JNK)
pathways
Experimental procedures
Antibodies and reagents
Mouse monoclonal antibodies against FLAG-peptides
(M2), Myc-peptides (9E10) and HA-peptides were
pur-chased from Sigma, Santa Cruz Biotechnology and Roche,
respectively.Rabbit Ig against c-Src were purchased from
Santa Cruz Biotechnology.Peroxidase-conjugated porcine
(rabbit IgG) and peroxidase-conjugated goat
anti-(mouse IgG) and anti-(rabbit IgG) were obtained from
Dako (Dako-Japan, Tokyo, Japan).Human recombinant
IL-1a (IL-1) was kindly provided by Dainippon
Pharma-ceutical Co.(Suitashi, Osaka, Japan).Src inhibitor, PP2,
JNK inhibitor I, and PI3-kinase inhibitor, LY294002, were
purchased from Calbiochem–Novabiochem Co.and Alexis
Biochemicals, respectively.SAM68 and histone H1 were
obtained from Santa Cruz Biotechnology and SigmaCell
Culture.A human glioblastoma cell line, T98G (JCRB,
Kamiyoga, Tokyo) and human embryonic kidney cells,
HEK293T were maintained in Dulbecco’s modified Eagle’s
medium (DMEM; Nissui Seiyaku, Tokyo) supplemented
with 10% heat-inactivated fetal bovine serum (Nippon
Bio-Supply Center, Tokyo, Japan), 4 mM glutamine, 100
unitsÆmL)1penicillin G and 100 lgÆmL)1streptomycin
Construction of plasmid vectors
A dominant negative mutant of TRAF6 (TRAF6 DN;
mouse where TRAF6 lacks amino acids 1–288) and the
full-length cDNA with the N-terminal FLAG sequence were
generated from the total RNA of a murine thymoma cell
line, EL4, by RT-PCR amplification as described by Cao
et al.[7].PCR products were purified and inserted into the
EcoRI and BamHI site of pCMV5.The cDNA encoding
mouse c-Src was also generated from the total RNA of EL4
by RT-PCR.A kinase dead mutant of c-Src cDNA (c-Src
KD) contained one point mutation (Arg296fiLys).PCR
products were inserted into the HindIII site of pCMV5.The
cDNA encoding human Csk with the N-terminal FLAG
sequence were generated by RT-PCR amplification and was
inserted into the EcoRI and BamHI site of pCMV5
Transient transfection and luciferase assay plasmid DNAs
were transfected into T98G or HEK293T cells by the
calcium phosphate precipitation method.Final amounts of
the transfected DNA were adjusted to 10 lg per 60-mm dish by addition of empty vector, pCMV5.pCMV5-FLAG-TRAF6, and/or pCMV5-c-Src or pCMV5-c-Src KD (1 lg each) were cotransfected with 0.1 lg of pRL-TK (Promega) and 1 lg of pNF-jB-Luc, pAP-1-Luc, or pIL-8-Luc ()133
to +46 [31,32]).Cells were exposed for 1 day to DNA precipitation in DMEM containing 10% fetal bovine serum.After 48 h of transfection, cells were harvested and the luciferase activities were measured by Lumat LB9501 (Bertold Japan, Tokyo [33,34]).Efficiency of transfection was normalized with sea pansy luciferase activities Immunoprecipitation and immunoblotting All cells were harvested and lysed in lysis buffer [10 mM Tris/HCl (pH 7.4), 158 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM EGTA, 1 mM Na3VO4,
2 lgÆmL)1 aprotinin, 2 lgÆmL)1 leupeptin] on ice and cleaned by centrifugation at 13 000 g to obtain whole cell extracts.Aliquots (250 lg) of cell lysate were mixed with protein G-sepharose (Pharmacia-LKB Biotechnologies, Uppsala, Sweden) with each antibody for 2 h at 4C Immune complexes were precipitated by centrifugation at
13 000 g and washed three times with lysis buffer and were boiled in Laemmli sample buffer.Boiled samples were separated by SDS/PAGE and the proteins were transferred to nitrocellulose membranes.Immunoblot and visualization using the enhanced chemiluminescence Western blotting detection system (Amersham) was described elsewhere [33,34]
In vitro kinase assay The immunoprecipitates were washed twice with lysis buffer and three times with kinase buffer (25 mM Hepes/NaOH (pH 7.5), 20 mM MgCl2, 20 mM b-glycerophosphate, 0.1 mM Na3VO4, 2 mM dithiothreitol, 20 mM p-nitrophe-nylphosphate).The kinase reaction (20 lL of kinase buffer,
10 lM ATP and 0.5 lg SAM68 for c-Src, Histon H1 for Akt, or GST-c-Jun for JNK as substrates) was carried out with [32P]ATP for 15 min at 30C.Samples were resolved
by 15 or 12% PAGE and phosphorylated SAM68, histone H1 and GST-c-Jun were visualized by autoradio-graphy
RNA isolation and PCR analysis Total RNA separation and RT–PCR analysis were per-formed according to the manufacturer’s protocols (Takara Shuzo, Shiga, Japan) using an oligo(dT) 20-primer and 1 lg total RNA for first strand cDNA synthesis.PCR was performed at an annealing temperature of 57C and 20 amplification cycles.The PCR products were resolved and electrophoresed on a 1% agarose gel in Tris/borate/EDTA The primers used were as follows: human IL-8, 5¢-GAG
(down-stream); human c-Fos, 5¢-CCGGGGATAGCCTCTCT TAC-3¢ (upstream) and 5¢-CTTCTCCTTCAGCAGGT TGG-3¢ (downstream); human c-Jun, 5¢-TTCTATGAC GATGCCCTCAA-3¢ (upstream) and 5¢-GTTGCTGAG GTTTGCGTAGA-3¢ (downstream); human GAPDH,
Trang 35¢-GTCAGTGGTGGACCTGACCT-3¢ (upstream) and
5¢-TGAGGAGGGGAGATTCAGTG-3¢ (downstream)
Electrophoretic mobility shift assay (EMSA)
EMSAs were carried out as described previously [31,32]
The consensus double-strand oligodeoxynucleotide probes
for AP-1 (Santa Cruz Biotechnology, Inc.) were
radio-actively labeled using [c-32P]ATP and T4 polynucleotide
kinase using standard procedures.Nuclear proteins (10 lg)
prepared from cells were incubated with [c-32P]labeled
double-strand oligonucleotide probe.The binding reaction
was carried out at room temperature for 30 min in a total
volume of 25 lL.Bound complexes were separated on 5%
Tris-Glycine EDTA gel by electrophoresis in Tris-Glycine
EDTA buffer, dried and visualized by autoradiography
Results
IL-1-induced c-Src activation: c-Src kinase activity is
required for IL-1-induced AP-1 but not NF-jB activation
The IL-1/IL1R-mediated signaling pathway has been
extensively explored and many signaling molecules have
been identified so far.However, the cross talk between these
signaling molecules is complicated and less well defined.To
explore the possible involvement of tyrosine kinase and
c-Src in IL-1 signaling, we first explored whether IL-1
induced activation of c-Src.Cell lysates of IL-1-treated
T98G cells were assayed for c-Src kinase activity in vitro
using SAM68, a known Src-family kinase substrate.As
shown in Fig.1A,B c-Src activity was detected at 15–30 min
after IL-1 stimulation and the activity returned to basal
levels by 60 min.In addition, most of the IL-1-induced Src
activity was abrogated by pretreatment with PP2, a known
Src family inhibitor, after 20 min (Fig.1C)
While it has been established that IL-1 rapidly activates
the transcription factors, NF-jB and AP-1 [33,34], we
explored how c-Src gene expression modulated
IL-1-induced NF-jB and AP-1 activation.A luciferase reporter
gene assay for NF-jB and AP-1 demonstrated that cells
transfected with wild-type c-Src exhibited a substantial
increase in NF-jB activation ( 4-fold) almost comparable
to that by IL-1 stimulation alone ( sixfold;
Fig.2A).IL-1-induced NF-jB activation was markedly augmented by the
transfection of wild-type c-Src ( 14-fold) as well as by the
kinase-dead mutant of c-Src (c-Src KD) ( 15-fold).While
either IL-1 or wild-type c-Src expression alone induced a
marginal AP-1 activation, simultaneous stimulation with
c-Src and IL-1 enhanced AP-1 activation substantially
( 2-fold) (Fig.2B).Interestingly, in contrast to its effect on
the NF-jB activation, c-Src KD virtually abrogated
IL-1-induced AP-1 activation to the unstimulated level.Thus,
these results suggested that c-Src kinase is more prominently
involved more in AP-1 rather than NF-jB activation in IL-1
signaling
c-Src-induced AP-1 activation is augmented
synergistically by the coexpression of TRAF6
As TRAF6 functions as a major signal transducer in
IL-1signaling pathways [6–9], we attempted to explore how
c-Src affects the TRAF6-induced signaling by coexpression experiments.Therefore, we first determined the effect of c-Src on TRAF6-induced NF-jB activation.T98G cells transfected with TRAF6 alone displayed a 10 to 15-fold increase in NF-jB activation that was augmented additively
by the coexpression of c-Src (Fig.2C).Furthermore, both TRAF6 and IL-1-induced NF-jB activation ( 18-fold) was augmented further by c-Src coexpression ( 32-fold)
In contrast, TRAF6 or c-Src-transfection alone, as well as IL-1-stimulation alone, induced minimal levels of AP-1 activation (> twofold; Fig.2D).IL-1-induced AP-1 acti-vation was augmented additively, though minimally, by TRAF6 or by c-Src.Of note is that cotransfection with both TRAF6 and c-Src that exhibited a dramatic activation of AP-1 ( 13-fold), to a level that was not achieved by the combination of either TRAF6 and IL-1 or c-Src and IL-1 AP-1 activation induced by TRAF6 and c-Src was not further augmented by the addition of IL-1.These data indicated that both TRAF6 and c-Src appear to be essential for the full activation of AP-1
Fig 1 IL-1 induced c-Src activation (A) T98G cells were stimulated with IL-1 for the indicated periods and cell lysates were cipitated with anti-c-Src antibody.c-Src activity in the immunopre-cipitates was measured by an in vitro kinase assay using GST-SAM68 (B) c-Src activity in (A) was quantified by a BAS 2000 II (Fuji Bio-image analyzer) and fold increase was shown.Results are expressed as mean ± SD of three independent experiments.(C) c-Src activity was measured in the presence of PP2 (20 l M ) at 20 min after IL-1 stimu-lation (C).
Trang 4TRAF6 interacts with c-Src in an IL-1-dependent manner
To investigate the molecular mechanism of TRAF6 and
c-Src-induced synergistic AP-1 activation in the IL-1
signaling pathway, we examined whether TRAF6 physically
interacted with c-Src.As detected by a
coimmunoprecipi-tation assay in Fig.3A, endogenous c-Src was found to
associate with ectopically expressed FLAG-tagged TRAF6
in an IL-1-dependent manner.Similarly, endogenous
TRAF6 was coimmunoprecipitated with ectopic c-Src in
response to IL-1 stimulation (Fig.3B).We then examined
whether the kinase activity of c-Src is required for the
interaction with TRAF6 FLAG-tagged TRAF6 was
coex-pressed with wild-type or kinase-dead mutant of c-Src (c-Src
KD) in T98G cells, and their association was tested by
detecting c-Src in anti-FLAG immunoprecipitates.We
could detect the strong association of TRAF6 with c-Src
without IL-1 stimulation as shown in Fig.3C.We could not
detect interaction of TRAF6 with c-Src KD, in spite of the
similar expression amounts of these two molecules.These
data suggested that the kinase activity of c-Src is essential
for the interaction with TRAF6
Synergistic AP-1 activation by TRAF6 and c-Src
is induced through c-Fos expression
To further delineate the molecular mechanism of synergistic
AP-1 activation by Src and TRAF6, we used HEK293T
(293T) cells in the subsequent studies, because of its high
transfection efficiency.As shown in Fig.4A, 293T cells transfected with TRAF6 displayed a moderate increase, i.e
2-fold increase, in AP-1 activation compared to the vector-transfected cells.Similarly, c-Src-transfected 293T cells exhibited a substantial increase in AP-1 activation ( seven- to eightfold).Interestingly, simultaneous expres-sion of TRAF6 and c-Src induced a dramatic increase of AP-1 activity (> 20-fold).This enhanced AP-1 activation was suppressed partially by the cotransfection of Csk, C-terminal Src kinase (data not shown), which inhibits c-Src kinase activity by inducing conformational changes [19,20], suggesting that synergistic AP-1 activation by c-Src and TRAF6 requires c-Src kinase activity.To assess the effect of TRAF6 and c-Src on AP-1 activation further, we measured AP-1 DNA binding by an EMSA assay, using oligo-nucleotide probes for AP-1.Consistent with the results of luciferase assay, we confirmed that synergistic AP-1 activa-tion was induced by coexpression of TRA6 and c-Src (Fig.4B) This DNA binding activity was not detected using mutant oligonucleotide with a substitution in the AP-1 binding motif.Furthermore, the band of AP-1/DNA complex disappeared by adding a specific antibody against c-Fos to the nuclear extracts (Fig.4C).Thus, a supershift assay suggested that TRAF6- and c-Src-induced AP-1 DNA binding proteins contain c-Fos but not c-Jun The above observation was further confirmed at the mRNA level.Namely, either TRAF6 or c-Src transfection alone induced moderate c-Fos mRNA expression.Of note
is that the dramatic expression of c-Fos mRNA was induced
Fig 2 Synergistic activation of AP-1 by TRAF6 and c-Src:requirement of c-Src kinase activity for the IL-1-induced AP-1 activation T98G cells were transiently cotransfected with NF-jB-responsive reporter (pNF-jB-Luc) (A) or AP-1-responsive reporter (p AP-1-Luc) (B), with either an empty vector (none) or plasmids bearing wild-type (WT) or c-Src KD.T98G cells were transiently cotransfected with pNF-jB-Luc (C) or pAP-1-Luc (D), with expression plasmids bearing FLAG-TRAF6 and/or c-Src (1 lg each).Cell lysates were harvested at 48 h after transfection, and luciferase activity was assayed as described in Experimental procedures.Results are expressed as mean ± SD of three independent experiments.
Trang 5by the coexpression of TRAF6 and c-Src (Fig.4D) In
contrast, levels of c-Jun mRNA was changed little by the
overexpression of TRAF6 and/or c-Src.Thus, TRAF6 and
c-Src appeared to induce synergistic AP-1 activation
through enhancement of c-Fos expression
TRAF6 and c-Src induce AP-1 activation independently
Then, in order to investigate the mutual dependency of
TRAF6 and c-Src in AP-1 activation, we cotransfected
c-Src KD with TRAF6 and determined its effect on the TRAF6-induced AP-1 activation.As shown in Fig.5A c-Src KD did not down-regulate the TRAF6-induced AP-1 activation at all, suggesting that c-Src activity is not required for TRFA6 to induce AP-1 activation.Similarly, TRAF6 DN was cotransfected with c-Src, no reduction
in c-Src-induced AP-1 activation was observed as well This observation was further substantiated by the DNA binding and mRNA levels.Namely, as shown in Fig.5B, neither c-Src KD nor TRAF6 DN counteracted each-induced AP-1 DNA binding activity.Furthermore, both TRAF6 and c-Src-induced c-Fos mRNA expression were not inhibited by c-Src KD or TRAF6 DN, respectively (Fig.5C) Thus, the above data suggested that there is
no hierarchy between TRAF6 and c-Src in AP-1 activation, and that each works in an independent manner
JNK and PI3-kinase inhibitors down-regulate TRAF6 and c-Src-induced AP-1 activation
To further explore the signals involved in the synergistic AP-1 activation by TRAF6 and c-Src, we focused on the MAP kinase pathways involving ERK, JNK and p38 MAPK activation.In addition, involvement of PI3-kinase was also studied as it is known to be involved in TNFa or IL-1 signaling that leads to NF-jB and AP-1 activation [26,33,35].To date, no direct interplay between c-Src or TRAF6 and PI3-kinase or MAP kinases have been defined.To address this question, we tested several inhibitors for MEK/ERK, JNK, p38MAPK, and PI3-kinase on the TRAF6 and c-Src transfection system Using a luciferase assay, neither PD98059 (PD) nor SB203580 (SB), that are specific inhibitors for MEK/ERK
or p38MAPK, respectively, affected TRAF6- or c-Src-induced AP-1 activation (data not shown).In contrast, a JNK inhibitor I (JI) or LY294002 (LY), a specific PI3-kinase inhibitor, significantly reduced TRAF6- or c-Src-induced AP-1 activation by approximately 50% (data not shown).These two inhibitors attenuated synergistic AP-1 activation by the coexpression of TRAF6 and c-Src to approximately 40% (Fig.6A) Consistent with Fig.5A, both JI and LY significantly reduced AP-1 DNA binding activity induced by TRAF6 and c-Src (Fig.6B) In addition, both JI and LY significantly reduced c-Fos mRNA expression induced by TRAF6 and c-Src, while
PD or SB did not (Fig.6C)
These results suggested that c-Src and/or TRAF6-induced AP-1 activation is dependent predominantly on the JNK and PI3-kinase signaling pathways, but not on the ERK or p38MAPK pathways
Synergistic activation of AP-1 by TRAF6 and c-Src via PI3-kinase-Akt pathway
As PI3-kinase was presumed to be involved in the TRAF6 and c-Src–induced AP-1 activation as described above, we assumed that Akt/PKB, which is activated via PI3-kinase [26], was also involved in this pathway.To further confirm the direct involvement of Akt, 293T cells with AP-1-Luc were cotransfected with TRAF6 and/or c-Src, as well as with the dominant negative form of Akt
Fig 3 TRAF6 interacts with c-Src in response to IL-1 (A) T98G cells
were transiently transfected with expression plasmids bearing
FLAG-tagged TRAF6.FLAG-FLAG-tagged TRAF6 was immunoprecipitated from
the whole cell lysates (WCL) using anti-FLAG antibodies.(B) T98G
cells were transiently transfected with expression plasmids bearing
c-Src.c-Src was immunoprecipitated from the WCL using antic-Src
antibodies.(C) T98G cells were transiently cotransfected with
expression plasmids bearing FLAG-tagged TRAF6 with either
plas-mids bearing wild-type (WT) or c-Src KD.FLAG-tagged TRAF6 was
immunoprecipitated from the whole cell lysates using anti-FLAG
antibodies.Immunoprecipitates (IP) and WCL were analysed by
immunoblot analysis to detect c-Src and TRAF6 as indicated.
Trang 6(Akt DN).Virtually most of the TRAF6 or
c-Src-induced AP-1 activation was abrogated by the increasing
doses of Akt DN (data not shown).Similarly, synergistic
AP-1 activation by TRAF6 and c-Src was attenuated
thoroughly by the Akt DN (Fig.7A).Inability of
TRAF6 and/or c-Src to stimulate AP-1 activation in
the presence of the Akt DN was not due to the
suppression of TRAF6 and/or c-Src expression vectors,
since cell extracts displayed similar protein expression
levels of transfected cells (shown in the blots).In
addition, synergistic AP-1 DNA binding activity and
c-Fos expression induced by TRAF6 and c-Src
cotrans-fection was similarly reduced by the presence of Akt DN
(Fig.7B,C)
To further delineate how TRAF6 and/or c-Src modulate
Akt activation, Akt activity was measured in an in vitro
kinase assay using histone H1 as a substrate.Akt expression
alone induced slight phosphorylation of histone H1 as with
control vectors.When Akt was transfected with TRAF6 or
c-Src, significant Akt activity, i.e Akt-induced histone H1
phosphorylation, was observed (Fig.8A).Of note is that
coexpression of the three constructs, i.e Akt, TRAF6 and
c-Src, resulted in marked Akt activity that was significantly
blocked in the presence of LY but not JI (Fig.8B).These
data indicated that the PI3-kinase/Akt-dependent pathway
plays a critical role in the synergistic AP-1 activation by the
TRAF6 and c-Src
Synergistic activation of JNK by TRAF6 and c-Src: involvement of Akt pathway
To confirm the involvement of JNK as shown in Fig.6, we examined whether expression of TRAF6 and/or c-Src directly modulates JNK activity, by measuring in vitro kinase activity with GST-c-Jun as a substrate.When JNK was cotransfected, either with TRAF6 or c-Src, only moderate phosphorylation of c-Jun was detected.However, coexpression of the three constructs, i.e TRAF6, c-Src and JNK, resulted in a marked JNK activity (Fig.9A), which was inhibited by both JI and LY (Fig.9B).Furthermore, the expression of Akt DN inhibited TRAF6- and c-Src-induced JNK activation, while wild-type Akt enhanced JNK activation
These data in addition to Fig.6 support the notion that synergistic AP-1 activation induced by coexpression of TRAF6 and c-Src is mediated through a JNK-dependent signaling pathway.Thus, these data suggest that the PI3-kinase/Akt pathway also plays a critical role in TRAF6 and c-Src-induced JNK activation
TRAF6 and c-Src induced marked IL-8 production via JNK and PI3 kinase pathway
We finally examined how TRAF6 and c-Src would partici-pate in the IL-1-induced IL-8 expression using an IL-8
Fig 4 Synergistic AP-1 activation induced by TRAF6 and c-Src through significant expression of c-Fos (A) 293T cells were transiently cotransfected with pAP-1-Luc and expression plasmids bearing FLAG-TRAF6 and/or c-Src (1 lg each).Cell lysates were harvested at 48 h after transfection and luciferase activity was assayed as described in Experimental procedures.Results are expressed as mean ± SD of three independent experiments Total protein was isolated from representative transfection experiments, and immunoblot analysis was performed for transfected gene expression Protein samples were probed with a FLAG-specific antibody for FLAG-tagged TRAF6 and with a c-Src specific antibody for c-Src.(B) 293T cells were transiently cotransfected with expression plasmids bearing FLAG-TRAF6 and/or c-Src (1 lg each).Nuclear extracts were prepared for detection of AP-1 activity using EMSA with radiolabeled probe containing the consensus AP-1 binding site (AP-1 oligonucleotide 5¢-CGCTTGATGACTCAGCCGGAA-3¢, mutant oligonucleotide; AP-1 mut: 5¢-CGCTTGATGACTTGGCCGGAA-3¢ obtained from Santa Cruz).The arrows denote the specific AP-1-DNA complex.(C) For supershift assays, nuclear extracts were incubated in the presence of 2 lg of specific antibodies against each c-Fos and c-Jun.(D) Total RNA were prepared and analysed by RT-PCR.The PCR products were electrophoresed
on 2% agarose gels and visualized by ethidium bromide staining.
Trang 7promoter assay.Transfection of either TRAF6 or c-Src alone
induced a minimal level of IL-8 promoter activity when
compared to IL-1 stimulation (Fig.10A) Simultaneous
expression of both TRAF6 and c-Src induced much higher
IL-8 promoter activity (mean 22-fold) than IL-1 stimulation
(8-fold).Most prominent activation was obtained by the
coexpression of TRAF6 and c-Src with IL-1 stimulation
(50-fold).NF-jB and also AP-1 activation by TRAF6 and
c-Src are required for optimal and maximal IL-8 promoter activation.IL-8 promoter activity was partially, but signifi-cantly down-regulated by JI, LY, or by Akt DN (Fig.10B), indicating again that JNK and PI3-kinase-mediated path-ways play substantial roles in the TRAF6 and c-Src-induced IL-8 gene activation.IL-8 mRNA expression by RT-PCR (shown at the bottom panel) confirmed the results obtained
by the promoter assay
Fig 5 Src activates AP-1 independently with TRAF6 (A) 293T cells were transiently cotransfected with pAP-1-Luc and expression plasmids bearing FLAG-TRAF6, TRAF6 DN, and/or c-Src, c-Src KD (1 lg each).Cell lysates were harvested at 48 h after transfection, and luciferase activity was assayed as described in Experimental procedures.Results are expressed as mean ± SD of three independent experiments.(Blots) Total protein was isolated from representative transfection experiments, and immunoblot analysis was performed for the transfected gene expression, by probing a FLAG specific antibody for FLAG-tagged TRAF6 and TRAF6 DN, and by a c-Src specific antibody for c-Src and c-Src KD.(B) 293T cells were transiently cotransfected with expression plasmids bearing FLAG-TRAF6, TRAF6 DN, and/or c-Src, c-Src KD (1 lg each).Nuclear extracts were prepared for detection of AP-1 activity using EMSA.The arrows denote the specific AP-1-DNA complex.(C) Total RNA were prepared and analysed by RT-PCR.The PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.
Fig 6 Down-regulation of TRAF6 and c-Src-induced AP-1 activation by JNK and PI3 kinase inhibitors (A) 293T cells were transiently cotransfected with pAP-1-Luc and expression plasmids bearing FLAG-TRAF6 and/or c-Src (1 lg each).Cells were treated with PD98059 (10 l M ), SB203580 (10 l M ), JNK inhibitor I (10 l M ) and LY294002 (10 l M ) for 6 h.Results are expressed as means ± SD of three independent experiments.(Blots) Total protein was isolated from representative transfection experiments, and immunoblot analysis was performed for the transfected gene expression.Protein samples were probed with a FLAG specific antibody for FLAG-tagged TRAF6, with a c-Src specific antibody for c-Src.(B) 293T cells were transiently cotransfected with expression plasmids bearing FLAG-TRAF6 and c-Src (1 lg each).After the treatment of various inhibitors for 6 h, nuclear extracts were prepared for detection of AP-1 activity using EMSA.The arrows denote the specific AP-1-DNA complex (C) Total RNA were prepared and analysed by RT-PCR.The PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.
Trang 8Fig 7 Down-regulation of TRAF6 and c-Src induced activation of Akt by dominant negative mutant of Akt (Akt DN) (A) 293T cells were transiently cotransfected with pAP-1-Luc and expression plasmids bearing FLAG-TRAF6 and c-Src (1 lg each) and varying amounts of Akt DN.Cell lysates were harvested at 48 h after transfection, and luciferase activity was assayed.Results are expressed as means ± SD of three independent experiments.(Blots) Blots were probed with a FLAG specific antibody for FLAG-tagged TRAF6, with a c-Src specific antibody for c-Src, and Myc specific antibody for Akt DN.(B) 293T cells were transiently cotransfected with expression plasmids bearing FLAG-TRAF6, c-Src and Myc-Akt DN (1 lg each).Nuclear extracts were prepared for detection of AP-1 activity using EMSA.The arrows denote the specific AP-1–DNA complex.(C) Total RNA were prepared and analysed by RT-PCR.The PCR products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.
Fig 8 TRAF6 and c-Src induced marked Akt activation (A) 293T cells were transiently cotransfected with expression plasmids bearing FLAG-TRAF6, and/or c-Src and Myc-Akt (1 lg each).Cells were treated by LY294002 (10 l M ) and JNK inhibitor I (10 l M ) for 6 h (B).Cell lysates were harvested at 48 h after transfection, and the relative activity of Akt in the immunoprecipitates was measured by in vitro kinase assay using with histone H1 as substrate.Blots were probed with a FLAG specific antibody for FLAG-tagged TRAF6, with a c-Src specific antibody for c-Src and with Myc specific antibody for Myc-tagged Akt.
Trang 9These data suggested that TRAF6 and c-Src are
signi-ficant signal molecules involved in the AP-1 activation as
well as IL-1-induced IL-8 expression, acting particularly
through the PI3-kinase/Akt/JNK
Discussion
IL-1 plays an important role in immunological and
inflammatory reactions by rapidly activating transcription
factors, NF-jB and AP-1, that induce various
inflamma-tory genes [1–3].Recently, a number of molecules
involved in the IL-1 signaling pathway have been identified [4–11].While details of the IL-1/IL-1R-mediated signal cascade have been uncovered, the roles of many regulatory and interacting molecules remain to be eluci-dated, particularly those from the tyrosine kinase families Our initial observation revealed that IL-1-induced IL-8 production was attenuated in the presence of Src family kinase inhibitor, suggesting the involvement of Src family kinases in IL-1 signaling (data not shown).As NF-jB is
a master transcription factor involved in the inflammatory responses elicited by IL-1 and is necessary for IL-8
Fig 9 TRAF6 and c-Src induced marked JNK activation through PI3-kinase-Akt pathway 293T cells were transiently cotransfected with expression plasmids bearing FLAG-TRAF6, and/or c-Src (A, B), and/or Myc-Akt WT and Akt DN and HA-JNK (1 lg each) (C) Cells were treated with JNK inhibitor I (10 l M ) and LY294002 (10 l M ) for 6 h (B).Cell lysates were harvested at 48 h after transfection, and the relative activity of JNK in the immunoprecipitates was measured by in vitro kinase assay using with GST-c-Jun as substrate.(Blots) Protein extracts were probed with a FLAG specific antibody for FLAG-tagged TRAF6, with c-Src specific antibody for c-Src, with Myc specific antibody for Myc-tagged Akt WT and Akt DN, and with HA specific antibody for HA-tagged JNK.
Fig 10 TRAF6 and c-Src activate IL-8 promoter activity through JNK and PI3-kinase pathways 293T cells were transiently cotransfected with pIL-8-Luc ( )133 to +46) and FLAG-TRAF6, c-Src (A), and/or Akt DN (1 lg each), in the presence or absence of JNK inhibitor I (10 l M ) or LY294002 (10 l M ) (B).Cell lysates were harvested at 48 h after transfection, and luciferase activity was assayed.Results are expressed as means ± SD of three independent experiments.(Bottom) Total RNA were prepared and analysed by RT-PCR.PCR products were electro-phoresed on 1% agarose gels and visualized by ethidium bromide staining.
Trang 10production [31–34,36], we investigated how c-Src
influen-ces the IL-1-induced NF-jB activation.A marked c-Src
kinase activity was induced by IL-1, but both wild-type
and a mutant of c-Src, the latter lacking its kinase
activity, could activate NF-jB in the presence or absence
of IL-1.Thus, IL-1-induced c-Src kinase activity per se
was not necessarily required for the IL-1-induced NF-jB
activation, but c-Src appeared to activate NF-jB directly
or to act as a scaffold protein or an adapter protein.A
similar observation has been suggested elsewhere; namely,
while c-Src deficient mice exhibit severe osteopetrosis
(caused by an intrinsic defect in osteoclast function), it is
rescued by kinase-deficient c-Src mutants (Y416F,
K295M) as well as by a wild-type c-Src [20,26,27]
However, when expressed beyond normal levels, the
inactive c-Src kinase mutant fails to rescue c-Src deficient
mice by inducing osteoclasts to undergo apoptosis [37]
Therefore, c-Src kinase activity is likely to be important
for regulating at least some aspects of osteoclasts (such as
cell morphology and survival)
In this study, kinase activity of c-Src appeared to be
indispensable for IL-1-induced AP-1 activation and
bind-ing to TRAF6 (Figs 2 and 3).Furthermore, our surprisbind-ing
finding is that TRAF6 and c-Src augmented AP-1
activation dramatically (Figs 2 and 4).It is also the case
that the TNF-related activation-induced cytokine
(TRANCE), which plays an essential role in bone
resorption and osteoclast differentiation, utilizes TRAF6
and c-Src as important downstream molecules mediating
this effect [38,39].TRANCE activates a serine/threonine
kinase, Akt (through TRAF6), that appears capable of
binding to and activating c-Src [26].Activated c-Src could
then recruit PI3-kinase, which generates
D3-phospho-inositides for Akt activation.In addition, TRANCE and
CD40L-mediated Akt activation in dendritic cells was
regulated by cytoplasmic adapter molecules, Cbl family
proteins [40].The above observation, however, might be
different from the finding in our IL-1 study.That is,
although TRAF6 and c-Src could form the complex in an
IL-1-dependent manner and lead to significant AP-1
activation, the hierarchy between these two molecules
did not exist in IL-1-induced AP-1 activation.Although
the kinase activity of c-Src is required for IL-1-induced
AP-1 activation, a kinase-dead mutant of c-Src had no
effect on TRAF6-induced AP-1 activation
(Fig.5).More-over, a recent study by Zhang et al.[41] indicated that
TNF-a markedly accelerates osteoclastogenesis induced by
TRANCE, and deletion of TNF receptor 1 (TNFR1)
abrogates this response.Enhanced osteoclastogenesis was
thus associated with high expression of TNF and
TRANCE-signaling mediators, including c-Src, TRAF2,
TRAF6 and MEKK-1, and the levels of these signal
molecules were notably reduced in TNFR1 knockouts.In
addition, MEKK1 is a potential target for TRAF2 and
TRAF6, two related signal transducers to TNFa and
IL-1, respectively, and is critically required for JNK
activation in the study using MEKK1-deficient embryonic
stem cells [42–44].Although the detailed mechanisms of
osteoclastogenesis by these molecules is not clarified, these
studies also mentioned that c-Src, TRAF2, TRAF6 and
MEKK-1 work cooperatively as potent osteoclastogenic
factors.That is, TRAF6-c-Src complex leads to NF-kB
activation, whereas TRAF2-MEKK1 primarily activates ERK and JNK in the TRANCE and TNFa signaling pathway
Our next interest was the signaling pathways downstream
of TRAF6 and c-Src.As demonstrated in Fig.6, we found that PI3-kinase–Akt pathway and JNK pathway but not ERK and p38 MAP kinase pathways are important for AP-1 activation induced by TRAF6 and c-Src.Further-more, we presented that PI3-kinase inhibitor or Akt DN inhibited TRAF6 and c-Src-induced JNK activation, sug-gesting that Akt activates JNK at downstream of TRAF6 and c-Src.However, a recent study demonstrated that JNK activity could be antagonized by Akt in various cell systems Kim et al.[45] have reported that Akt1 inhibits JNK activation, where Akt1 interaction with JNK interacting protein 1 (JIP1) inhibited JIP1 binding to specific JNK pathway kinases, such as mixed-lineage kinase (MLK) and JNK.One possible interpretation of the difference in the function of Akt on JNK activation we presume is that JNK activation induced by TRAF6 and c-Src in our study may not be mediated by JIP.Actually, Kim et al.indicated that Akt1 specifically inhibited JNK1 activation induced by JIP1 but not JNK activation induced by expression of MKK7 or
by MLK3, thus, indicating that Akt1 specifically inhibits the JIP-mediated JNK activity.On the other hand, Yuan et al [46] have reported on the negative regulation mechanism of JNK that is required for kinase activity of Akt2.In their system, stress-induced Akt2 interacts with and phosphory-lates IKKa, leading to NF-jB activation.Moreover, Akt2-induced IKKa phosphorylation and NF-jB activation is required for inhibition of JNK activation.Thus, they have proposed that Akt2–NF-jB-upregulated XIAP inhibits the activation of MKK7/JNKK2, upstream of JNK [46].In our study, a kinase-dead mutant of c-Src was able to induce NF-jB activation (Fig.2A) but was unable to induce AP-1(Figs 2B and 5A) or Akt activation (data not shown) Therefore, it is presumed that activation of NF-jB by c-Src
is not induced through the activation of Akt.Thus, inhibition of JNK by Akt through the NF-jB pathway does not function in TRAF6 and c-Src-induced AP-1 activation
AP-1 is usually composed of a heterodimer of Jun and Fos proteins.AP-1 is activated by various stimuli such as growth factors, cytokines, neurotransmitters, cell–matrix interactions and bacterial infections as well as by physical and chemical stresses and its function appear complex.AP-1
is thus recognized as an important regulator of cell function and this has been revealed recently by studying mice lacking AP-1 components [36,43,44].AP-1 components can be activated through direct phosphorylation, i.e c-Jun is phosphorylated at its N-terminal region by JNK, which in turn is activated in response to growth factors and cytokines [47,48].An alternative mechanism for c-Jun activation is enhanced transcription of c-Jun mRNA by ERK and p38 MAP kinases [49].In addition, it is also reported that c-Fos protein expression, that is another typical component
of AP-1, is induced by ERK or JNK in growth factor or CD28 signaling pathway, and consequently, AP-1 DNA binding activity as a whole is enhanced [50].As AP-1 activation induced by the overexpression of TRAF6 and/or c-Src was inhibited more strongly by JNK inhibitor than by PD98059 or SB203580 [Fig.6], JNK rather than ERK1/2