From the B Cell Molecular Immunology Section, Laboratory of Immunoregulation, NIAID, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, November 19, 2002, and in revised form, February 11, 2003
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ABSTRACT |
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Tumor necrosis factor (TNF)-induced activation of
apoptosis signal-regulating kinase 1 (ASK1) and
germinal center kinases (GCKs) and the subsequent activation of
stress-activated protein kinases (SAPKs and c-Jun
NH2-terminal kinases) requires TNF
receptor-associated factor 2 (TRAF2). Although the TRAF2 TRAF domain
binds ASK1, GCK, and the highly related kinase GCKR, the RING finger
domain is needed for their activation. Here, we report that TNF
activates GCKR and the SAPK pathway in a manner that depends upon TRAF2 and Ubc13, a member along with Uev1A of a dimeric ubiquitin-conjugating enzyme complex. Interference with Ubc13 function or expression inhibits
both TNF- and TRAF2-mediated GCKR and SAPK activation, but has a
minimal effect on ASK1 activation. TNF signaling leads to TRAF2
polyubiquitination and oligomerization and to the oligomerization, ubiquitination, and activation of GCKR, all of which are sensitive to
the disruption of Ubc13 function. These results indicate that the
assembly of a TRAF2 lysine 63-linked polyubiquitin chain by Ubc13/Uev1A
is required for TNF-mediated GCKR and SAPK activation, but may not be
required for ASK1 activation.
The stress-activated protein kinases/c-Jun
NH2-terminal kinases
(SAPK1/JNKs) and the p38
mitogen activated protein kinases (MAPK) are activated by a variety of
inflammatory stimuli and stresses. Many of the cytokines of the tumor
necrosis factor (TNF) family, TNF in particular, potently activate the
p38 MAPKs and SAPKs. Like the other MAPKs, the SAPKs are regulated as
part of a three-tiered core of protein kinases. At least two
MAPK/extracellular signal-regulated kinase (ERK) kinases (MEKs) lie
upstream of the SAPKs, namely SAPK/ERK kinase1/MAPK kinase 4 (SEK1/MKK4) and MKK7, while multiple protein kinases, including the MEK
kinases (MEKK) and the mixed lineage kinases (MLK), have been
implicated as proximal elements in the core SAPK pathway (reviewed in
Refs. 1 and 2).
The cytoplasmic domains of the TNF receptors (TNF-R) serve as docking
sites for signaling molecules that link activated receptors to
downstream signaling pathways. The TNF-R uses two classes of cytoplasmic adaptor proteins, i.e. death domain (DD)
molecules and TRAFs (TNF-R-associated factors) (reviewed in Ref. 3). The type 1 TNF-R (TNF-R1) recruits the death domain protein TRADD and a
TRAF (TRAF2), a critical step in TNF-induced activation of nuclear
factor Several members of the germinal center kinase (GCK) family, a group of
kinases homologous to the Saccharomyces cerevisiae Ste20p, a
direct upstream activator of the yeast MAP3K Ste11p, are also potent
and selective activators of the SAPK pathway, suggesting that they may
act in an similar fashion as proximal activators of the core SAPK
pathway (8). TNF Additional insights into how the recruitment of TRAF molecules to TNF
family receptors activates downstream signaling pathways arose from
studies of the interleukin-1 receptor, TRAF6, and the I These data suggested that TNF may trigger the Ub modification of TRAF2,
thereby activating intermediaries in the NF- Cell Lines, Plasmids, and Constructs--
The human embryonic
kidney 293 and HeLa cell line was obtained from the American Tissue
Culture Collection (Manassas, VA). The cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. pMT2T-TRAF2, pcDNA3-ASK1, Ubc13 and Ubc13(C87A), and
pEBG GST-SEK1-KR constructs were kindly provided by Dr. U. Siebenlist
(NIAID, National Institutes of Health), Dr. E. Nishida (Kyoto
University), Dr. Zhijian J. Chen (University of Texas), and Dr.
John Kyriakis (Harvard University), respectively. The Uev1A and Ub
cDNAs were obtained by PCR from a cDNA library created from
HeLa cells. The PCR products were inserted into the pCR3.1 vector. The
veracity of the coding sequence was checked by DNA sequencing. The
Ub(K63R) mutant was created using QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA). The HA-GCKR, FLAG-GCKR, and pCR3-TRAF2
(87-501) constructs and the GCKR polyclonal antiserum were described
previously (9, 11). The ASK1 and I In Vitro Kinase Assays--
HEK 293 cells were seeded in 6-cm
dishes, and the following day the cells were transfected with the
appropriate expression vectors using SuperFect (Qiagen, Valencia, CA).
The DNA was incubated with the cells for 4 h in serum-free media.
Subsequently, the media were replaced with media containing 2.5%
serum, and the cells were maintained overnight. The following day, the
cells were serum starved for 2 h and then treated with TNF (100 ng/ml for 15 min). HA-SAPK, FLAG-GCKR, GCKR, and ASK1
immunoprecipitates were subjected to in vitro kinase assays
using myelin basic protein (MBP; Sigma) for GCKR, c-Jun-(1-79)
(Santa Cruz Biotechnology, Santa Cruz, CA) for SAPK, and GST-SEK1-KR
for ASK1 as substrates (9, 16). To check the activation of endogenous
SAPK, an anti-SAPK (pT183/pY185) phospho-specific antibody (BioSource
International, Camarillo, CA) was used to determine pSAPK levels.
Immunoprecipitations, GST Pull Downs, and Immunoblots--
HeLa
cells were seeded on a 10-cm plate and transfected with expression
vectors for HA-GCKR or TRAF2 in the presence or absence of expression
constructs for Ubc13(C87), Ub(K63R), Ubc13 and Ub, or Ubc13(C87A) and
Ub(Lys63). 24 h later, the cells were serum starved
overnight and, in some cases, treated the following day with TNF (150 ng/ml) for 15 min. In some instances, untransfected HeLa or HEK 293 cells were used. Following cell lysis (lysis buffer contained 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton 100, and 10% glycerol). GCKR, HA-GCKR, HA-SAPK, or TRAF2
immunoprecipitates were collected with the appropriate goat anti-Ig
magnetic beads, washed 4-8 times with lysis buffer and, in some
instances, twice with the same buffer containing 500 mM
NaCl. The immunoprecipitates were subjected to in vitro
kinase assays (see above) or fractionated by SDS-PAGE and transferred
to nitrocellulose for immunoblotting with anti-TRAF2, anti-HA, or
anti-Ub antibodies. The GST-TRAF2 construct was transfected into HEK
293 cells as indicated above. The harvested cells were suspended in a
lysis buffer, and GST-TRAF2 was isolated using glutathione-Sepharose
beads (Amersham Biosciences). The beads were washed twice with the
lysis buffer and three times in a similar buffer but with 500 mM NaCl. The collected GST-TRAF2 samples were boiled for 5 min in SDS-sample buffer and size fractionated by SDS-PAGE prior to immunoblotting.
To test whether TNF triggers rapid TRAF2 ubiquitination, we
examined endogenous TRAF2 immunoprecipitates from HeLa cells, which had
been treated or not treated with TNF for 15 min, for the presence of a
ladder of molecules reactive with the TRAF2 antibody, a result
consistent with Ub modification. Following TNF treatment, such a ladder
of molecules appeared that ranged from ~60 to 180 kDa (Fig.
1a). Because TRAF2 shares a
similar molecular mass as the immunoglobulin heavy chain (IgH) used to immunoprecipitate it, unmodified TRAF2 merged with the IgH band. To
verify that Ub accounts for the higher molecular weight TRAF2 molecules
that we had detected, we stripped and re-probed the above blot with a
Ub-specific antibody. A similar ladder of molecules resulted, although
it extended to a higher molecular mass than that observed with the
TRAF2 antibody, perhaps because the multiple Ub molecules needed to
achieve the higher molecular masses can be more readily detected with
the Ub antibody than with the TRAF2 antibody (Fig. 1a,
bottom panel).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B (NF-
B) and SAPKs (4, 5). Genetic and biochemical studies
implicate the MEKK, MEKK1, as an effector in TNF-induced SAPK
activation (6, 7). TRAF2 and MEKK1 co-immunoprecipitate following TNF
treatment, and TRAF2 activates MEKK1 in vivo. However, the
mechanism by which TRAF2 activates MEKK1 remains obscure.
potently activates GCK and GCKR (germinal center
kinase-related) and facilitates their interaction in vivo
with TRAF2 (9-11). Although the activation of GCK and GCKR depends
upon the RING domain of TRAF2, both required the TRAF domain to
efficiently interact with TRAF2. In addition, GCK associates with MEKK1
in vivo, and purified active GCK plus TRAF2 activates MEKK1
in vitro (12). The RING domain of TRAF2 is needed for the
activation of MEKK1, although the kinase domain of GCK is dispensible.
GCK and, by analogy, GCKR may function by promoting the oligomerization
of MEKK1, resulting in MEKK1 autophosphorylation and activation.
B kinase
complex (IKK), an intermediary in NF-
B activation (13, 14). TRAF6
requires the following two factors to activate IKK: (i) a dimeric
ubiquitin (Ub)-conjugating enzyme composed of Ubc13 and Uev1A and (ii)
the TAK1 kinase complex. Ubc13/Uev1A and TRAF6 catalyze the
formation of lysine 63-linked polyubiquitin (polyUb) chains triggering
the activation of the TAK1 kinase complex, which, in turn,
phosphorylates and activates IKK. Ubc13, an E2 family member, forms a
dimer with Uev1A, which is structurally similar to that of an E2 but
lacks a catalytic cysteine residue. Ubc13/Uev1A along with TRAF6, which
functions as an E3 ligase in this reaction, facilitate the synthesis of
Lys63-linked polyUb chains (13). This contrasts with
Lys48-linked polyUb chain formation catalyzed by many other
E2/E3 complexes, a modification that often targets proteins for
degradation (reviewed in Ref.15). The E3 ligase activity of TRAF6
requires an intact TRAF6 RING finger domain, and one of the targets of
the interleukin-1-induced Lys63-linked ubiquitination is
TRAF6 itself (13, 14).
B and the SAPK pathways.
Here we show that TNF triggers rapid Lys63-linked
ubiquitination of TRAF2. Inhibiting this modification blocks
TNF-induced GCKR and SAPK activation. In contrast, it has little effect
on the activation of the MAP3K, ASK1, which has also been implicated as
an upstream activator in the signaling pathway leading from TRAF2 to
SAPK activation (16, 17). In addition, we show that GCKR is likely a
substrate for the E3 ligase activity of TRAF2, as TNF triggers
Lys63-linked ubiquitination of GCKR and GCKR
oligomerization. This may promote MEKK1 oligomerization and activation
of the SAPK-signaling module.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B polyclonal antibodies were
purchased from Santa Cruz Biotechnology. The TRAF2 polyclonal antibody
was kindly provided by Dr. U. Siebenlist. A Ub-specific monoclonal
antibody (Santa Cruz Biotechnology) was used to detect Ub. The HA
antibody is a mouse monoclonal antibody attached to beads (Covance,
Berkley, CA).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
TRAF2 undergoes TNF-induced ubiquitination
that depends upon Ubc13 and Ub(K63R). a, 10-cm plates
of HeLa cells were transfected with constructs that express
Ubc13(C87A), 10 µg (lane 3) and 5 µg (lane
6); Ub(K63R), 10 µg (lane 4) and 5 µg (lane
6); Ubc13, 5 µg (lane 5); Ub, 5 µg (lane
5), or an empty vector, 10 µg (lanes 1 and
2). The cells were treated with TNF for 15 min or not as
indicated. TRAF2 immunoprecipitates were fractionated on SDS-PAGE and
immunoblotted with the immunoprecipitating antibody to detect TRAF2
(top). The IgH and TRAF2 bands merge. The same blot was
stripped and re-probed with a Ub-specific antibody to detect Ub
containing proteins (bottom). b, HeLa cells
transfected with constructs expressing Ub(K63R), 4 µg, or Ub(K48R), 4 µg, were treated with TNF or not. TRAF2 immunoprecipitates were
subjected to Ub immunoblotting. In addition, the cell lysates were
immunoblotted for I B and for GCKR as a loading control.
c, HeLa cells were transfected with constructs expressing
TRAF2, 1 µg, or TRAF2(C34A), 1 µg, in the presence of either 4 µg
of Ub, Ub(K48R), or Ub(K63R) as indicated. Some of cells were also
transfected with constructs expressing Ubc13 or Ubc13(C87A), 4 µg,
together with Uev1A, 1 µg. TRAF2 immunoprecipitates were subjected to
Ub immunoblotting. In addition the cell lysates were immunoblotted for
TRAF2 and HA to detect HA-tagged Ubc13 or Ubc13(C87A).
Although this result indicates that TRAF2 undergoes Ub modification, it
did not address the type of Ub modification. Therefore, to determine
whether Ubc13 and Ub Lys63 participated in the rapid
ubiquitination of TRAF2 observed following TNF treatment, we used
constructs expressing either Ubc13(C87A) or Ub(K63R). These constructs
express proteins that antagonize the activities of endogenous Ubc13 and
Ub, respectively. Ubc13(C87A) no longer functions as an E2 but acts to
interfere with wild type Ubc13 activity. The expression of Ubc13(C87A)
blocked TRAF2-, TRAF6-, and TNF-induced NF-B activation as assessed
by a reporter gene assay without affecting the activation of NF-
B by
NF-
B-inducing kinase (NIK) or Tax (13). Ub(K63R) cannot be used to
create Lys63-linked polyUb and thereby competes with wild
type Ub for the generation of Lys63 but not
Lys48-linked polyUb chains (13). We found that the
expression of high amounts of either Ubc13(C87A) or Ub(K63R)
significantly blocked both the appearance of the slower mobility TRAF2
molecules as well as the appearance of Ub in the TRAF2
immunoprecipitates (Fig. 1a). The origin of the ~100 kDa
band detected with the TRAF2 antibody and not with the Ub antibody is
unknown, although its mobility is that of a TRAF2 dimer (Fig.
1a, lane 3). Surprisingly, the simple
overexpression of Ubc13 and Ub also caused TRAF2 polyubiquitination. In
contrast, the overexpression of Ubc13(C87A) and Ub(K63R) did not (Fig.
1a). As a further specificity control, we compared the effects of expressing Ub(K63R) to that of Ub(K48R), which should inhibit Lys48-linked polyUb chain formation. As before,
Ub(K63R) blocked, whereas Ub(K48R) enhanced the detection of
polyUb-modified endogenous TRAF2 (Fig. 1b). Interestingly,
both mutants interfered with TNF-induced I
B degradation. Presumably,
the expression of Ub(K63R) inhibited TRAF2 signaling, whereas Ub(K48R)
interfered with the targeting of I
B for proteasomal destruction.
Ub(K63R), but not Ub or Ub(K48R) also interfered with the TRAF2
polyubiquitination that occurred following overexpression of Ubc13
(Fig. 1c). Underscoring the importance of the E3 ligase
activity of TRAF2, a mutation in TRAF2 at a site predicted to cripple
its E3 ligase activity (14) resulted in a TRAF2 protein that failed to
undergo Ub modification following Ubc13 overexpression.
Having established that TRAF2 likely undergoes Lys63-linked
polyubiquitination, we tested whether this modification contributes to
the ability of TRAF2 to activate the SAPK pathway and GCKR. We
transfected constructs that express HA-SAPK, FLAG-GCKR, and TRAF2 into
HEK 293 cells, a highly transfectable cell line that requires GCKR for
TNF-induced SAPK activation (9). To assess GCKR and SAPK kinase
activities, we performed in vitro kinase assays with
immunoprecipitated GCKR or SAPK using MBP and c-Jun-(1-79) as
substrates, respectively (Fig.
2a). By intention, we
expressed relatively low amounts of FLAG-GCKR to avoid significant SAPK activation by GCKR alone. As expected, the addition of TRAF2 enhanced both GCKR and SAPK activation. When we co-expressed low amounts of
Ubc13(C87A) and Ub(K63R) together or antisense Ubc13 along with
Ub(K63R), we blocked the in vitro kinase activities of GCKR and SAPK (Fig. 2a). Whereas the expression of low amounts of
Ub(K63R) partially blocked GCKR and SAPK activation, the addition of
either the antisense Ubc13 or Ub13(C87A) further enhanced the
inhibition. In contrast, the expression of the inhibitors had little
effect on GCKR-mediated SAPK activation even when we expressed higher amounts of GCKR (data not shown). We also showed that, although the
expression of Ub(K63R) blocked TRAF2-induced GCKR and SAPK activation,
the expression of Ub(K48R) did not (Fig. 2b).
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Having relied on a transfection system, we checked the importance of Ubc13 in TNF-induced activation of endogenous GCKR and SAPK. We treated HEK 293 cells with TNF or not, immunoprecipitated endogenous GCKR, checked its activity by an in vitro kinase assay, and immunoblotted for pSAPK. As expected, the treatment of HEK 293 cells with TNF increased the activity of endogenous GCKR and elevated the levels of pSAPK. The expression of either Ubc13(C87A) and Ub(K63R) or antisense Ubc13 along with Ub(K63R) nearly abolished it (Fig. 2c; both isoforms of pSAPK were resolved in panel b, whereas a non-gradient gel failed to resolve the two isoforms in panel c). Finally, just as the expression of the RING finger domain deleted a version of TRAF2 (9), the expression of the E3 ligase-crippled TRAF2, TRAF2(C34A), also failed to activate GCKR (Fig. 2d).
Next, we examined the effect of Ubc13(C87A) on TRAF2- and TNF-mediated
ASK1 activation. ASK1 has also been implicated in linking TRAF2 to the
SAPK pathway in TNF signaling (16, 17). We transfected constructs
expressing ASK1 and TRAF2 in the presence or absence of the Ubc13(C87A)
construct into HEK 293 cells or treated the cells with TNF rather than
transfecting the TRAF2 construct. Afterward, we performed an ASK1
in vitro kinase assay using ASK1 immunoprecipitates with the
substrate GST-SEK1-KR. Although the Ubc13(C87A) construct again blocked
TRAF2-mediated GCKR activation, it had a very minor effect on TRAF2- or
TNF-induced ASK1 activation (Fig. 3). We
repeated the experiments but substituted MBP, also an ASK1 substrate,
for GST-SEK1-KR with similar results (data not shown). We had expected that the activations of ASK1 by TRAF2 would require Ubc13; however, these results suggest otherwise. Perhaps TNF triggers the dissociation of thioredoxin, a negative regulator of ASK1 but not GCKR (18, 19), via
a mechanism independent of Ubc13 activity. Alternatively, very low
levels of Ubc13 activity may be sufficient for ASK1 activation.
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It has been suggested that oligomerization of TRAF2 or TRAF6 through
their carboxyl-terminal TRAF domains is needed to activate IKK and the
SAPK pathway by proinflammatory cytokines (6). To determine whether
TRAF2 ubiquitination enhances TRAF2-TRAF2 and TRAF2-GCKR interactions,
we constructed a GST-TRAF2 expression vector. When we transfected HEK
293 cells with this construct, the GST-TRAF2 pull downs contained low
amounts of Ub, whereas similar pull downs prepared from HEK 293 cells
transfected with GST alone lacked Ub (Fig.
4a). The GST-TRAF2 pull downs
also contained low amounts of endogenous TRAF2 and GCKR. Consistent
with our initial results, TNF treatment resulted in a dramatic
dose-dependent increase in the amount of Ub in the
GST-TRAF2 pull downs, but not the GST pull downs. Of note, the amount
of GST-TRAF2 detected in each of the lanes appeared similar, indicating
that only a minority of the GST-TRAF2 became ubiquitinated. The
expression of GST-TRAF2 also potently enhanced the TNF-induced
appearance of pSAPK in HEK 293 cells as compared with those expressing
GST alone. TNF treatment enhanced the recruitment of endogenous TRAF2 and GCKR to GST-TRAF2, and blocking Ubc13 activity strikingly reduced
it. Expression of Ubc13(C87A) also decreased the appearance of
polyUb-modified proteins in the GST-TRAF2 pull downs and the presence
of pSAPK in the TNF-treated HEK 293 cells (Fig. 4a). Because
the GST-TRAF2 pull downs included endogenous TRAF2 and GCKR, the
polyUb-modified proteins detected in the Ub immunoblot may not only
include GST-TRAF2 but also modified endogenous proteins as well.
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To test whether TRAF2 ubiquitination also led to GCKR oligomerization, we co-transfected constructs expressing HA-GCKR and FLAG-GCKR along with TRAF2 or treated the cells with TNF. We found significantly higher amounts of FLAG-GCKR in the HA-GCKR immunoprecipitates following either expression of TRAF2 or TNF treatment, which Ubc13(C87A) expression blocked (Fig. 4b). Thus, the assemblies of stable higher order TRAF2 and GCKR complexes and the optimal recruitment of GCKR to TRAF2 following TNF treatment depends, at least in part, upon Ubc13 and the E3 ligase activity of TRAF2.
The interaction of GCKR with TRAF2 suggested that GCKR might serve as a
TRAF2 substrate. To test that possibility, we transfected HeLa cells
with constructs that expressed either HA-GCKR or FLAG-GCKR in the
presence or absence of constructs expressing TRAF2, Ubc13, and Ub. We
extensively washed HA immunoprecipitates to remove any
co-immunoprecipitating proteins and immunoblotted for the presence of
Ub. From those cells transfected with the construct expressing HA-GCKR,
but not FLAG-GCKR, we observed a smear of molecules between 110 and 200 kDa that reacted with the Ub antibody (Fig.
5a). A similar although less
intense smear appeared when we re-blotted with the HA antibody. These
results indicate that GCKR can undergo Ub modification. To test the
involvement of Ubc13 and Ub(K63R), we transfected HeLa cells with
constructs expressing TRAF2 and HA-GCKR in the presence of constructs
expressing either Ubc13 and Ub, Ubc13(C87A) and Ub, Ubc13 and Ub(K48R),
or Ubc13 and Ub(K63R). We found that the expression of Ubc13(C87A) or
Ub(K63R) inhibited the detection of a polyUb ladder in the HA-GCKR
immunoprecipitates, whereas Ub(K48R) enhanced it (Fig. 5b).
Similarly, the expression of Ubc13(C87A), Ub(K63R), or TRAF2-(87-501)
inhibited the appearance of a polyUb ladder in the HA-GCKR
immunoprecipitates following 15 min of TNF treatment. As expected, TNF
enhanced the presence of pSAPK, and each of the three inhibitors
reduced its induction. Besides these data, a Ub immunoblot of
endogenous GCKR immunoprecipitated from TNF-treated cells also contains
a smear of molecules consistent with Ub-modified GCKR; however, the
GCKR antibody has not been useful for re-blotting
GCKR.2 Finally, we tested
whether the impairment of the E3 ligase activity of TRAF2 would
interfere with the appearance of the polyUb ladder detected in HA-GCKR
immunoprecipitates. In contrast to the overexpression of TRAF2, the
expression of a similar amount of TRAF2(C34A) failed to induce GCKR
polyUb or SAPK activation. Furthermore, the same construct inhibited
TNF-induced SAPK activation and GCKR polyUb.
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How TRAF2 activates GCKR remains unknown, although it does depend upon the E3 ligase activity of TRAF2, which we show promotes or perhaps stabilizes the oligomerization of TRAF2 and GCKR. This, in turn, likely facilitates GCKR trans-autophosphorylation, which may alter the conformation of GCKR, thereby facilitating MEKK1 oligomerization and its activation of subsequent steps in the SAPK pathway as has been recently proposed for GCK (12). Alternatively, TRAF2-mediated ubiquitination of GCKR may have a direct role in kinase activation, resulting in a similar scenario. The identification of the specific lysines in TRAF2 that undergo a Lys63-linked chain modification should assist in discriminating the relative importance of TRAF2 self-ubiquitination versus its modification of other substrates such as GCKR for TNF-mediated GCKR and SAPK activation. Besides functioning as a target and catalyst for Lys63-linked Ub chains, TRAF2 also undergoes tagging with Lys48-linked chains. Signaling through the TNF-RII, where the anti-apoptotic molecules c-IAP1 and c-IAP2 function as E3 ligases, generates polyUb-modified TRAF2 (20). In addition, TRAF2 may also use a classical E2 for self-ubiquitination and proteosomal dependent degradation (21). Thus, TRAF2 may undergo either Lys48- or Lys63-linked Ub modification, each with a far different functional consequence. Supporting that concept, the overexpression c-IAP1 significantly inhibited TRAF2-mediated GCKR activation.2
In this report we demonstrate a requirement for Ubc13 in TNF-mediated
activation of GCKR and SAPK and for the optimal recruitment of GCKR to
TRAF2. Ubc13/UevA1 along with TRAF2 catalyze the synthesis of
Lys63 polyUb chains that modify TRAF2 and likely GCKR. In
the absence of these modifications, TNF treatment fails to activate the
SAPK pathway due to a failure to recruit and/or activate upstream
kinases that lead to pathway activation. Polyubiquitination through
Lys63-linked ubiquitin has emerged as a general
modification used to activate upstream kinases in the SAPK and NFB pathways.
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ACKNOWLEDGEMENTS |
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We thank Ms. Mary Rust for editorial assistance and Dr. Anthony Fauci for continued support
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: B Cell Immunology
Section, Laboratory of Immunoregulation, NIAID, National Institutes of
Health, Bldg. 10, Rm. 11B-08, 10 Center Dr., MSC 1876, Bethesda, MD
20892-1876. E-mail: Jkehrl@niaid.nih.gov.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M211796200
2 C.-S. Chi, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
SAPK, stress-activated protein kinase;
MAPK, mitogen-activated protein
kinase;
ERK, extracellular signal-regulated kinase;
MEK, MAPK/ERK
kinase;
MEKK, MEK kinase;
SEK, SAPK/ERK kinase;
TNF, tumor necrosis
factor;
TNF-R, TNF receptor;
TRAF2, TNF-R-associated factor 2;
DD, death domain;
NF-B, nuclear factor
B;
GCK, germinal center
kinase;
GCKR, GCK-related (protein kinase);
IKK, I
B kinase complex;
ASK1, apoptosis signal-regulating kinase 1;
IgH, immunoglobulin heavy
chain;
Ub, ubiquitin;
polyUb, polyubiquitin;
HA, hemagglutinin;
MBP, myelin basic protein;
GST, glutathione S-transferase.
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