By
* Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku,
Tokyo 162, Japan; Department of Oncology, The Institute of Medical Science, The University of
Tokyo, 4-6-1, Shiroganedai, Minato-ku, Tokyo 108, Japan; § Department of Immunology, School of
Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113, Japan;
CREST (Core
Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST),
and ¶ Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai,
Suita-shi, Osaka-fu 565, Japan
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Abstract |
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CD40 activates nuclear factor kappa B (NFB) and the mitogen-activated protein kinase
(MAPK) subfamily, including extracellular signal-regulated kinase (ERK). The CD40 cytoplasmic tail interacts with tumor necrosis factor receptor-associated factor (TRAF)2, TRAF3,
TRAF5, and TRAF6. These TRAF proteins, with the exception of TRAF3, are required for
NF
B activation. Here we report that transient expression of TRAF6 stimulated both ERK
and NF
B activity in the 293 cell line. Coexpression of the dominant-negative H-Ras did not
affect TRAF6-mediated ERK activity, suggesting that TRAF6 may activate ERK along a Ras-independent pathway. The deletion mutant of TRAF6 lacking the NH2-terminal domain
acted as a dominant-negative mutant to suppress ERK activation by full-length CD40 and suppress prominently ERK activation by a deletion mutant of CD40 only containing the binding
site for TRAF6 in the cytoplasmic tail (CD40
246). Transient expression of the dominant-negative H-Ras significantly suppressed ERK activation by full-length CD40, but marginally suppressed ERK activation by CD40
246, compatible with the possibility that TRAF6 is a
major transducer of ERK activation by CD40
246, whose activity is mediated by a Ras-independent pathway. These results suggest that CD40 activates ERK by both a Ras-dependent pathway and a Ras-independent pathway in which TRAF6 could be involved.
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Introduction |
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CD40 is a cell-surface glycoprotein on B lymphocytes,
dendritic cells, follicular dendritic cells, and thymic
epithelial cells (1), and a member of the TNF- receptor
superfamily that includes 55- and 75-kD TNF receptors
(TNFR1 and TNFR2, respectively), CD30 receptor, low-affinity nerve growth factor receptor, lymphotoxin
receptor (LT
R), and Fas antigen (1).
The cytoplasmic domain of the TNFR superfamily
members lacks sequences indicative of catalytic activity, but
is associated with a signal transducer, TNFR-associated factor (TRAF; reference 2). The cytoplasmic domain of human CD40 consists of 62 amino acids at positions 196-257
and is associated with TRAF2, TRAF3, TRAF5, and
TRAF6 (3) and Janus kinase (Jak)3 (10); the membrane proximal region of the cytoplasmic tail of CD40 contains a
proline-rich region at positions 202-209 that is crucial for
Jak3 binding (10). TRAF6 binds to the NH2-terminal cytoplasmic tail of CD40 at positions 210-225, although the
possibility can not be excluded that full association of
TRAF6 with CD40 may also require the COOH-terminal part at positions 226-249 (9). TRAF2, TRAF3, and TRAF5
bind to the COOH-terminal CD40 cytoplasmic domain at
positions 226-249 (9), containing a minimum element,
designated TIMct, responsible for TRAF2 and TRAF3
binding and signal transduction mediating nuclear factor
kappa B (NFB) activation (7).
Stimulation of CD40 results in activation of protein tyrosine kinases (PTKs), NFB, the mitogen-activated protein kinase (MAPK), and Jak3/signal transducers and activators of transcription (STAT)3 (10), and it mediates
critical biological effects in B cell growth, survival, and differentiation (19). It is known that TRAF2 and TRAF5
play a role in NF
B activation in signaling through CD40, as
well as TNFR1, TNFR2, CD30, and lymphotoxin
receptor (6, 28). TRAF6 participates in NF
B activation signaled by CD40 and IL-1 receptor (9, 33). The
TRAF family is characterized by a homologous COOH-terminal TRAF-COOH (TRAF-C) domain, an
-helical TRAF-NH2 (TRAF-N) domain, and an NH2-terminal
RING finger with the exception of TRAF1 (2, 8, 9, 30,
33). The effector function of TRAF2 and TRAF5 toward
NF
B activation is mediated by its NH2-terminal RING
finger domain (6, 8, 30), whereas that of TRAF6 is mediated by the RING finger and zinc fingers (9, 33).
It has been reported that TRAF2 stimulates c-jun NH2-terminal kinase (JNK) activity in TNFR1 signaling (34), leading to the idea that TRAF2 may also play a role in JNK activation by CD40 (15, 16). However, the signaling pathway coupling CD40 to extracellular signal-regulated kinase (ERK) activation has remained unknown. To investigate which TRAF proteins might participate in ERK activation, we have performed transient transfection experiments in the human embryonic kidney 293 cell line. In the present study, we demonstrate that TRAF6 plays a role as a signal transducer in ERK activation by CD40, probably along a Ras-independent pathway.
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Materials and Methods |
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Cell Culture.
Human embryonic kidney cell line 293 was maintained in DME supplemented with 10% FCS, 200 mM L-glutamine, and penicillin/streptomycin.Plasmid Construction.
TRAF2, TRAF3, and human CD40 cDNA were obtained by the reverse transcription PCR (RT-PCR) by use of messenger RNA purified from B cell lymphomas, WEHI231 and Raji, with primers flanking the entire coding region, and then cloned into pMIKHygB, a gift from Dr. K. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan). Deletion mutants of TRAF2 that lack a RING finger motif (amino acids [aa] 87-501) and the TRAF-C domain (aa 352-501) were constructed by PCR. Isolation of TRAF5 and TRAF6 cDNAs and construction of the expression vectors coding full-length TRAF5, full-length TRAF6, and the TRAF-C of TRAF6 were reported previously (9, 30). A deletion mutant of human CD40, designated CD40In Vitro MAPK Assay.
An in vitro kinase assay for endogenous ERK activity was performed as previously described (17). For estimating exogenous ERK2 kinase activity, 293 cells were cotransfected with 1 µg of GST-ERK2 and various amounts of plasmid constructs by the calcium phosphate coprecipitation method. The total amount of DNA was adjusted with empty vector at 10 µg. When 293 cells were cotransfected with 1 µg of CD40 cDNA, the cells were stimulated with anti-CD40 mAb (2 µg/ml) 24 h after transfection. In some experiments, cells were analyzed for the expression of CD40 on the surface by FACScan® (Becton Dickinson, Mountain View, CA) with FITC-conjugated anti-human CD40 mAb (5C3; PharMingen, San Diego, CA). The cell lysates were incubated with glutathione-coupled Sepharose 4B (Pharmacia, Uppsala, Sweden) at 4°C for 1 h. The Sepharose beads were pelleted down by centrifugation and washed twice with lysis buffer and with Tris-buffered saline (10 mM Tris-HCl, pH 7.2, 100 mM NaCl) supplemented with 5 mM benzamidine and 1 mM Na3VO4. The GST-ERK2 immobilized on Sepharose was directly subjected to a kinase reaction as previously described (17).Luciferase Assay.
Cells were cotransfected with either full-length CD40 or CD40 ![]() |
Results and Discussion |
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Overexpression of TRAF2, TRAF5, and TRAF6
results in its aggregation and causes activation of the NFB
signaling pathway, which could be similar to that induced
by ligand-triggered receptor aggregation (6, 8, 9, 30, 33).
Therefore, to investigate which TRAF proteins might participate in ERK activation, we cotransfected human embryonic kidney 293 cells with various TRAF expression vectors and an GST-ERK2 fusion construct. After transfection, the GST-ERK2 fusion protein was precipitated by use
of glutatione Sepharose 4B from the cell lysate, and the
ERK activity was analyzed by an in vitro kinase assay, with
myelin basic protein (MBP) used as an exogenous substrate.
As shown in Fig. 1 A, transient expression of full-length
TRAF6, but not TRAF2, TRAF3, or TRAF5, stimulated exogenous ERK activity approximately fivefold above the
vector control, suggesting that transient expression of
TRAF6 stimulates ERK activity in the 293 cell line.
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ERK is known to phosphorylate target protein within the
cytosol (42), whereas, under certain conditions, ERK translocates to the nucleus to phosphorylate the nuclear substrate
(41, 43). To investigate the effect of various TRAF proteins on ERK activity in the nucleus, we used an ERK-
dependent activator and reporter system, established by
Davis and coworkers (40, 41, and see Materials and Methods).
Human 293 cells were transiently transfected with various
TRAF expression vectors, together with an ERK-dependent activator, a fusion construct of the GAL4 DNA-binding domain and the nuclear localization signal/NH2-terminal transactivation domain of c-Myc, and a luciferase reporter plasmid containing GAL4 binding sites adjacent to a thymidine
kinase promoter, as an ERK-dependent reporter (40, 41). In
addition, to confirm the activity of TRAF proteins, we
cotransfected 293 cells with various TRAF expression vectors and the NFB-dependent luciferase reporter.
As shown in Fig. 1 B a, transient expression of full-length
TRAF6 triggered activation of the cotransfected ERK-dependent reporter in a dose-dependent manner to a maximum of approximately fourfold above the vector control,
whereas a deletion of the RING finger and zinc fingers of
TRAF6 (TRAF-C) abolished the activity of ERK activation.
Consistent with previous reports (9, 33), transient expression of full-length TRAF6, but not TRAF-C of TRAF6, caused NFB activation in the 293 cell line (Fig. 1 B b),
suggesting that TRAF6 stimulates both ERK and NF
B
activity through its NH2-terminal region containing the
RING finger and zinc fingers. Parallel experiments showed
that transient expression of full-length TRAF2 and TRAF5 had no effect on ERK activity, whereas it stimulated NF
B
activity efficiently (Fig. 1 C; 6, 8, 30-32). In this context,
previous reports also ruled out the role of TRAF2 in ERK
activation (34, 36). Transient expression of TRAF3 stimulated neither ERK nor NF
B activity, in agreement with
previous reports (7, 9, 34). Taken together, these results
suggest that TRAF6, but not TRAF2, TRAF3, or TRAF5, is
a transducer of both ERK and NF
B activation.
The Ras-dependent protein kinase cascade leading from various receptors to ERK is dependent on the protein kinase Raf-1, which activates a dual-specificity kinase, MEK (42). To investigate the role of Ras, Raf-1, and MEK1 in TRAF6-mediated ERK activation, we cotransfected 293 cells with a full-length TRAF6 and ERK-dependent activator and reporter, or GST-ERK, together with a dominant-negative mutant of H-Ras (N17Ras; 38), c-Raf-1, which contains only the NH2-terminal regulatory domain (DNRaf; 37), or MEK1, in which Asp208 was replaced with Ala (MEK1DN; 39). Some experiments were performed in parallel with the experiments depicted in Fig. 4. As shown in Fig. 2, coexpression of N17Ras had no effect on the activity of the cotransfected ERK-dependent reporter (Fig. 2 A) or on the exogenous ERK kinase activity (Fig. 2 B), which were increased five- to sixfold above the vector control by transient expression of TRAF6. The results suggest that TRAF6 may stimulate ERK activity along a Ras-independent pathway.
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The dominant-negative DNRaf and MEK1DN suppressed TRAF6-mediated ERK activity in the 293 cell line, but not entirely (Fig. 2, A and B). Although ERK activation is essentially induced by growth factors via a Ras-mediated pathway (42), the result could be interpreted as evidence that the diversified signal cascades regulate ERK activity, sometimes in a cell-type-specific manner, via c-Raf-1 and other possible MAPK kinase kinase in a Ras-independent fashion (46).
TRAF6 Is Involved in ERK Activation in CD40 Signaling.The truncated mutant of TRAF proteins that lack the
RING finger or the RING finger and zinc fingers acts as a
transdominant-negative mutant to suppress NFB activation in signaling through TNFR1, TNFR2, IL-1R, CD30,
and CD40 receptor (6, 8, 9, 28, 30, 31, 33), in which the
dominant-negative mutant may act specifically by replacing
the corresponding endogenous wild-type TRAF protein.
To investigate whether TRAF6 could be involved in
ERK activation by CD40, we cotransfected 293 cells with
the TRAF-C of TRAF6 and ERK-dependent activator
and reporter, or GST-ERK, together with full-length CD40
cDNA. Because a previous report suggested that the NH2-terminal CD40 cytoplasmic tail at positions 210-225 could
be essential for TRAF6 binding and could function for
NFB activation (9), the involvement of TRAF6 in ERK
activation by CD40 was also investigated by transient transfection of the TRAF-C of TRAF6 and ERK-dependent
activator and reporter, or GST-ERK, into 293 cells coexpressing a deletion mutant of CD40, CD40
246 (9), that
removes the COOH-terminal cytoplasmic tail at positions 226-257. Previous results showed that TRAF6 interacts
with the CD40
246 construct in vivo (9).
As shown in Fig. 3, A and B, cross-linking of full-length
CD40 and CD40246 caused ERK activation approximately four- to sixfold above the level of unstimulated
cells, comparable to the magnitude of the endogenous
ERK activity that was transiently increased in 293 transfectants which constitutively expressed CD40 after stimulation
with anti-CD40 mAb (data not shown). As shown in Fig. 3, A (a) and B both ERK-dependent reporter activity (Fig.
3 A) and the exogenous ERK kinase activity (Fig. 3 B) mediated by full-length CD40 were suppressed by coexpression of TRAF-C of TRAF6 in a dose-dependent manner
to a maximum of 30-35% of inhibition, compatible with
the notion that TRAF6-dependent and -independent pathways could be involved in ERK activation by CD40 (see
below). Coexpression of the TRAF-C of TRAF6 caused
prominent suppression of ERK activity mediated by cross-linking of CD40
246 in a dose-dependent manner, suggesting a major role of TRAF6 in ERK activation by
CD40
246. It is not likely that the transdominant-negative
mutant of TRAF6 nonspecifically diminished the activity
of the ERK-dependent activator and reporter, because activation of the ERK-dependent reporter mediated by coexpression of catalytically active MEK1 (39) in the 293 cell
line was not prevented by coexpression of TRAF-C of
TRAF6 (data not shown).
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In control experiments, transient expression of the
TRAF-C of TRAF6 caused prominent suppression of the
activity of cotransfected NFB-dependent reporter mediated by CD40
246 in the 293 cell line (Fig. 3 C b), consistent with previous reports (9). In contrast to ERK activation, the activation of NF
B activity by full-length CD40
was greater than that by CD40
246, and ~50% of the activity was suppressed by coexpression of the TRAF-C of
TRAF6 (Fig. 3 C a). Because of a similar frequency and the
level of expression of full-length CD40 and CD40
246 in
the 293 cell line in transient transfection assays (data not
shown), the results seem compatible with the notion that
distinct regions of the CD40 cytoplasmic domain could be
required for full activation of NF
B in the 293 cell line.
To investigate the role of Ras, Raf-1, and MEK in ERK
activation by CD40, we cotransfected 293 cells with the
ERK-dependent activator and reporter, or NFB-dependent reporter as a control, and with N17Ras, DNRaf, or
MEK1DN, together with either full-length CD40 or
CD40
246. The experiments were performed in parallel
with some of the experiments depicted in Fig. 2 A. Cotransfection of N17Ras, DNRaf, and MEK1DN caused ~70%
inhibition of ERK activity mediated by cross-linking of full-length CD40 (Fig. 4 A a). In contrast, coexpression of N17Ras and MEK1DN did not affect NF
B activation by
both full-length CD40 and CD40
246, whereas DNRaf
marginally suppressed NF
B activation (Fig. 4 B). In conjunction with the previous observation indicating activation of the Ras-mediated pathway by CD40 stimulation in
B cells (52), the results support the possibility that the Ras-Raf-MEK-mediated pathway could be involved in ERK
activation, but not NF
B, by cross-linking of CD40. Transient expression of N17Ras resulted in ~30% inhibition of
ERK activity mediated by CD40
246, compatible with
the notion that TRAF6 is a major transducer of ERK activation by CD40
246, whose activity is mediated by a Ras-independent pathway.
Taken together, these results support the possibility that
CD40 may activate ERK via Ras-dependent and -independent pathways in which TRAF6 could be involved. The
inhibition by a dominant-negative mutant of MEK1 in ERK
activity by full-length CD40 was more prominent than in
ERK activity mediated by TRAF6 and by CD40246, in
which TRAF6 may play a major role. Because the MEK
isoforms MEK1 and MEK2 are an ERK-specific kinase (53),
these results might imply that preferential usage of MEK
isoforms would differ in TRAF6-dependent and -independent pathways in CD40 signaling. In this context, it has
been suggested that the signaling pathways leading to activation of MEK isoforms appear to differ, and that each
MEK isoform could be preferentially used in a particular
external stimulus in a certain type of cell (54). Further
study is needed to clarify the signaling pathway coupling
TRAF6 to ERK activation.
Given that full ERK activity is stimulated through
TRAF6-dependent and -independent pathways in CD40
signaling, a similar extent of ERK activation by full-length
CD40 and CD40246 in the 293 cell line led to the idea
that ERK activity could be regulated in full-length CD40
signaling in 293 cells, probably via activation of MAPK
phosphatase or downregulation of MEK activity (57). In contrast to ERK activation in murine splenic resting B
cells (17, 18), it was reported that CD40 stimulation did
not cause ERK activation in human tonsil B cells (15, 16).
Whether ERK activation is regulated in CD40 signaling,
depending on the maturation stage of B cells, would be
worth analyzing.
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Footnotes |
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Address correspondence to Dr. T. Takemori, Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-Ku, Tokyo 162, Japan. Phone: 81-3-5285-1156; Fax: 81-3-5285-1156; E-mail: ttoshi{at}nih.go.jp
Received for publication 12 September 1997.
1 Abbreviations used in this paper:We are grateful to Drs. G.M. Cooper (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA), K. Maruyama (Tokyo Medical and Dental University, Tokyo, Japan), and K. Takishima (National Defense Medical College, Saitama, Japan) for reagents. We are also grateful to Drs. Tsunetsugu-Yokota and Z. Matsuda (NIID, Tokyo, Japan) for helpful discussions and reading of the manuscript, and to Mr. H. Shimizu, Ms. M. Takizawa, and K. Ishii for technical help.
This work was supported by a grant from the Agency of Technology and Science of the Japanese government and partly from the Ministry of Education and Science and Japanese Human Sciences Foundation to T. Takemori.
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