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* National Research Laboratory for Bone Metabolism and Research Center for Proteineous Materials, Chosun University School of Dentistry, Kwangju;
Immunomodulation Research Center, Ulsan;
Yonsei University College of Science, Seoul; and
Department of Signal Transduction, Division of Molecular Life Science, Pohang University of Science and Technology, Pohang, Korea
Correspondence: Hong-Hee Kim, Ph.D., Chosun University School of Dentistry, 375 Seosuk-Dong, Dong-Ku, Kwangju 501-759, Korea. E-mail: hhkim{at}mail.chosun.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: tumor necrosis factor receptor-associated factor • apoptosis • tumor necrosis factor receptor superfamily
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B
(NF-B) and JNK, perhaps through association with NIK and MEKK1,
kinases that can phosphorylate IKKs and JNK kinases
[3
4
5
]. Although the mechanism by which TRAFs activate
NIK and MEKK1 is not clear, oligomerization of TRAF2 and TRAF6 has been
demonstrated to be sufficient to induce the activation of NF-B and
JNK [6
].
TRAF3 was initially identified as an adaptor molecule that binds CD40
and LMP-1 [7
, 8
], but later found to also
associate with other TNFR family proteins CD27, CD30, 4-1BB, Ox40,
LT-ßR, ATAR, AITR, and RANK [2
]. In in
vitro culture systems, TRAF3 has been demonstrated to play diverse
roles for different receptor functions. An amino-terminal deletion
mutant of TRAF3 that displaced endogenous TRAF3 from LT-ßR was shown
to inhibit the LT-ß-induced cell death, suggesting a pro-apoptotic
role of TRAF3 in LT-ß signaling [9
, 10
].
TRAF3 blocks the activation of NF-B induced by CD40 and TNFR2
overexpression [11
]. TRAF3 negatively regulates the
CD40-stimulated antibody secretion from a mouse B cell line, however,
whether the inhibition of NF-B mediates this response is not clear
[12
]. The in vivo function of TRAF3 has been
revealed by a gene knockout study [13
].
TRAF3-/- mice displayed defects in postnatal development
that correlated with low glucose, high corticosterone, and white cell
depletion. TRAF3-/- mice also showed defect in
antigen-induced T cell proliferation and reduction of B cell
precursors, suggesting that TRAF3 plays a role in activation, growth,
and/or survival of lymphocytes.
In addition to TRAF3, other TRAF family members have been implicated in the regulation of cell death. TRAF2, TRAF4, and TRAF6 were reported to modulate apoptosis induced by the neutrophin receptor p75NTR [14 ]. Biochemical connections between TRAF proteins and apoptosis may be suggested by the physical associations of TRAFs with caspase-8 and its homologs, caspase-10 and MRIT [15 ]. However, effects of these interactions on the function of either TRAF proteins or that of caspases are not known. In this study, we found that TRAF proteins, particularly TRAF3, may be subject to proteolysis during cell death. TRAF3 was cleaved by FasL- and anti-CD3-treatment of Jurkat-T cells, and this cleavage was blocked by caspase inhibitors. Furthermore, the amino-terminal part of TRAF3 seemed to have a different intracellular localization from full-length TRAF3 with a higher nuclear concentration and preferential distribution to detergent-insoluble fractions after cell lysis. These findings suggest that TRAF3 may be regulated by caspases during apoptosis of T cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin
(no. CLT9002) was from Cedarlane (Hornby, Ontario). Anti-TRAF3
antiserum was generated in rabbits through the use of the GST fusion
protein of full-length TRAF3 as the immunogen and purified with a
protein A column. As the source of FasL and anti-CD3, culture
supernatants of the CHO-K1 stable transfectant [16
] and
OKT3 hybridoma cells were used, respectively.
Expression plasmids
Flag-tagged full-length human TRAF1, -2, -3, and -6 and murine
TRAF5 plasmids were generously provided by Dr. H. Y. Song (Lilly
Co. Center, Indianapolis, IN). TRAF3 deletion and point mutants were
generated by polymerase chain reaction (PCR) and subcloned into the
same vector as the wild-type TRAF3. The GFP-TRAF3 constructs were made
by creating EcoRI and SalI sites by PCR and subcloning into pEGFP-C2
vector (Clontech, Palo Alto, CA). The sequences of PCR products were
verified by automated DNA sequencing (Perkin Elmer, ABI 310).
Transfection, immunoprecipitation, and Western blotting
293-T cells were transfected by the calcium phosphate method and
COS1 cells by the DEAE-dextran method. HeLa and 293-T cells were
transfected using Superfect (Qiagen, Hilden, Germany) as described
[17
]. HeLa cells were also sometimes transfected using
Lipofectamine (GIBCO-BRL, Grand Island, NY) and Fugene (Boehringer
Mannheim, Mannheim, Germany) following manufacturers’ instructions.
Jurkat-T cells were electroporated as described [18
].
Briefly 2 x 107 cells suspended in 400 µL RPMI 1640
medium were mixed with 20 µg DNA and pulsed at 260 V, 900 µF, and
481 Ohms (Easyject Plus, EquiBio, Kent, UK). Twenty to thirty-six hours
after transfection, cells were harvested and lysed directly in sodium
dodecyl sulfate (SDS) sample buffer. Alternatively, cells were lysed in
a lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 0.5% Triton X-100, 1 mM
EDTA, 2 mM sodium orthovanadate, 50 mM sodium fluoride, 2 µg/mL
pepstatin A, 10 µg/mL aprotinin, 10 µg/mL leupeptin, pH 7.4) and
centrifuged for 10 min at 10,000 g. For Fas or CD3
stimulation, 24 h after transfection cells were treated with a
1/10 volume of the FasL or a 1/2 volume of OKT3 culture supernatants.
The caspase inhibitors were added 1 h before the FasL or OKT3
treatment. Immunoprecipitation and Western blotting were performed as
previously described [17
].
Flow cytometry
The flow cytometric analysis of apoptosis was performed as
previously described [19
]. Briefly, 1 x
106 cells washed with PBS/5 mM EDTA were fixed in 50%
ethanol for 30 min at 20°C. Cells were then treated with RNase A (40
µg/mL final concentration) for 30 min at 20°C and mixed with an
equal volume of propidium iodide solution (50 µg/mL final
concentration). FACS analyses were carried out in FL2 linear mode and
percentages of cells with sub Go/G1 DNA content were considered
apoptotic.
In vitro cleavage of TRAFs by recombinant caspase 3
Immunoprecipitated beads were resuspended in the caspase
reaction buffer (20 mM HEPES, pH 7.3, 1 mM EDTA, 1 mM EGTA, 4 mM
dithiothreitol), mixed with recombinant caspase 3 (200 nM final
concentration), and incubated for 2 h at 37°C. Recombinant
caspase 3 was prepared as follows. A fragment corresponding to amino
acids 66–277 of human caspase 3 cDNA was cloned into the pRSET vector
using XbaI(NheI)/BamHI sites. After transformation, BL21 cells were
induced by addition of IPTG (1 mM) for 4 h at 30°C. Cells were
harvested and washed twice with phosphate-buffed saline (PBS) followed
by extraction with a lysis buffer (20 mM Tris-HCl, pH 7.4, 0.4 M NaCl,
10% glycerol, 1 mM dithiothreitol, 1% Triton X-100, 50 mM imidazole).
Cell extracts were applied to Ni+-NTA column (Amersham
Pharmacia, Piscataway, NJ) and the column was washed with 100 column
volumes of the lysis buffer. Bound proteins were eluted on a linear
gradient of imidazole (0.05–1 M). An aliquot of each fraction was
analyzed by Western blotting with anti-caspase 3. Fractions containing
caspase 3 were pooled, dialyzed against buffer A (20 mM Tris-HCl, pH
8.0, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol), and then applied to a
Mono Q FPLC column (Amersham Pharmacia). Bound proteins were eluted on
a linear gradient of NaCl (0–1 M). The caspase 3 peak was confirmed by
Western blotting. The purity of preparations was usually 
90%. The
catalytic activity of each preparation was verified using specific
caspase 3 substrate peptides.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40-kDa band in addition to the 60-kDa full-length
protein (data not shown). Generation of cleaved fragments was specific
for TRAF3 because cleavage of TRAF1, -2, -5, and -6 that were expressed
from the same expression vector was not detected under the same
experimental conditions. To exclude the possibility that the selective
TRAF3 cleavage was an artifact resulting from impurity of the DNA
preparation, DNAs prepared by different methods were tested, and
similar results were obtained. In addition, different transfection
techniques employing various kinds of liposomes, calcium phosphate, and
DEAE-dextran were applied and no difference was observed. The selective
TRAF3 cleavage was also observed in 293T and COS1 cells. In HeLa cells,
in addition to TRAF3 cleavage, a cleaved fragment of TRAF1 (29 kDa)
was erratically detected. These observations made us reason that the
TRAF3 protein might be subjected to an action of specific proteolytic
enzymes such as caspases. To explore the possibility of caspase-mediated cleavage of TRAF proteins, we immunoprecipitated each Flag-tagged TRAF protein from the lysate of cells after transient transfection and cleaved the precipitated proteins with purified recombinant caspase 3. As shown in Figure 1 , treatment with caspase 3 generated various fragments (indicated by an arrowhead) of TRAF 1 (lane 3), TRAF2 (lane 6), TRAF3 (lane 12), and TRAF5 (lane 15), whereas the same treatment did not result in any cleavage of TRAF6 (lane 9). The TRAF3 fragment generated by the in vitro incubation of caspase 3 (indicated by an arrowhead in lane 12) exhibited the size close to that observed in 293-EBNA cells. In COS1 cells, the TRAF3 fragment was sometimes resolved as a doublet (indicated by the double arrowhead in lane 11). The upper band disappeared upon caspase 3 incubation (lane 12), indicating that the larger fragment of TRAF3 could be further cleaved by caspase3. In line with this, treatment of COS1 cells with caspase 3 inhibitor Ac-DEVD-CMK resulted in a significant reduction of the lower band (data not shown). These results suggest that some TRAF family proteins could be a target of caspases and TRAF3 might be more prone to caspase-mediated cleavage than other TRAF molecules in the cell.
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40-kDa
amino-terminal fragment of TRAF3 (lanes 11 and 12). However, no
cleavage of TRAF2, -5, and -6 was detected under the same experimental
conditions (Fig. 2A) . The 40-kDa fragment of TRAF3 was sometimes
present in untreated cells (see Fig. 4A
, right panel, lane 1), perhaps
as a result of autologous interaction between Fas and FasL present in
these cells. An 29-kDa band of TRAF1 fragment was generated by
either FasL or OKT3 treatment in a few cases (data not shown), whereas
the fragmentation of TRAF3 was detected consistently. This occasional
observation of TRAF1 cleavage in Jurkat-T cells together with that in
HeLa cells (see above) suggests that TRAF1 cleavage may not be fully
induced by FasL or OKT3 treatment and may be influenced by cellular
states. Comparable loading of total cellular proteins was verified by
Western blotting of a cytoskeletal component, -tubulin (Fig. 2A
,
bottom panel). No cleaved fragment of -tubulin was generated by
FasL- or OKT3-treatment of these cells.
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40-kDa
amino-terminal fragment accompanied generation of the corresponding
carboxy-terminal fragment (25 kDa) of TRAF3, we constructed a
plasmid for TRAF3 tagged with T7 at the carboxy terminus (TRAF3-T7).
FasL- or OKT3 treatment did not result in any detectable
carboxy-terminal fragments in Western blotting analyses with anti-T7
(Fig. 2D)
. One speculation for the absence of carboxy-terminal fragment
detection might be that the carboxy-terminal fragments once generated
become further degraded by machinery such as the proteasome. The
reduction of the full-length TRAF3 in FasL-treated cells (Fig. 2D
, lane
3) may reflect this notion. These results demonstrated that certain
apoptotic signals such as FasL and anti-CD3 can result in an activation
of cellular process that led to cleavage of the TRAF3 protein.
Intracellular location of the cleaved amino-terminal part of TRAF3
We next examined whether the intracellular location of TRAF3 can
change after cleavage by apoptotic stimuli. Jurkat-T cells stimulated
with FasL or OKT3 were lysed in a buffer containing nonionic detergent,
and both the soluble and insoluble fractions were analyzed by Western
blotting. FasL treatment of Jurkat cells led to reduction of the
full-length TRAF3 in the detergent-soluble fraction (Fig. 3A
, lane 4) and appearance of 40-kDa fragment in the pellet
fraction (lane 9). OKT3 treatment also resulted in detection of the
cleaved fragment of TRAF3 in the detergent-insoluble fraction (lane
10). This result may be interpreted as translocation of the TRAF3
amino-terminal fragment after cleavage. Alternatively, TRAF3 proteins
in the particulate fraction might be somehow more susceptible to the
cleavage reaction. This proposition, however, might be less likely in
the case of FasL stimulation, considering little change of the
full-length TRAF3 band in the pellet fraction (lane 9) in contrast to a
significant reduction of that in the supernatant fraction (lane 4).
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Involvement of caspases in FasL- and OKT3-induced TRAF3 cleavage
The finding that TRAF3 could be cleaved by in vitro
incubation with caspase 3 (Fig. 1)
and in Jurkat-T cells challenged
with FasL or OKT3 (Fig. 2)
prompted us to test whether caspases are
involved in the FasL- or OKT3-stimulated fragmentation of TRAF3.
Treatment of Jurkat-T cells with either a broad-spectrum caspase
inhibitor Z-VAD-FMK or a caspase 3-selective inhibitor Z-DEVD-FMK
abrogated the FasL and OKT3-induced TRAF3 cleavage (Fig. 4A
). The treatment of caspase inhibitors also reduced death of these
cells (data not shown). These results indicate that TRAF3 may be
proteolyzed by caspases or caspase-dependent proteases during FasL- or
anti-CD3-induced apoptosis. Disappearance of the cleaved fragment of
TRAF3 in the pellet fraction coincided with an increase in the
intensity of the full-length TRAF3 band (left panel, lanes 3, 4, 6, 7).
This observation may further support the notion (see above) that after
cleavage the amino-terminal part of TRAF3 translocates into
detergent-insoluble compartments such as the nucleus.
We next sought to determine the cleavage site of TRAF3. A study using a
positional scanning synthetic combinatorial library revealed that most
caspases prefer glutamate at the -2 position from the aspartate residue
at the cleavage site [23
], although sequences not
obeying this rule and still cleaved by caspases have been reported
[24
, 25
]. The TRAF3 protein contains two
sequences (KELD332 and EEAD347) of the XEXD
motif and an additional less preferred but possible caspase target site
(ESVD367) that would produce an 40-kDa amino-terminal
fragment. However, sequences with a lysine residue at the -3 position
were shown to have almost no probability of cleavage in the
combinatorial study [23
]. Thus, we excluded the
KELD332 sequence and focused on the other two sites.
Aspartate-to-alanine mutations were introduced separately into either
the EEAD347 or ESVD367 sequences or in both
sequences simultaneously, and the cleavage of the mutant TRAF3 proteins
was then compared with that of the wild-type TRAF3. As shown in Figure 4B
, either the mutation of D347 or that of D367
abrogated the FasL-induced cleavage of TRAF3 (lanes 5 and 8). The
cleavage of TRAF3 induced by anti-CD3 was also inhibited by the
mutation at D347 (lane 6) and to a lesser degree by the
D367A mutation (lane 9). The double point mutant D347/367A was not
cleaved upon FasL- or OKT3-treatment (lanes 11 and 12). This result
together with that from the caspase inhibitor experiment provides
evidence that TRAF3 is cleaved by caspases in Jurkat-T cells during
FasL- or anti-CD3-induced apoptosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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OKT3 treatment of Jurkat-T cells also led to the cleavage of TRAF3 in
our study. Repeated triggering of TCR/CD3 complexes is known to result
in apoptosis of T cells and in this process the interaction of Fas and
its ligand provides the death signal [26
]. Jurkat cells
have been shown to constitutively express Fas and undergo apoptosis
upon anti-CD3 treatment, which was ascribed to the induction of FasL
expression [21
]. The OKT3-induced cleavage of TRAF3 may
therefore be dependent on the Fas pathway. It is also feasible that
anti-CD3 induces other death-promoting ligands such as TNF- TRAIL,
and THANK.
The cleavage of TRAF3 in Jurkat-T cells appeared to occur in the
EEAD347 and ESVD367 sequences (Fig. 4B)
.
Similar sequences are not present in other TRAF proteins except TRAF5.
The human TRAF5 sequences contain EAVD350 and
ENND363 (corresponding to DAVD350 and
EADS363 in mouse TRAF5) analogous to the TRAF3 cleavage
sites. However, TRAF5 was not cleaved in the FasL- or CD3-stimulated
Jurkat-T cells in our study. The reason for this selective cleavage of
TRAF3 is not clear. It is tempting to speculate that this difference
may account for the opposite effects on NF-B and JNK activation by
TRAF3 and TRAF5 despite the high overall sequence homology between the
two proteins. Mutation of either D347 or D367
as well as the double point mutation potently blocked the TRAF3
cleavage (Fig. 4B)
. If the two sites were independently cleaved, single
mutations would have only weakly affected the cleavage. One possible
explanation for the strong effect by either single mutation may be that
the conformation of the protein is important for recognition as the
cleavage target site and the alanine substitution at one site affects
the conformation surrounding the other site. In this regard it is
intriguing that D347 and D367 are present in
the coiled-coil structural domain of TRAF3 [10
].
Several groups of cellular proteins have been identified as substrates
of caspases. These include components of apoptotic machinery,
regulators of apoptosis, structural proteins, and proteins involved in
cellular signaling and repair [27
]. Caspase cleavage of
many of these proteins has been shown to contribute to the promotion of
apoptosis. In other cases, such as PITSLRE kinase and cytosolic
phospholipase A2, the function of caspase cleavage is
uncertain. Moreover, unexpected cell proliferation-inducing effects of
the caspase activation have recently been proposed [28
,
29
], which imply that the function of caspase substrate
cleavage is more complex than simply being either apoptotic or
anti-apoptotic. The role of Fas-induced TRAF3 cleavage is under
investigation. Based on the intracellular location studies (Fig. 3B)
and our observation of TRAF3 binding to a protein involved in nuclear
translocation (data not shown), we speculate that TRAF3 cleavage may
result in translocation of the amino-terminal fragment into the
nucleus. In support of this notion, Krajewski et al.
[22
] observed nuclear immunostaining of TRAF3 in neurons
showing degenerative morphology, and suggested that the nuclear
translocation of TRAF3 might be induced by local cytotoxic cytokine
production. The ring and zinc finger motifs in the amino-terminal of
TRAF3 may bind DNA to regulate transcription factors. In this regard
the amino-terminal part of TRAF3 appeared to be sufficient for
suppression of NF-B activity in our preliminary experiments (data
not shown).
Another possibility of the function of TRAF3 cleavage is the regulation of JNK activation. Fas-induced JNK activation has been shown to involve both caspase-dependent and -independent pathways depending on the cell type and magnitude of stimulation [30 , 31 ]. A recent study demonstrated a correlation between the distribution to detergent-insoluble pellet fractions of cell lysates and the JNK-activating potency of TRAF proteins [32 ]. Thus the preferential localization of the cleaved amino-terminal fragment of TRAF3 in the pellet fraction in our study (Figs. 3A and 4A) implies that the TRAF3 cleavage by Fas stimulation may lead to triggering of the JNK pathway. Whether caspase-mediated fragmentation of TRAF3 may constitute a new mechanism by which Fas induces JNK activation is an intriguing question. The answer to this question could illuminate our understanding of the function of TRAF3 in death receptor signaling.
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ACKNOWLEDGEMENTS |
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Received August 28, 2000; revised November 7, 2000; accepted November 9, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B induction by TNF, CD95 and IL-1 Nature 385,540-544[Medline]
B and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2 Proc. Natl. Acad. Sci. USA 94,9792-9796B recruits multiple TRAF family adaptors and activates c-Jun N-terminal kinase FEBS Lett 443,297-302[Medline]
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