From the Department of Pathology and
Comprehensive Cancer Center,
Department of Pediatrics, The
University of Michigan Medical School, Ann Arbor, Michigan 48109, the
¶ Division of Molecular Medicine, Aichi Cancer Center Research
Institute, Nagoya, Japan, and the ** Department of Microbiology, Tokyo
Medical and Dental University, School of Medicine, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan
Received for publication, November 2, 2000, and in revised form, March 13, 2001
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ABSTRACT |
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At least two distinct recurrent chromosomal
translocations have been implicated in the pathogenesis of MALT
lymphoma. The first, t(1;14), results in the transfer of the entire
Bcl10 gene to chromosome 14 wherein Bcl10 expression is
inappropriately stimulated by the neighboring Ig enhancer. The second,
t(11;18), results in the synthesis of a novel fusion protein,
API2-MALT1. Until now, no common mechanism of action has been proposed
to explain how the products of these seemingly unrelated translocations
may contribute to the same malignant process. We show here that Bcl10 and MALT1 form a strong and specific complex within the cell, and that
these proteins synergize in the activation of NF- B cell lymphomas of the mucosa-associated lymphoid tissue
(MALT)1 were first described
in 1983 by Isaacson and Wright (1). It is now recognized that these are
the most common form of lymphoma arising in extranodal sites; most
cases originate in the gastric mucosa where they are strongly
associated with chronic Helicobacter pylori infection (2).
However, MALT lymphomas may also occur in other sites such as the lung,
salivary glands, and thyroid. Again, when the lymphoma arises in these
sites, it is frequently associated with a prior chronic inflammatory
process such as lymphoid interstitial pneumonia in the lung,
Sjögren's disease in the salivary glands, and Hashimoto's
thyroiditis. These findings have lead to the notion that, at least in
early stages of tumor development, growth of the neoplastic cells is
facilitated by antigen-driven T cells. Based on histopathologic
criteria, MALT lymphomas are classified as either low- or high-grade.
Nevertheless, it is likely that for most cases there is a gradual
progression in the pathogenesis of the disease, such that untreated
borderline cases progress to low-grade lymphomas which ultimately
develop into high-grade, aggressive lymphomas. Consistent with this
notion is the finding that many early cases of gastric MALT lymphoma
can be effectively treated solely by eradication of H. pylori with antibiotics (3). More established, higher-grade
lymphomas may require more aggressive treatment, as the progression of
such tumors appears to no longer depend on a coexisting chronic
inflammatory process.
The molecular events responsible for the development and progression of
MALT lymphomas are still poorly understood. Several recurrent
chromosomal abnormalities have been observed in these lymphomas, but no
unifying molecular model has been proposed to explain how these
distinct abnormalities may promote tumorigenesis. Perhaps the best
characterized chromosomal abnormality is the recurrent t(1;14)
(p22;q32) which was recently shown to result in the translocation of
the entire coding region of the Bcl10 gene to chromosome 14, wherein its expression is inappropriately placed under the control of
the strong Ig transcriptional enhancers (4, 5). We and
others have shown that overexpression of Bcl10 (also called CIPER,
mE10, c-CARMEN, CLAP, and c-E10) activates NF- A second major chromosomal translocation, seen in up to 50% of MALT
lymphomas, is the t(11;18) (q21;q21). In this case, a fusion gene is
created which encodes a chimeric protein consisting of the N-terminal
portion of API2 (one of the inhibitors of apoptosis proteins, also
known as c-IAP2, Hiap1, and MIHC) linked to the C terminus of a
novel protein, MALT1 (MLT) (11-13). Although MALT1 contains a
caspase-like domain in its C terminus, which is preserved in the
API2-MALT1 chimera, no physiologic function has been ascribed to this protein.
Although it is known that Bcl10 activates NF- Plasmids--
The expression plasmids pcDNA3-Myc (-HA
and -Flag), pcDNA3-p35, pEF1-BOS- Transfections and NF- Immunoprecipitations and Western Blotting--
HEK293T cells
were harvested 24 h following transfection and lysed in 0.2%
Nonidet P-40 lysis buffer (22). Immunoprecipitations were carried out
using rabbit polyclonal anti-Myc antibody (Santa Cruz) as described (6,
14). The products were then resolved by 12% SDS-PAGE, and detected by
Western blotting with mouse monoclonal anti-Myc (Santa Cruz).
Co-immunoprecipitated proteins were detected by probing blots with
either mouse monoclonal anti-Flag (M2) (Sigma) or anti-HA 12CA5 (Roche
Molecular Biochemicals). Total lysates were similarly analyzed for
transgene expression by Western blotting, both in experiments where
immunoprecipitations were performed and in experiments where NF- Gel Mobility Shift Assay--
Nuclear extracts were prepared
following transfection of HEK293T cells by an established method (23).
Gel shift assays were then performed as described (23) using a
radiolabeled, double-stranded oligonucleotide containing a single Bcl10 Activates NF-
Next, Bcl10 was co-expressed with mutant forms of I
Because IKK Bcl10 Does Not Interact Directly with IKK
In a search to identify signaling proteins which may bridge the gap
between Bcl10 and IKK Bcl10 and MALT1 Synergize in the Activation of NF- Bcl10 Binds to the MALT1 Ig-like Domains through a Region
Critically Important for NF-
Next, we made use of previously published reports which together
implicated a short 20-amino acid region in Bcl10 as a critical component of the Bcl10 NF-
To determine whether the loss of function produced by deleting these 13 amino acids correlated with a loss of MALT1 binding, we again used the
co-immunoprecipitation assay. The MALT1 mutant containing the Ig-like
domains (MALT1-(1-330)), which shows full binding to Bcl10, was used
for the analysis because this truncated form of the protein can be
expressed at significantly higher levels than the wild-type MALT1, and
is thereby more amenable for analysis in immunoprecipitation
experiments. Again, Bcl10 was seen to efficiently bind to this MALT1
construct, while Bcl10 Bcl10 Mediates the Oligomerization and Activation of the MALT1
Caspase-like domain, a Step Which Is Sufficient to Activate
NF-
Having established a role for the MALT1 caspase-like domain in NF-
If oligomerization of the MALT1 caspase-like domain results in its
activation, and this activated domain represents the effector portion
of the Bcl10·MALT1 signaling complex, then we would expect that
artificial oligomerization of the isolated caspase-like domain, in the
absence of Bcl10 or other MALT1 domains, may be sufficient to activate
NF- The API2-MALT1 Fusion Protein Activates NF- A Unifying Model for the Pathogenesis of MALT Lymphoma--
The
data presented herein suggest that Bcl10 serves to oligomerize and
activate the MALT1 caspase-like domain. Whether the caspase-like domain
of MALT1 is capable of cleaving an unknown substrate that is then able
to activate the IKK complex through direct or indirect interaction with
IKK
A second major chromosomal translocation that is seen in up to 50% of
MALT lymphomas is the t(11;18) which creates a fusion gene encoding the
chimeric API2-MALT1 protein. We have now shown that this chimeric
protein can efficiently activate NF-
In addition to creating a chimeric protein which potentially possesses
a mechanism for self-activation, fusion of the API2 and
MALT1 genes may result in a second advantage with regard to NF-
In summary, we have provided evidence that two independent targets of
chromosomal translocation in MALT lymphoma, Bcl10 and MALT1, bind to
one another and cooperate in the activation of NF-B. The data support
a mechanism of action whereby Bcl10 mediates the oligomerization and
activation of the MALT1 caspase-like domain. This subsequently
activates the IKK complex through an unknown mechanism, setting in
motion a cascade of events leading to NF-
B induction. Furthermore,
the API2-MALT1 fusion protein also strongly activates NF-
B and shows
dependence upon the same downstream signaling factors. We propose a
model whereby both the Bcl10·MALT1 complex and the API2-MALT1
fusion protein activate a common downstream signaling pathway that
originates with the oligomerization-dependent activation of
the MALT1 caspase-like domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B, a potential
pro-survival signal in B cells, and have suggested this to be a major
mechanism whereby unregulated Bcl10 expression contributes to
transformation and tumor progression (6-10).
B, little is known
about the precise signaling pathway utilized by Bcl10 to accomplish
this effect. In this study, we further characterized this pathway, and
in so doing, discovered that MALT1 participates with Bcl10 in a novel
mechanism for NF-
B activation. The data indicate that Bcl10 and
MALT1 form a tight complex which serves to oligomerize and activate the
caspase-like domain of MALT1. Through an unknown mechanism, this
appears to subsequently activate the downstream I
B kinase (IKK)
complex, leading to the induction of NF-
B. In similar fashion, we
found that the API2-MALT1 fusion protein also potently activates
NF-
B, again through activation of the IKK complex. These results
provide a unifying model for the molecular pathogenesis of MALT
lymphoma and also suggest a novel pathway for activation of NF-
B
through the involvement of a caspase-like enzyme.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-gal, pcDNA3-Flag-IKK
,
pcDNA3-RIP-(1-580)-Myc, pcDNA3-Myc-RICK, pcDNA3-Nod1-HA/Flag,
pcDNA3-Apaf-1-(1-559)-Myc, pcDNA3-Myc-NIK, pcDNA3-IKK
-Myc,
RSVMad-3MSS (I
B
-S32A/S36A), pRK7-Flag-IKK
-K44A,
pRK7-IKK
-K44A, pcDNA3-IKK
-(134-419),
pcDNA3-Ask1-(K709R), pcDNA3-Flag-DAPK-(K42A), pc-TBK1-Myc,
pcDNA3-TNFR1-Flag, pcDNA3-Flag-IKKi, and
pcDNA3-Flag-IKKi-(K38A) have been described previously (6, 14-19).
The plasmid pcDNA3-Bcl10-Myc (previously referred to as pcDNA3-CIPER-Myc) encodes the mouse Bcl10 protein with a C-terminal Myc tag, and has been described (6). pcDNA3-Bcl10
107-119 was created by adjoining two Bcl10 polymerase chain reaction
fragments which together encompassed the entire coding region except
for that corresponding to amino acids 107-119. The pcDNA3-MALT1-HA (and -Myc) vectors were constructed by inserting the coding region of
MALT1 into the BamHI/XhoI sites of the parental
tagged vectors using a polymerase chain reaction method. Site-directed
mutagenesis of MALT1 was carried out using the Stratagene
QuickchangeTM method as directed by the manufacturer's
protocol. To construct pcDNA3-MALT1-Casp-FKBPx3, a polymerase chain
reaction fragment encoding amino acids 324 to 813 of MALT1 was inserted
into the Acc65I/XhoI sites of
pcDNA3-FKBPx3-Myc (20) which encodes three tandemly repeated FKBP
dimerizer domains with a Myc tag. The pcDNA3-Flag-API2-MALT1 plasmid was created by inserting the coding region of the API2-MALT1 fusion gene into the XbaI/ApaI sites of
pcDNA3-Flag. To construct pcDNA3-Flag-API2-MALT1-(1-762), the
full-length API2-MALT1 insert was digested with Acc65I and
XbaI to liberate the truncated fragment which was then
subcloned into the parental tagged vector in the same fashion as the
full-length cDNA. Likewise, pcDNA3-Flag-API2-MALT1-(1-700) was
constructed by digesting with Acc65I and SspI,
with subsequent subcloning into
Acc65I/EcoRV-digested pcDNA3.
B Activation Assays--
2 × 105 HEK293T cells were transfected with the reporter
constructs pEF1-BOS-
-gal and pBVI-Luc, and indicated expression
plasmids, using a calcium phosphate method as described (6, 14).
NF-
B activation was assessed by measuring luciferase activity
(normalized for
-galactosidase expression) in cell extracts after
24 h, as described (6, 14). pcDNA3-p35 was also transfected in
all cases to prevent cell death. For transfections of the Rat-1 and 5R
fibroblast cell lines, as well as the wild type and RelA-deficient MEFs
(14, 21), the same procedures were followed except that LipofectAMINE
(Life Technologies, Inc.) was utilized, as recommended by the
manufacturer, instead of the calcium phosphate method. In experiments
where FKBP containing proteins were expressed, the media was removed
6 h following transfection and replaced with media containing the
indicated amounts (50-800 nM) of the cell permeable
ligand, AP1510 (Ariad Pharmaceuticals). As for other transfections,
cell extracts were prepared 24 h after transfection.
B
activity was being assessed.
B
site (14) as a probe. For competition experiments, the indicated
amounts of unlabeled, double-stranded oligonucleotide, containing
either the wild-type or mutant
B site (14), were included in the
binding reactions. Supershift analyses were performed by including
anti-RelA antibody (Santa Cruz) in binding reactions.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B by Signaling through the IKK
Complex--
Many inducers of NF-
B are now known to signal through
the IKK complex, which is composed of two catalytic subunits (IKK
and IKK
) and a regulatory subunit, IKK
(NEMO/FIP3/IKKAP1) (Fig. 1A). Two related kinases, RIP
and RICK, are prototypical examples of such inducers, and are now
thought to activate the IKK complex through direct interaction with
IKK
(14, 24, 25). However, an emerging theme is that signaling
pathways induced by other stimuli may bypass components of the IKK
complex, particularly IKK
, or even the entire IKK complex itself
(Fig. 1A) (16, 17, 26-28). In order to delineate the
downstream signaling pathway utilized by Bcl10, we analyzed the
requirement for each step in the prototypical pathway mapped for
inducers such as RIP and RICK. Starting with the most distal step in
the signaling pathway, we first tested whether the well characterized
functional activation of NF-
B by Bcl10 is dependent on one of the
principle Rel family members, RelA (p65). Wild type and RelA-deficient
MEFs were co-transfected with an expression plasmid encoding Bcl10 and
an NF-
B-responsive luciferase reporter construct; while Bcl10
expression resulted in ~11-fold induction of NF-
B in the wild type
MEFs, almost no induction was seen in cells lacking RelA (Fig.
1B). As a control, activation of NF-
B by another inducer,
Nod1 (14, 15), was also seen to depend upon RelA. In contrast,
expression of Apaf-1, a protein involved in apoptosis signaling, had no
effect on NF-
B signaling in either cell line. We next used the gel
mobility shift assay to test whether expression of Bcl10 results in
nuclear translocation of NF-
B and its binding to DNA containing the
B consensus sequence. Expression of Bcl10 in 293T cells resulted in
the formation of a DNA-protein complex identical to that seen when
cells were stimulated with TNF-
(Fig. 1C). Furthermore,
formation of the complex was abolished by competition with unlabeled
oligonucleotide containing the
B consensus, but not by
oligonucleotide containing a mutant
B site. Finally, a specific
supershift complex was seen when antibody to the p65 subunit of NF-
B
was included in the binding reaction (Fig. 1C). Taken
together, these results indicate that the activation of NF-
B by
Bcl10 is indeed the result of enhanced nuclear translocation and DNA
binding by the NF-
B transcription factor.
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Fig. 1.
Downstream signaling pathway utilized by
Bcl10 in the activation of NF- B.
A, schematic of known NF-
B signaling pathways.
B, 1 × 105 wild type and RelA-deficient
MEFs were transfected in triplicate with 33 ng of pcDNA3,
pcDNA3-Nod1-HA, pcDNA3-Bcl10-Myc, or
pcDNA3-Apaf-1-(1-599)-Myc in the presence of 33 ng of
pcDNA3-p35, 0.3 ng of pBVI-Luc (an NF-
B-responsive luciferase
reporter), and 33 ng of pEF1-BOS-
-gal (used as a control for
transfection efficiency). For this and all transfections described in
this article, the total amount of transfected DNA was adjusted to be
equal for each condition by supplementing with pcDNA3. 24 h
post-transfection, cell extracts were prepared and NF-
B activation
assessed by measuring the luciferase:
-galactosidase ratio as
described (14). C, radiolabeled oligonucleotide containing
the consensus
B site was incubated for 20 min with ~10 µg of
nuclear extract from 2 × 106 293T cells transfected
with 30 µg of either pcDNA3 vector or
pcDNA3-Bcl10(119+L)-Myc, which encodes our most active form of
Bcl10. DNA-protein complexes were resolved by 6% nondenaturing PAGE.
Unbound DNA is not shown in the figure. For competition experiments,
unlabeled wt or mutant oligonucleotide was included in the binding
reaction at 10-, 25-, or 50-fold excess. For antibody supershit
analysis, 0.2, 2, or 20 ng of polyclonal antibody to the p65 subunit of
NF-
B (Santa Cruz) was included. As a control, the mobility shift
pattern was compared with that obtained with extract from 293T cells
treated for 1 h with 10 ng/ml TNF-
. Equivalency in the quality
of extracts was confirmed by the constant presence of a nonspecific
DNA-binding protein. D, 2 × 105 293T cells
were transfected with 75 ng of pcDNA3-Bcl10-Myc in the presence of
30 ng of pEF1-BOS-
-gal and 3 ng of pBVI-Luc. For co-transfections,
75 ng of pRK7-Flag-IKK
-K44A, pRK7-IKK
-K44A,
pcDNA3-IKK
-(134-419), or pcDNA3-Flag-IKKi-(K38A) was also
included. 24 h post-transfection, extracts were prepared and
NF-
B induction assessed by measuring the
luciferase:
-galactosidase ratio. Western blots were performed on
total extracts using polyclonal anti-Myc (Santa Cruz). WB,
Western blot. E, transfections were performed as described
above using the indicated amounts of expression vector for the IKK
dominant negative (pcDNA3-IKK
-(134-419)) along with 75 ng of
pcDNA3-Bcl10-Myc, pcDNA3-Myc-RICK, or pcDNA3-Flag-IKKi.
24 h post-transfection, cells were harvested and NF-
B induction
measured as described above. F, induction of NF-
B was
determined from triplicate cultures of 2 × 105 Rat-1
or 5R cells transfected with the indicated plasmids. Cells were
harvested after 24 h and NF-
B activity was determined as above.
NS, nonspecific; WT, wild type.
B
, IKK
,
IKK
, and IKK
which have been shown to act as dominant inhibitors of their corresponding endogenous counterparts (14, 29). Expression of
all four of these dominant negative mutants resulted in near complete
inhibition of Bcl10-responsive NF-
B activation (Fig. 1D).
Western blotting confirmed that none of these mutants interfered significantly with the expression of Bcl10, so that inhibition was
likely to have occurred through functional interference with the
Bcl10-responsive signaling pathway. As a control, a dominant negative
mutant of IKK-i (IKK
) (16) had no effect on
Bcl10-dependent NF-
B activation. These results support
the notion that, similar to RIP and RICK, Bcl10 acts to stimulate a
signaling pathway which depends upon the IKK complex.
may represent a critical link between Bcl10 and
downstream factors, we sought to more stringently test its involvement in Bcl10 signaling. First, we performed a dose-response study to
analyze the potency of the IKK
dominant negative in inhibiting Bcl10
activity. This inhibitor blocked Bcl10 induction of NF-
B with
precisely the same effectiveness as it blocked induction by RICK (Fig.
1E). In contrast, the IKK
dominant negative had no effect
on induction of NF-
B by IKK-i; this was an expected finding, as it
has been shown that IKK-i appears to act as an IKK complex-independent
I
B kinase (see Fig. 1A) (16, 26). Using a different
methodology, we confirmed the requirement for IKK
in Bcl10 signaling
by testing NF-
B induction in the IKK
-defective 5R cell line, a
derivative of the Rat-1 fibroblast line (21). While in the parental
Rat-1 cells, Bcl10 effectively induced NF-
B, we observed no
Bcl10-responsive NF-
B activation in the IKK
-deficient 5R cells
(Fig. 1F). As a control, expression of RIP resulted in similar findings, consistent with the previously documented
critical role for IKK
in RIP signaling (14). To demonstrate that 5R cells are capable of mounting a robust NF-
B response when
transfected with factors downstream of IKK
, we also expressed IKK
in both cell lines and confirmed that this factor could activate
NF-
B irrespective of the presence of functional IKK
.
, but Binds to
MALT1--
It has been previously shown that both the RIP and RICK
signaling proteins must bind directly to IKK
in order to stimulate the kinase activity of the IKK complex (14, 24, 25). To test if Bcl10
acts in a similar way, we first analyzed whether Bcl10 binds directly
to IKK
using a co-immunoprecipitation assay. Myc-tagged Bcl10 was
co-expressed with Flag-tagged IKK
in 293T cells, extracts were
prepared, and Bcl10 was immunoprecipitated with anti-Myc antibody (Fig.
2A). Subsequent Western
blotting failed to detect any significant co-immunoprecipitation of
IKK
. As a positive control, Flag-tagged IKK
effectively
co-immunoprecipitated with Myc-tagged RIP. These results suggest that
there are one or more missing signaling factors which mediate the
connection between Bcl10 and the IKK complex.
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Fig. 2.
Bcl10 does not interact directly with
IKK but binds to MALT1. A,
2 × 106 293T cells were transfected with 350 ng of
pcDNA3-Flag-IKK
and 7 µg of pcDNA3, pcDNA3-Bcl10-Myc,
or pcDNA3-RIP-(1-580)-Myc. 24 h post-transfection, cells were
lysed with 0.2% Nonidet P-40 lysis buffer and Myc-tagged proteins were
immunoprecipitated with polyclonal anti-Myc antibody. The products were
resolved by 12% SDS-PAGE and visualized by probing with either
monoclonal anti-Myc or monoclonal anti-Flag M2 antibodies.
B, similar to panel A, 293T cells were
transfected with 13 µg of pcDNA3-MALT1-HA and 4.4 µg of
pcDNA3-Bcl10-Myc, 4.4 µg of pcDNA3-Myc-RICK, 3.5 µg of
pcDNA3-Myc-NIK, or 3.5 µg of pc-TBK1-Myc. 24 h
post-transfection, extracts were prepared and immunoprecipitations
carried out as described above. Western blots were probed with either
monoclonal anti-Myc or monoclonal anti-HA 12CA5 antibodies.
, we decided to test the possible involvement
of MALT1. MALT1 is a newly discovered protein of unknown function
which contains a C-terminal caspase-like domain, including the
universally conserved cysteine-histidine catalytic diad (11-13). The MALT1 gene was recently demonstrated to be the target of
the recurrent t(11;18) (q21;q21) seen in a subset of MALT lymphomas. We
therefore wondered whether the seemingly disparate translocations which
target Bcl10 and MALT1 might, in fact, influence the same signaling
pathway. To test for an interaction between Bcl10 and MALT1, we again
performed co-immunoprecipitation experiments using Myc-tagged Bcl10 and
HA-tagged MALT1. When co-expressed in 293T cells, MALT1 bound to Bcl10
but not to a series of other proteins involved in NF-
B
signaling (Fig. 2B). Quantitative immunoprecipitation experiments demonstrated that the interaction is remarkably strong, with more than 20% of expressed MALT1 being co-immunoprecipitated in
experiments where direct immunoprecipitation of Myc-Bcl10 is less than
50% efficient.2 These results indicate that
Bcl10 and MALT1 form a tight and specific complex when co-expressed in cells.
B--
To
explore the functional consequence of the interaction between Bcl10 and
MALT1, we tested whether MALT1 influences Bcl10-responsive NF-
B
activation. In a dose-dependent manner, expression of Bcl10 alone resulted in NF-
B activation; a maximal level of ~35-fold induction was achieved with 75 ng of transfected expression plasmid (Fig. 3A). In contrast,
transfection of up to 500 ng of MALT1 expression plasmid had absolutely
no effect on NF-
B (Fig. 3E). However, when MALT1 was
co-expressed with Bcl10, there was a remarkable synergy, with close to
150-fold induction of NF-
B achieved when maximal levels of Bcl10
were used (Fig. 3A). The effect of MALT1 was specific for
Bcl10-responsive NF-
B activation, as a similar dose-response study
showed no influence of MALT1 on RICK activity (Fig. 3B).
Western blotting confirmed that neither Bcl10 nor RICK expression
levels were affected by co-expression of MALT1, indicating that MALT1
was specifically influencing the function of the Bcl10 signaling
pathway (Fig. 3, C and D). Further specificity
was demonstrated by testing the affect of MALT1 on either low or high
levels of co-expressed IKK-i, TBK-1/NAC, and TNFR1 (Fig. 3,
E and F). In all cases, MALT1 had no significant
effect on the level of NF-
B induction achieved with these signaling
proteins.
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Fig. 3.
Specific synergy between Bcl10 and MALT1 in
the activation of NF- B. A and
B, 1 × 105 293T cells were transfected as
described in the legend to Fig. 1 with the indicated amounts of
expression vector encoding wt Bcl10 (panel A) or RICK
(panel B), either in the presence or absence of 0.5 µg of
co-transfected pcDNA3-MALT1-Myc. NF-
B induction was measured as
described in the legend to Fig. 1. C and D,
extracts used to measure NF-
B induction in panels A and
B of this figure were subjected to Western blotting with
polyclonal anti-Myc antibody. E and F, 293T cells
were transfected with either low levels (10, 10, or 2.5 ng, panel
E) or high levels (50 ng, panel F) of expression vector
for IKK-i, TBK-1, or TNFR1, respectively. Experiments were carried out
in the presence or absence of 0.5 µg of co-transfected
pcDNA3-MALT1-Myc as indicated. NF-
B induction was measured as
described in the legend to Fig. 1. Results were compared with
transfections in which 75 ng of Bcl10 expression vector was transfected
in the presence or absence of MALT1 expression plasmid.
B Activation--
To test whether the
binding between Bcl10 and MALT1 is functionally related to the observed
synergy in activation of NF-
B, we analyzed several mutants of each
protein. First, we mapped the region of MALT1 which is responsible for
binding Bcl10. Three truncation mutants were constructed which together
encompassed the entire MALT1 protein, but which isolated the three
known domains of the protein, the death domain (DD), two adjacent
Ig-like domains, and the caspase-like domain (Fig.
4A). All mutants were
HA-tagged and coexpressed with Myc-tagged Bcl10. Following
immunoprecipitation with anti-Myc antibody, Western blots were probed
with anti-HA antibody to identify co-immunoprecipitated MALT1 mutants.
The results clearly showed that only the mutant containing both Ig-like domains could interact with Bcl10 (Fig. 4B). Identification
of the two Ig-like domains as the composite Bcl10-binding domain is
compatible with the known role of the Ig-like domain as a protein recognition motif (30).
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Fig. 4.
Correlation between binding and function for
the Bcl10·MALT1 complex. A, schematic representation
of MALT1 mutants used for binding domain analysis. B, 2 × 106 293T cells were transfected with 2 µg of
pcDNA3-Bcl10-Myc and 12 µg of pcDNA3-MALT1-(1-139)-HA, 8 µg of pcDNA3-MALT1-(1-330)-HA, 6 µg of
pcDNA3-MALT1-(324-813)-HA, or pcDNA3. Myc-tagged Bcl10 was
immunoprecipitated as described in the legend to Fig. 2, and Western
blots of both total lysates and immunoprecipitated proteins were
carried out using anti-Myc and anti-HA antibodies. The
asterisk represents a supershifted MALT1 band which was
occasionally seen; it is unclear whether this indicates phosphorylation
or some other modification of MALT1. C, schematic
representation of wild-type and mutant Bcl10 proteins used in the
following functional and binding studies. D, 2 × 105 293T cells were transfected with either 75 ng of
pcDNA3-Bcl10-Myc or 225 ng of pcDNA3-Bcl10 107-119-Myc.
Transfections were carried out either in the presence or absence of 0.5 µg of added pcDNA3-MALT1-Myc, as indicated. Cell lysates were
prepared and analyzed for both NF-
B activation and transgene
expression as described previously. E, 2 × 106 293T cells were transfected with 2 µg of
pcDNA3-Bcl10-Myc, 6 µg of pcDNA3-Bcl10
107-119-Myc, or
pcDNA3 control. As indicated, transfections were carried out in the
presence or absence of 6 µg of added pcDNA3-MALT1-(1-330)-HA.
Myc-tagged Bcl10 proteins were immunoprecipitated and both total
lysates and immunoprecipitated products were analyzed by Western blot
(WB) as described above.
B activation domain. Our laboratory had
shown that a truncated Bcl10 protein consisting of only the N-terminal
CARD (amino acids 1-103) plus the adjoining 16 amino acids of
C-terminal sequence (amino acids 104-119) was active at inducing
NF-
B (6). Others, however, had shown that the CARD alone was
ineffectual (9). These results suggested that the region of Bcl10
between amino acids 104 and 119 is critically important for NF-
B
activation. We therefore targeted this region by selectively deleting
13 amino acids from this domain, residues 107-119 (Fig.
4C). When expressed in 293T cells, this mutant indeed showed
almost a complete loss of function with regard to NF-
B activation
(Fig. 4D). Importantly, there was also complete absence of
functional synergy between the mutant and MALT1. Despite the lack of
activity, however, Western blotting confirmed that the mutant was
expressed at least as well as the wild-type counterpart (Fig.
4D).
107-119, when expressed at similar levels,
showed absolutely no interaction with MALT1 (Fig. 4E). These
results demonstrate a correlation between the Bcl10 residues required
for NF-
B activation and those required for binding to MALT1. In
summary, the analysis suggests that the binding observed between Bcl10
and MALT1 may be mechanistically related to the functional synergy seen
with the two proteins.
B--
To explore the mechanistic relationship between Bcl10 and
MALT1, we hypothesized that MALT1 may represent the downstream factor in a Bcl10·MALT1 signaling complex. The C-terminal domain of MALT1 shows homology to the proteolytic domain of caspases, and includes the
universally conserved cysteine-histidine catalytic diad (31). For
caspases such as caspase-9, activation is mediated through oligomerization directed by upstream regulators (Apaf-1 in the case of
caspase-9). If MALT1 behaves in an analogous manner, then it is
possible that binding to Bcl10 serves to oligomerize and activate the
caspase-like domain of MALT1. This activated domain may then function
as the effector in the NF-
B signaling pathway, at a point upstream
of the IKK complex. To test this hypothesis, we first analyzed the role
of the caspase-like domain in the synergistic enhancement of NF-
B by
the combination of Bcl10 and MALT1. Two mutants were used for the
analysis. For the first, MALT1-(1-330), we removed the entire
caspase-like domain. This deletion completely abolished enhancement of
Bcl10 activity (Fig. 5), indicating that the caspase-like domain is a critical component of the NF-
B
signaling pathway. For the second mutant, the conserved cysteine
(Cys453), which is critical for catalytic activity of
traditional caspases (31), was mutated to alanine (Fig. 5).
Importantly, in six separate experiments, the C453A mutant showed only
an approximate 40% reduction (p = 0.01) in the
synergistic enhancement of NF-
B (Fig. 5). These results indicate
that while the caspase-like domain is essential for Bcl10/MALT1
signaling, this domain may differ significantly in its function from
that of traditional caspases; either this caspase-like domain possesses
a proteolytic active site that does not depend as critically on the
cysteine residue, or there may be some other function of this domain
that is important for this NF-
B signaling pathway, but which does
not involve catalytic activity. Indeed, others have now suggested that
MALT1 does not behave as a traditional caspase in that it does not
induce apoptosis and cannot cleave a range of known caspase substrates
(32). Structural modeling suggests that the active site may in fact show specificity toward an uncharged residue, rather than aspartic acid, in the substrate P1 position (32). Because of these properties, it may be most accurate to classify MALT1 under a broader category of
cysteine proteases, and not as a caspase per se.
Nevertheless, for lack of a better term, we shall continue to refer to
the protease domain of MALT1 as a caspase-like domain.
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Fig. 5.
The MALT1 caspase-like domain is
critical for signaling through the Bcl10·MALT1 complex. 2 × 105 293T cells were transfected with 75 ng of
pcDNA3-Bcl10-Myc and 500 ng of pcDNA3-MALT1-Myc,
pcDNA3-MALT1-(C453A), pcDNA3-MALT1-(1-330), or pcDNA3 control.
24 h post-transfection, cell lysates were prepared and analyzed
for both NF- B activation and transgene expression as described
previously. The difference between values marked by an
asterisk was statistically significant (p = 0.01), as measured by the Student's t test. WB,
Western blot.
B
activation, we next tested whether Bcl10 is capable of inducing the
oligomerization of MALT1. To this end, we expressed both HA- and
Myc-tagged versions of MALT1 in 293T cells, either in the presence or
absence of several different Flag-tagged proteins (Fig.
6A). Myc-tagged MALT1 was
immunoprecipitated and Western blots were probed for the presence of
co-immunoprecipitated HA-tagged MALT1. While no oligomerization was
observed when the MALT1 proteins were expressed by themselves, the
presence of Bcl10 resulted in strong MALT1 oligomerization (Fig.
6A). As negative controls, coexpression of either IKK
or
Nod1 was ineffective at promoting MALT1 oligomerization. The Nod1
control was particularly relevant, as this protein contains a CARD, as
does Bcl10, and has been shown to mediate a similar oligomerization of
the signaling protein RICK (14).
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Fig. 6.
Oligomerization of the MALT1 caspase-like
domain is sufficient for NF- B activation.
A, 2 × 106 293T cells were transfected as
indicated with 6 µg of pcDNA3-MALT1-(1-330)-HA or -Myc, and 3 µg of pcDNA3-Bcl10-Flag, 4 µg of pcDNA3-IKK
-Flag, or 4 µg of pcDNA3-Nod1
LRR-Flag. Myc-tagged MALT1-(1-330) was
immunoprecipitated and both total lysates and immunoprecipitated
products were analyzed by Western blot as described previously.
B, 2 × 105 293T cells were transfected
with 1000 ng of pcDNA3-MALT1-Caspase-FKBPx3-Myc, 500 ng of
pcDNA3-MALT1-Caspase-Myc, or 250 ng of the parental
pcDNA3-FKBPx3-Myc vector. 6 h post-transfection, cells were
treated with the indicated concentrations of the AP1510 dimerizer for
the next 18 h, after which lysates were prepared and analyzed for
NF-
B activation. Western blotting confirmed that treatment with the
drug had no effect on expression of any of the FKBP/MALT1 transgenes
(see Footnote 3).
B. To test this notion, we constructed a chimeric expression
plasmid, encoding the MALT1 caspase-like domain linked to three
tandemly repeated FKBP dimerization domains which can be oligomerized
by the cell-permeable artificial ligand, AP1510 (Fig. 6B)
(20). Expression of the resulting fusion protein, MALT1-Casp-FKBPx3,
resulted in ligand-dependent activation of NF-
B, while
neither the caspase-like domain alone nor the FKBP domains showed any
ligand-responsive activity (Fig. 6B). Taken together, the
above results support a model whereby Bcl10 promotes the
oligomerization of MALT1, thereby activating MALT1 caspase-like domains
whose activity in some way stimulates the IKK complex and sets in
motion the downstream steps leading to NF-
B activation.
B--
As shown
above, MALT1 was unable to activate NF-
B on its own, in the absence
of its oligomerization which is specifically mediated by co-expressed
Bcl10 (Figs. 3E and 6A). However, the possibility
remained that the API2-MALT1 fusion protein, created as a consequence
of the t(11;18) translocation, might represent a gain-of-function
mutant which is active at inducing NF-
B, independent of
Bcl10-mediated oligomerization. Several different breakpoints have
been identified for this translocation, producing several variants of
the fusion protein, the principle difference between the variants being
the presence or absence of the Ig-like domains of MALT1. In all cases,
however, the caspase-like domain of MALT1 is preserved and linked to
the three BIR domains present in the N terminus of API2 (Fig.
7A). To test the hypothesis
that the API2-MALT1 chimera may display gain-of-function activity, we
transfected 293T cells with an expression plasmid encoding the chimera
depicted in Fig. 7A, and measured NF-
B activation.
Indeed, this fusion protein was able to induce a potent NF-
B
response (Fig. 7B). Furthermore, this response was
completely dependent on an intact caspase-like domain, as deletion
mutants which removed the region corresponding to the small subunit of
the domain showed no significant activity. Western blots confirmed that
these mutants were expressed at levels comparable to that of the
full-length fusion protein.3
To test whether the API2-MALT1 protein utilizes the same downstream signaling pathway as Bcl10, we analyzed the effect of co-expressing dominant negative mutants of the principle IKK complex components. Similar to the results obtained for Bcl10, dominant negative mutants of
IKK
, IKK
, and IKK
all blocked API2-MALT1-responsive NF-
B activation (Fig. 7C). As a control, dominant negative
mutants of two unrelated kinases, Ask1 and DAPK, had no effect.
Finally, we confirmed the essential role of IKK
in API2-MALT1
signaling by testing NF-
B activation in the IKK
-deficient cell
line, 5R. Again, similar to Bcl10, API2-MALT1 was unable to activate
NF-
B in these cells while in the parental Rat-1 cells, the NF-
B
response was fully intact (Fig. 7D).
View larger version (21K):
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Fig. 7.
The API2-MALT1 fusion protein activates
NF- B through the IKK complex, in a caspase
domain dependent fashion. A, schematic representation
of an API2-MALT1 fusion protein and related deletion mutants.
B, 1 × 105 293T cells were transfected
with either 50 ng of expression vector encoding the full-length
API2-MALT1 fusion protein or 125 ng of expression vector encoding the
two deletion mutants depicted in panel A. 24 h
post-transfection, extracts were prepared and NF-
B induction was
measured as described in the legend to Fig. 1. C, 293T cells
were transfected with 50 ng of expression vector encoding the
full-length API2-MALT1 fusion protein along with 125 ng of either
pcDNA3 or expression plasmids for the dominant negative mutants of
IKK
, IKK
, IKK
, Ask1, or DAPK. 24 h post-transfection,
extracts were prepared and NF-
B induction was measured as described
in the legend to Fig. 1. D, the Rat-1 and 5R cell lines were
transfected as described in the legend to Fig. 1E with
expression plasmids encoding either the full-length API2-MALT1 or the
RIP and IKK
proteins as controls. Extracts were prepared and NF-
B
induction was measured as described in the legend to Fig. 1.
Ig, Ig-like domain.
is an open question which must be tested further. However, in
our hands we have not seen a direct interaction between MALT1 and
IKK
,2 suggesting that at
least one signaling step may separate MALT1 from the IKK complex.
Importantly, the MALT1 caspase-like domain may differ significantly in
its function from that of traditional caspases, as site-directed
mutagenesis of the conserved catalytic cysteine had only a modest
effect on NF-
B activation. Thus, the MALT1 caspase-like domain may
serve more than one function, such that the catalytic activity of the
domain might not be required for NF-
B activation. Alternatively,
active catalysis may not rely as much on the conserved cysteine as it
does for traditional cysteine proteases.
B. As described previously,
several variants of the API2-MALT1 fusion gene have been
characterized, but the only domain from MALT1 which is consistently
preserved in all API2-MALT1 variants is the caspase-like domain
(11-13). This is consistent with the data that implicates the MALT1
caspase-like domain as the effector arm of the Bcl10·MALT1 signaling
complex. However, unlike wild-type MALT1 which appears to depend upon
an interaction with Bcl10 as a mechanism for oligomerization and
autoactivation, the API2-MALT1 fusion protein may possess a mechanism
for self-oligomerization; the three BIR domains contributed by
the API2 portion of the chimera could fulfill this role. These domains
are thought to represent protein interaction domains (33, 34), and a
recent report has shown oligomerization of BIR-containing proteins
through direct BIR-BIR interactions (35). Alternatively, the API2-MALT1
protein might be oligomerized through BIR-dependent interactions with a second, ubiquitous cellular component. Thus, the
API2-MALT1 fusion protein may possess a mechanism for efficient, Bcl10-independent autoactivation of the MALT1 caspase-like domain.
B signaling. The API2 gene promoter is known to be NF-
B
responsive, so that expression of the API2-MALT1 fusion is likely to
also be stimulated by NF-
B (36). This could set in motion a positive feedback-loop resulting in unrelenting NF-
B induction.
B. As NF-
B is
known to direct the expression of several survival-related genes, it is
now thought that unregulated activation of this pathway may contribute
to malignant transformation and/or progression (37). As this article
was completed, Uren et al. (32) published a report which
compliments the findings described herein. These authors used a yeast
two-hybrid system to screen for binding partners of the MALT1 protease.
Multiple positive clones were identified and found to encode Bcl10; the
validity of this interaction was further tested by demonstrating an
interaction between endogenous MALT1 and Bcl10 in a number of cell
lines (32). Although the functional significance of the interaction was
not explored in their study, the authors did demonstrate that two
API2-MALT1 fusion proteins, slightly different from that used in our
studies, could activate NF-
B. Taken together, the results of their
study and ours support a unifying model for understanding how two
distinct translocations may impact the same signal transduction
pathway, and thereby contribute to the development of a single
clinicopathologic entity, MALT lymphoma.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank R. O'Brien, L. Bischof, K. Orth, and T. Koseki for numerous suggestions and advice throughout this project; F. Mercurio, H. Ichijo, and A. Kimchi for plasmids; Victor Rivera (Ariad Pharmaceuticals) for providing FKBP plasmids and the dimerization agent AP1510; A. Berg for the kind gift of RelA-deficient MEFs; and all members of the Núñez lab for their helpful thoughts.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant CA84064 and Michigan Life Sciences Corridor Fund Grant 1506 (to G. N.).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.
§ Supported by a University of Michigan Cancer Biology Training Program Grant and individual NRSA Grant F32-CA88470-01 from the National Institutes of Health.
To whom correspondence should be addressed: Dept.
of Pathology, University of Michigan Medical School, Ann Arbor, MI
48109. Tel.: 734-764-8514; Fax: 734-647-9654; E-mail:
bclx@umich.edu.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M009984200
3 N. Inohara and G. Núñez, unpublished results.
2 P. C. Lucas and G. Núñez, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MALT, mucosa-associated lymphoid tissue;
Ig, immunoglobulin;
NF-B, nuclear
factor
B;
I
B, inhibitor of NF-
B;
IKK, I
B kinase;
B,
chain B site;
TNF-
, tumor necrosis factor
;
TNFR1, tumor necrosis
factor-
receptor 1;
CARD, caspase recruitment domain;
MEF, mouse
embryonic fibroblast;
FKBP, FK506-binding protein;
PAGE, polyacrylamide
gel electrophoresis.
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