Correspondence to: Pasquale Vito, Basel Institute for Immunology, Grenzacherstrasse 487, Postfach CH-4005, Basel, Switzerland. Tel:061-605-1349 Fax:061-605-1364 E-mail:Vito{at}bii.ch.
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Abstract |
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Mucosa-associated lymphoid tissue (MALT) lymphomas are associated with overexpression and constitutive activity of bcl10, a caspase recruitment domain (CARD)-containing protein that activates NF-B. Here, we show that arrangement of overexpressed bcl10 protein in cytoplasmic filaments is essential for recruitment of signal transducer molecules-involved NF-
B activation. We also show that cytoskeleton elements regulate bcl10 signaling.
Thus, organized assemblage of proteins in ordered structures linked to the cytoskeleton network may represent a general mechanism for intracellular signaling.
Key Words:
bcl10, filaments, mucosa-associated lymphoid tissue, NF-B, scaffold
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Introduction |
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Mucosa-associated lymphoid tissue (MALT)1 lymphomas with t(1;14)(p22;q32) display a recurrent breakpoint upstream of bcl10 that juxtaposes the gene under the control of the Ig heavy chain enhancer (Binducing activity, transform primary rat embryonic fibroblasts (
Here, we show that bcl10 protein assemblates in cytoplasmic filaments that serve as scaffold for recruitment of NF-Bactivating signal transduction molecules. Cytochalasin D, a potent inhibitor of actin filament function, disassembles bcl10 filaments and specifically inhibits bcl10-induced NF-
B activation. Thus, assemblage of bcl10 in filaments connected to the cytoskeleton network is essential for bcl10-mediated NF-
B induction.
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Materials and Methods |
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Cell Culture, Reagents and Luciferase Assay
HeLa, HEK 293, and Rat-2 cells were cultured in DME 10% FCS. HeLa and Rat-2 cells were transfected using lipofectamine (GIBCO BRL); 293 cells were transfected by calcium phosphate precipitation. To assess NF-B activation, HeLa and Rat-2 cells were transfected with 2 µg of the indicated cDNAs, together with pNF-
B-luc in 12-well plates. Cells were then lysed and luciferase activity was determined with the Luciferase Assay System (Promega). A plasmid expressing ß-galactosidase was added to the transfection mixture for normalization of the efficiency of transfection. Cytochalasin D was obtained from Sigma Chemical Co.
Plamids Construction and Reagents
PCR-based random mutagenesis of bcl10 was carried out in the following reaction buffer: 10 mM Tris-HCl, pH 8, 50 mM KCl, 0.5 mM MnCl2, 125 µM dNTPs. Reaction was performed using 2.5 U standard Taq polymerase, 200 ng of plasmidic template, and the oligos 5'-AAGAATTCCATGGAGCCCACCGCACC (forward) and 5'-AACTCGAGTCATGGAAAAGGTTCACAACTGCT (reverse). PCR products were purified and cloned in pcDNA3 expression vector (Invitrogen) provided with an HA epitope. The vectors pNF-B-luc and pCMV-ß-gal were from Clontech.
Immunofluorescence
HeLa cells were grown and transfected in chamber slides. 16 h after transfection, cells were fixed in 4% paraformaldehyde for 15 min at room temperature and then permeabilized in PBS/0.1% Triton X-100. Primary antibodies were incubated for 30 min in 5% FCSPBS, followed by several washes with 5% FCSPBS, and then incubating for 30 min with secondary antibody in 5% FCSPBS. All steps were done at room temperature. Sources of antibodies and reagents for immunofluorescence were: anti-HA (Roche Molecular Biochemicals); anti-FLAG and anti-actinin (Sigma Chemical Co.); anti-TRADD and anti-RIP (Santa Cruz).
Two-Hybrid Screen and ß-Gal Assays
The two-hybrid screening was conducted using the Matchmaker system (Clontech) according to the manufacturer's instructions. In brief, yeast strain HF7c, expressing GAL4-DR4 fused protein, was transformed with a human peripheral blood leukocyte cDNA library cloned in the pGAD10 vector (Clontech) by lithium acetate/PEG 4000 procedure. 2 x 106 clones were analyzed. Transformed yeast were selected on SD/agar plates lacking leucine, tryptophane, and histidine for 5 d at 30°C. Selected colonies were blotted on filter paper, permeabilized in liquid nitrogen, and placed on another filter soaked in Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1mM MgSO4, and 37.5 mM ß-mercaptoethanol) containing 1 mM 5-bromo-4-chloro-3-indolyl-ß-D-galactoside. Colonies that developed color were restreaked on selective plates to allow plasmid segregation and tested again for ß-galactosidase activity. The liquid ß-galactosidase assay was performed according to the manufacturer's instructions using CPRG as substrate.
In Vitro Binding and Immunoblot Analysis
Recombinant histidine-tagged proteins were made in Escherichia coli BL21 strain using the pET expression system (Novagen). Proteins were purified with Ni-NTA agarose beads (Quiagen) and mixed with recombinant -actinin (Sigma Chemical Co.) in E1A buffer (50 mM Hepes, 500 nM NaCl, 0.1% NP-40, and 10% glycerol). Samples were incubated for 12 h at 4°C, extensively washed by pulse centrifugation with E1A buffer and resuspended in 40 µl sample buffer. 5 µl of the reaction was loaded for SDS-PAGE and Western blot analysis.
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Results and Discussion |
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To determine the cellular localization of oncogenic bcl10, we transfected HeLa cells with an HA-tagged vector encoding the full-length bcl10, and the expressed protein was detected using a monoclonal anti-HA antibody. The results in Fig 1 show that bcl10 exhibits a clear pattern of discrete and interconnecting cytoplasmic filaments resembling the death-effector filaments (
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The NH2-terminal region of bcl10 contains a CARD motif (B induction (
Binducing activity, these experiments suggested a correlation between filaments formation and bcl10-mediated NF-
B activation. To further explore this possibility, we generated the point mutants bcl10(L41Q) and bcl10(G78R), because mutations of corresponding residues in the CARD of the cell death-inducing proteins, CED-3 and RAIDD, abrogate their functional activity (
B (Fig 2 B).
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bcl10 filament formation is not an effect of NF-B activation because when bcl10 was cotransfected with dominant negative forms of I
B and NIK, which abrogate bcl10-induced NF-
B activation (
To determine whether bcl10 filaments are unequivocally linked to NF-B activation, we generated a panel of random mutations in the CARD region of the protein. Microscopy and functional analysis of these mutants indeed revealed that only bcl10 mutants still able to form filaments could activate NF-
B (Fig 3).
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Previously, we and others have shown that bcl10 activates NF-B through a molecular pathway involved in tumor necrosis factor receptor 1 (TNF-R1) signaling (
B activation (
B signaling (
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To assess whether cytoskeleton dynamics influence bcl10 activity, HeLa cells expressing bcl10 were treated with cytochalasin D, a potent inhibitor of actin filament function, and bcl10 localization was determined. As shown in Fig 5, cytochalasin D disassembles bcl10 filaments and specifically inhibits bcl10-induced NF-B activation, whereas NF-
B activation induced by TNF stimulation or NIK overexpression is not affected. Thus, assemblage of bcl10 in filaments is essential for bcl10-mediated NF-
B induction.
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bcl10 filament formation could either reflect an intrinsic property of that protein or be ordered via interactions with preexisting filamentous proteins. The observation that cytochalasin D disassembles bcl10 filaments prompted us to explore the possibility these filaments might be organized by CARD-mediated association with cytoskeletal components. To test for this, we performed a two-hybrid screen fusing the CARD of bcl10 to the DNA binding domain of GAL4 and searched a plasmid library of fusion between the GAL4 transcription activation domain and cDNAs from peripheral blood leukocytes. 13 independent clones were isolated that activated the ß-galactosidase reporter gene when ~2 x 106 transformants were analyzed. Restriction mapping and partial sequencing of these 13 cDNAs revealed that six positive clones had the same ~2-kb cDNA insert encoding for the polypeptide Pro355Leu892 of -actinin, a member of the actin-binding proteins superfamily that is thought to cross-link actin filaments. As summarized in Table 1, a library plasmid encoding for Pro355Leu892 of
-actinin did not activate ß-galactosidase by itself, when coexpressed with the empty GAL4BD vector, or with unrelated control plasmids. Conversely, it strongly interacted with the CARD of bcl10. However, both wild-type and mutant forms of bcl10 bind similarly to
-actinin in yeast and in vitro (Table 1 and Fig 6 A). Together with cytochalasin D experiments, the two-hybrid data suggest that interaction of bcl10 with cytoskeleton components is necessary, but not sufficient for filament formation.
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Next, we tested whether filament formation could result from bcl10 multimerization. Immunoprecipitation analysis shown in Fig 6 B revealed that wild-type bcl10 self-associates via a CARD-mediated homophilic interaction, however, both mutants bcl10(L41Q) and bcl10(G78R) did not dimerize, suggesting that bcl10 filaments result from self-assembly. Thus, both dimerization of bcl10 and binding to -actinin are necessary for filament formation and NF-
B activation.
bcl10 is expressed in many normal tissues and overexpression of the gene causes cellular transformation (B activation both derive from disregulated level of expression of the protein. Indeed, in Rat-2 cells, endogenous bcl10 displays a diffuse cytosolic distribution, whereas overexpression of the gene induces filament formation and NF-
B activation (Fig 7). These results, however, do not exclude the possibility that local micro-assembling of the protein may physiologically occur in normal conditions or after specific stimulation.
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Independent evidence suggests that the transforming capability of bcl10 is the result of a nonreceptor-mediated NF-B activation arising from deregulated expression of the gene (
Binducing activity of the protein. Similarly to scaffolding and anchoring proteins that determine selective activation of mitogen-activated protein kinases by sequestration and localization of signaling complexes, filamentous arrangement of bcl10 is required for recruitment of TRADD, and may be essential for local concentration and activation of other downstream signaling proteins. Hence, as recruitment of TRADD to TNF-RI complex initiates a signal transduction pathway leading to NF-
B activation, the same signaling pathway can be activated upon TRADD recruitment to different cellular compartments.
Cytoskeletal-like shape of bcl10 filaments and direct interaction of bcl10 with cytoskeletal proteins suggest that cytoskeleton dynamics may regulate bcl10 signaling. Indeed, activation of the NF-B transcription factors is directly influenced by changes in the cytoskeleton network (
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Footnotes |
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1 Abbreviations used in this paper: CARD, caspase recruitment domain; MALT, mucosa-associated lymphoid tissue; TNF, tumor necrosis factor; TNF-R1, TNF receptor 1.
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Acknowledgements |
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We thank Drs. J. Andersson, J. Pieters and L. D'Adamio for comments and critical review of the manuscript.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche and Co. Ltd., CH-4005 Basel, Switzerland.
Submitted: 29 November 1999
Revised: 27 January 2000
Accepted: 11 February 2000
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References |
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