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Cloning and characterization of a cDNA encoding the human homolog of tumor necrosis factor receptor-associated factor 5 (TRAF5)
Gene 207 (1998) 135–140
Cloning and characterization of a cDNA encoding the human homolog of tumor necrosis factor receptor-associated factor 5 (TRAF5) Sei-ichi Mizushima a, Mikako Fujita b, Takaomi Ishida b, Sakura Azuma b, Katsuaki Kato a, Momoki Hirai c, Masami Otsuka d, Tadashi Yamamoto b, Jun-ichiro Inoue b,* a Biosciences Research Laboratory, Mochida Pharmaceutical Co. Ltd, 1-1-1 Kamiya, Kita-ku, Tokyo 115, Japan b Department of Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan c Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan d Laboratory of Synthetic Medicinal Chemistry, Faculty of Pharmaceutical Science, Kumamoto University, 5-1 Ohehonmachi, Kumamoto-shi, Kumamoto 862, Japan Received 3 June 1997; received in revised form 22 October 1997; accepted 27 October 1997; Received by I.M. Verma
Abstract A cDNA encoding the human homolog of the tumor necrosis factor receptor-associated factor 5 ( TRAF5) protein has been molecularly cloned from a cDNA library of Human Daudi B cell line. The sequence analysis revealed that the cDNA encoded a protein of 557 aa residues with a calculated molecular weight of 64 236. The encoded protein has typical structural characteristics shown in the TRAF family of proteins and binds to the cytoplasmic region of lymphotoxin-b receptor more efficiently than to that of CD40 and CD30. The TRAF5 gene was mapped to the human chromosome 1q32.3-q41.1. Overexpression of human TRAF5 activates NFkB transcription factor in human 293T kidney cells. These results suggest that the human TRAF5 protein could be involved in the signal triggered by various members of the tumor necrosis factor receptor (TNFR) superfamily including CD40, CD30 and lymphotoxin-b receptor. © 1998 Elsevier Science B.V. Keywords: Signal transduction; CD40; Protein–protein interaction; Nuclear factor kB; Chromosomal localization
1. Introduction The tumor necrosis factor ( TNF ) receptor-associated factor ( TRAF ) family of proteins is involved in transducing signals from various members of the TNF receptor ( TNFR) superfamily (Bazzoni and Beutler, 1995). We have recently cloned cDNAs encoding the mouse TRAF5 protein (mTRAF5) and TRAF6 protein (mTRAF6) via a yeast two-hybrid system using the cytoplasmic domain of CD40 as bait (Ishida et al., 1996a,b). In vitro binding study revealed that TRAF5 binds to the cytoplasmic domain of CD40, CD30 and lymphotoxin-b receptor (Ishida et al., 1996b; Aizawa
et al., 1997; Nakano et al., 1996). Furthermore, TRAF5 lacking a RING finger domain acts as a dominant negative mutant to suppress CD40-mediated induction of CD23 expression in the WEHI231 mouse premature B cell line, suggesting that TRAF5 mediates CD40 signaling (Ishida et al., 1996b). To elucidate the role of TRAF5 in human and compare the structure of mouse and human TRAF5, we have moleculary cloned a cDNA for human TRAF5 and characterized its product.
2. Experimental and discussion 2.1. Structure of human TRAF5
* Corresponding author. Tel.: +81 3 54495302; Fax: +81 3 54495413; e-mail: [email protected] Abbreviations: CAT, chloramphenicol acetyltransferase; GST, glutathione-S transferase; NFkB, nuclear factor kB; TNFR, tumor necrosis factor receptor; TRAF, tumor necrosis factor receptor-associated factor. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 6 16 - 1
From 1.2×106 clones of a human Barkitt’s lymphoma Daudi cDNA library, nine independent clones were hybridized with 32P-labeled mouse TRAF5 cDNA ( Ishida et al., 1996b). Since restriction mapping analysis revealed that clone 5 included the sequences of the other
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Fig. 1. Structure of the human TRAF5 protein. (A) Homology between the human and mouse TRAF5 protein. The RING finger, Zn fingers, coiled-coil and TRAF-C domains are indicated by arrows. The amino acids sequences were compared using ‘Gap’, as implemented in the University of Wisconsin GCG program (Devereux et al., 1984). Identity and similarity of amino acids are indicated by vertical lines and dots, respectively. A full-length mouse TRAF5 cDNA was used as a probe to screen a Burkitt’s B cell lymphoma line Daudi cDNA library in lgt 11 (Clontech). After hybridization, the filters were washed with 1×SSC/0.1% (w/v) SDS at 50°C for 30 min. (B) Diagram of the domain structure of the human TRAF5 protein. Identities and similarities (parentheses) of entire molecule and those of each domain between the human and mouse TRAF5 protein are shown.
eight clones, cDNA clone 5 was subjected to further analysis. The complete nucleotide sequence has been deposited in the GenBank database (Accession No.
AB000509). An open reading frame encoding a predicted protein of 557 aa (calculated molecular weight of 64 236) was found. The encoded protein has typical
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Fig. 2. Northern blot analysis of the human TRAF5 mRNA. Twelve micrograms of poly(A)+ RNA extracted from human B cell lines Daudi and Raji, were separated by 1% formaldehyde denaturing agarose gel and transferred onto nylon membrane (Hybond N, Amersham) as described (Ishida et al., 1995). The filter was incubated with 32P-labeled DNA fragment corresponding to the first 500 bp of human TRAF5 cDNA. The filter was finally washed with 0.5×SSC/0.2% SDS at 65°C for 30 min.
characteristics previously shown in the TRAF family of proteins: it has the RING finger, Zn finger, coiled-coil and TRAF-C domains (Rothe et al., 1994). The alignment of amino acid sequence of mouse TRAF5 with that of the encoded protein is shown in Fig. 1A. Overall identity is 79.8%, which is less than that between mouse and human TRAF3 (95.0% identity) (Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Sato et al., 1995). However, identities of the RING finger and that of the TRAF-C domain are 92.5% and 91.6%, respectively ( Fig. 1B). Thus, we conclude that the cDNA cloned could encode the human TRAF5 homolog. Identities of the Zn finger and coiled-coil domain are 69.3% and 72.7%, respectively, which are less conserved than the RING finger and TRAF-C domain. The length of human TRAF5 cDNA obtained was 3993 bp, including a long 3∞-untranslated region of approx. 2300 bp. Northern blotting of poly(A)+ RNA from human B cell lines, Daudi and Raji, revealed that the human TRAF5 mRNA could be 4.2 or 7.5 kb (Fig. 2). Thus, the TRAF5 cDNA obtained could be derived from 4.2 kb mRNA. Whether the longer transcript contains additional untranslated sequences or encodes a TRAF5-related protein is not known. 2.2. Chromosomal mapping of the human TRAF5 gene Chromosomal localization of the cDNA probe for the human TRAF5 gene was performed by fluorescence in situ hybridization (FISH ) as described previously (Hirai et al., 1996). In total, 63 metaphase spreads were
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Fig. 3. Chromosomal localization of the human TRAF5 gene. Partial metaphase showing the hybridization signals of the human TRAF5 cDNA on chromosome 1 at band q32.3-q41.1 (arrow). The biotinylated cDNA probe for human TRAF5 was hybridized to R-banded chromosomes prepared from PHA-stimulated lymphocytes of normal donors. After overnight hybridization at 37°C, the slides were washed in 50% formamide/2×SSC at 42°C for 10 min, followed by a wash in 1×SSC at room temperature for 15 min. Hybridization signals were amplified using rabbit antibiotin ( ENZO), fluorescein-labeled goat anti-rabbit IgG ( ENZO) and Cy2-labeled donkey anti-goat IgG (Amersham). The chromosomes were counterstained with propidium iodide.
analyzed. Thirty-five specific hybridization signals were localized to chromosome 1 at band q32.3-q41.1, with peak distribution at 1q32.3 (Fig. 3). No other hybridization sites were found. From these results, the TRAF5 gene was assigned to 1q32.3-q41.1. Although several human disorders including van der Woude syndrome (van der Woude, 1954), cardiomyopathy (Durand et al., 1995), Ripping muscle disease 1 ( Torbergsen, 1975) and Usher syndrome 2A ( Kimberling et al., 1995), are mapped to this locus, the relationship of the TRAF5 gene with these disorders is not known. 2.3. Expression of human TRAF5 mRNA in various tissues To analyze the expression level of the TRAF5 mRNA in various human tissues, quantitative RT–PCR was performed, since the amount of TRAF5 mRNA in human tissue is not abundant enough to be measured by Northern blotting. The TRAF5 fragment was amplified using the oligo-dT-primed cDNA pools synthesized from poly(A)+ RNA of various human tissues (Clontech, Palo Alto, CA) as templates ( Fig. 4A). To characterize the quality of cDNA pools used for templates, the b-actin fragment was also amplified ( Fig. 4B).
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Fig. 4. Analysis of TRAF5 mRNA expression in human tissues. One nanogram of the oligo-dT-primed cDNA pool synthesized from poly(A)+ RNA of various human tissues (Clontech) was used for PCR reaction (30 s denaturation at 94°C, 30 s annealing at 55°C, 1 min elongation at 72°C, 30 cycles). The TRAF5 fragment (1074 bp) was amplified by a sense primer (nucleotide 750–769) and an antisense primer (nucleotide 1804–1823) (A). The b-actin fragment (838 bp) was amplified from 0.1 mg of cDNA by a 5∞ primer (nucleotide 294–325) and a 3∞ primer (nucleotide 1100–1131) purchased from Clontech (B). The reaction mixture was analyzed on a 1.5% agarose gel. l phage DNA digested with EcoT14 I was used as a molecular weight marker (M.W.).
The human TRAF5 mRNA is easily detected in various tissues except brain. It is highly expressed in adrenal gland and thyroid gland as well as spleen, suggesting that TRAF5 could mediate signals triggered by some receptors which play a role in the specific function of these organs. Additional bands amplified from leukocyte cDNA could be alternate spliced products, since similar pattern was observed when we changed the 5∞-primer for amplification (data not shown).
2.4. Binding of human TRAF5 to various members of the TNF receptor superfamily The mouse TRAF5 protein could be involved in the signal derived from CD40, CD30 and lymphotoxin-b receptor (Ishida et al., 1996b; Aizawa et al., 1997; Nakano et al., 1996). To examine whether human TRAF5 could also mediate these signals, binding of human TRAF5 to the cytoplamic tail of CD40, CD30 and the lymphotoxin-b receptor was analyzed. The fusion protein of human TRAF5 with the maltose binding protien (MBP) was expressed in bacteria and was purified by passing through an amylose resin column. The MBP–TRAF5 was mixed with the fusion protein of glutathione-S-transferase (GST ) with the cytoplasmic tail of indicated receptors immobilized to agarose beads, and the amount of TRAF5 bound to each receptor was analyzed by Western blotting using anti-TRAF5 antibody (Fig. 5). TRAF5 bound to the lymphotoxin-b receptor more efficiently than to CD40 and CD30.
2.5. Human TRAF5 overexpression activates NFkB It has been demonstrated that previously identified TRAF family proteins except TRAF3 and TRAF1, mediate signals linked to NFkB activation (Rothe et al., 1995; Nakano et al., 1996; Ishida et al., 1996a,b). To examine the possible role of human TRAF5 in NFkB activation, transient transfection experiments were performed to determine whether human TRAF5 expression might lead to activation of transcription from a kB-sitedependent reporter gene. The kB-site-dependent reporter construct ([kB] TK-CAT ) (Inoue et al., 1991) was cot6 ransfected with a human TRAF5 expression vector (pME-FLAG-TRAF5) into human 293T cells. To confirm the specificity of transcription, the same reporter construct carrying mutant kB sites ([kBM ] TK-CAT ) 6 was transfected under the same conditions as the wild type reporter construct. The human TRAF5 protein activates kB-site-dependent transcription in a dosedependent manner ( Fig. 6), indicating that human TRAF5 could mediate the signal linked to NFkB activation. It has been shown that signal emanating from CD40 (Berberich et al., 1994), CD30 (Biswas et al., 1995) or the lymphotoxin-b receptor (Nakano et al., 1996) is linked to NFkB activation. Furthermore, human TRAF5 binds to the cytoplasmic tail of CD40, CD30 and the lymphotoxin-b receptor, as demonstrated in this paper. Thus, our data strongly suggest that human TRAF5 could mediate signals emanating from the various members of the TNFR superfamily including CD40, CD30 and the lymphotoxin-b receptor. During the preparation of this manuscript, Nakano et al. (1997)
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Fig. 6. NFkB activation by human TRAF5 in 293T cells. 293T cells (106) were transfected with 1 mg of [kB] TK-CAT (open circles) or 6 [kBM ] TK-CAT (closed circles), 1 mg of pbactin–bgal, the indicated 6 amounts of pME-FLAG-hTRAF5 and enough pME18S control plasmid to give 10 mg of total DNA by the calcium phosphate method. CAT assays were performed as described (Inoue et al., 1991). The percentage values of conversion were calculated from the results which were in the linear range of CAT activity (<60%). Experiments were performed three times and the representative result was shown.
Fig. 5. Binding of the human TRAF5 protein to the cytoplasmic tail of CD40, CD30 and lymphotoxin-b receptor. (A) Binding of human TRAF5 to the cytoplasmic tail of various receptors. The full-length human TRAF5 protein was expressed as fusion protein with the maltose binding protein (MBP) in bacteria. The MBP–TRAF5 protein recovered in the soluble fraction was purified by passing through amylose resin column. The purified MBP–TRAF5 was incubated in the presence (+) or absence (−) of the fusion protein of GST with the cytoplasmic tail of indicated receptors attached to beads. After washing, the amount of TRAF5 bound was analyzed by Western blotting using anti-TRAF5 antibody (sc-6195, Santa Cruz). An asterisk denotes background bands derived from the GST–LTbR fraction. (B) Analysis of GST fusion proteins. Each GST fusion protein used was analyzed on 10% acrylamide–SDS gel. Dots indicate the full-length GST or GST fusion proteins.
reported the molecular cloning of the human TRAF5 cDNA including chromosomal aassignment. However, we have shown here that human TRAF5 binds to various members of the TNFR family with a different affinity using the purified human TRAF5 protein and receptors.
3. Conclusions (1) A human cDNA encoding the TRAF5 protein was isolated. (2) The human TRAF5 gene is localized on chromosome 1q32.3-q41.1. (3) Human TRAF5 is expressed in various tissues.
(4) Human TRAF5 binds more efficiently to the cytoplasmic tail of lymphotoxin-b receptor than to that of CD40 and CD30. (5) Human TRAF5 mediates signal linked to NFkB activation.
Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan, a Grant-in-Aid from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists and a Grant-in-Aid for AIDS Research from the Japan Health Sciences Foundation.
References S. Aizawa, H. Nakano, T. Ishida, R. Horie, M. Nagai, K. Ito, H. Yagita, K. Okumura, J. Inoue, T. Watanabe, 1997. TRAF5 and TRAF2 are included in CD30 mediated NFkB activation. J. Biol. Chem. 272 2042-2045 Bazzoni, F., Beutler, B., 1995. How do tumor necrosis factor receptors work? J. Inflammation 45, 221–238. Berberich, I., Shu, G.L., Clark, E.A., 1994. Crosslinking of CD40 on B cells rapidly activates nuclear factor-kB. J. Immunol. 153, 4357–4366. Biswas, P., Smith, C.A., Goletti, D., Hardy, E.C., Jackson, R.W., Fauci, A.S., 1995. Cross-linking of CD30 induces HIV expression in chronically infected T cells. Immunity 2, 587–596. Cheng, G., Cleary, A.M., Ye, Z.-e., Hong, D.I., Lederman, S., Baltimore, D., 1995. Involvement of CRAF1, a relative of TRAF, in CD40 signaling. Science 267, 1494–1497. Devereux, J., Haeberli, P., Smithies, O., 1984. A comprehensive set of
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sequence analysis program for the VAX. Nucl. Acids Res. 12, 387–395. Durand, J.-B., Bachinski, L.L., Bieling, L.C., Czernuszewicz, G.Z., Abchee, A.B., Yu, Q.T., Tapscott, T., Hill, R., Ifegwu, J., Marian, A.J., Brugada, R., Daiger, S., Gregoritch, J.M., Anderson, J.L., Quinones, M., Towbin, J.A., Roberts, R., 1995. Localization of a gene responsible for familial dilated cardiomyopathy to chromosome 1q32. Circulation 92, 3387–3389. Hirai, M., Kusuda, J., Hashimoto, K., 1996. Assignment of human ADP ribosylation factor (ARF ) genes ARF1 and ARF3 to chromosomes 1q42 and 12q13, respectively. Genomics 34, 263–265. Hu, H.M., O’Rourke, K., Boguski, M.S., Dixit, V.M., 1994. A novel RING finger protein interacts with the cytoplasmic domain of CD40. J. Biol. Chem. 269, 30069–30072. Inoue, J., Kerr, L.D., Ransone, L.J., Bengal, E., Hunter, T., Verma, I.M., 1991. c-rel activates but v-rel suppresses transcription from kB sites. Proc. Natl. Acad. Sci. USA 88, 3715–3719. Ishida, T., Kobayashi, N., Tojo, T., Ishida, S., Yamamoto, T., Inoue, J., 1995. CD40 signaling-mediated induction of Bcl-X , Cdk4 and L Cdk6: implication of their cooperation in selective B cell growth. J. Immunol. 155, 5527–5535. Ishida, T., Mizushima, S., Azuma, S., Kobayashi, N., Tojo, T., Suzuki, K., Aizawa, S., Watanabe, T., Mosialos, G., Kieff, E., Yamamoto, T., Inoue, J., 1996a. Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region. J. Biol. Chem. 271, 28745–28748. Ishida, T., Tojo, T., Aoki, T., Kobayashi, N., Ohishi, T., Watanabe, T., Yamamoto, T., Inoue, J., 1996b. TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc. Natl. Acad. Sci. USA 93, 9437–9442. Kimberling, W.J., Weston, M.D., Moller, C., van Aarem, A., Cremers,
C.W.R., Sumegi, J., Ing, P.S., Connolly, C., Martini, A., Milani, M., Tamayo, M.L., Bernal, J., Ayuso, C., 1995. Gene mapping of Usher syndrome type IIa: localization of the gene to a 2.1-cM segment on chromosome 1q41. Am. J. Hum. Genet. 56, 216–223. Mosialos, G., Birkenbach, M., Yalamanchili, R., VanArsdale, T., Ware, C., Kieff, E., 1995. The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family. Cell 80, 389–399. Nakano, H., Oshima, H., Chung, W., Williams-Abbott, L., Ware, C.F., Yagita, H., Okumura, K., 1996. TRAF5, an activator of NFkB and putative signal transducer for the lymphotoxin-b receptor. J. Biol. Chem. 271, 14661–14664. Nakano, H., Shindo, M., Yamada, K., Yoshida, M.C., Santee, S.M., Ware, C.F., Jenkins, N.A., Gilbert, D.J., Yagita, H., Copeland, N.G., Okumura, K., 1997. Human TNF receptor-associated factor 5 ( TRAF5): cDNA cloning, expression and assignment of the TRAF5 gene to chromosome 1q32. Genomics 42 ( TRAF5), 26–32. Rothe, M., Sarma, V., Dixit, V.M., Goeddel, D.V., 1995. TRAF2-mediated activation of NF-kB by TNF receptor 2 and CD40. Science 269, 1424–1427. Rothe, M., Wong, S.C., Henzel, W.J., Goeddel, D.V., 1994. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681–692. Sato, T., Irie, S., Reed, J., 1995. A novel member of the TRAF family of putative signal transducing proteins binds to the cytosolic domain of CD40. FEBS Lett. 358, 113–118. Torbergsen, T., 1975. A family with dominant hereditary myotonia, muscular hypertrophy, and increased muscular irritability, distinct from myotonia congenita Thomsen. Acta Neurol. Scand. 51, 225–232. van der Woude, A., 1954. Fistula labii inferioris congenita and its association with cleft lip and palate. Am. J. Hum. Genet. 6, 244–256.
Cloning and characterization of a cDNA encoding the human homolog of tumor necrosis factor receptor-associated factor 5 (TRAF5) Sei-ichi Mizushima a, Mikako Fujita b, Takaomi Ishida b, Sakura Azuma b, Katsuaki Kato a, Momoki Hirai c, Masami Otsuka d, Tadashi Yamamoto b, Jun-ichiro Inoue b,* a Biosciences Research Laboratory, Mochida Pharmaceutical Co. Ltd, 1-1-1 Kamiya, Kita-ku, Tokyo 115, Japan b Department of Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan c Department of Biological Science, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan d Laboratory of Synthetic Medicinal Chemistry, Faculty of Pharmaceutical Science, Kumamoto University, 5-1 Ohehonmachi, Kumamoto-shi, Kumamoto 862, Japan Received 3 June 1997; received in revised form 22 October 1997; accepted 27 October 1997; Received by I.M. Verma
Abstract A cDNA encoding the human homolog of the tumor necrosis factor receptor-associated factor 5 ( TRAF5) protein has been molecularly cloned from a cDNA library of Human Daudi B cell line. The sequence analysis revealed that the cDNA encoded a protein of 557 aa residues with a calculated molecular weight of 64 236. The encoded protein has typical structural characteristics shown in the TRAF family of proteins and binds to the cytoplasmic region of lymphotoxin-b receptor more efficiently than to that of CD40 and CD30. The TRAF5 gene was mapped to the human chromosome 1q32.3-q41.1. Overexpression of human TRAF5 activates NFkB transcription factor in human 293T kidney cells. These results suggest that the human TRAF5 protein could be involved in the signal triggered by various members of the tumor necrosis factor receptor (TNFR) superfamily including CD40, CD30 and lymphotoxin-b receptor. © 1998 Elsevier Science B.V. Keywords: Signal transduction; CD40; Protein–protein interaction; Nuclear factor kB; Chromosomal localization
1. Introduction The tumor necrosis factor ( TNF ) receptor-associated factor ( TRAF ) family of proteins is involved in transducing signals from various members of the TNF receptor ( TNFR) superfamily (Bazzoni and Beutler, 1995). We have recently cloned cDNAs encoding the mouse TRAF5 protein (mTRAF5) and TRAF6 protein (mTRAF6) via a yeast two-hybrid system using the cytoplasmic domain of CD40 as bait (Ishida et al., 1996a,b). In vitro binding study revealed that TRAF5 binds to the cytoplasmic domain of CD40, CD30 and lymphotoxin-b receptor (Ishida et al., 1996b; Aizawa
et al., 1997; Nakano et al., 1996). Furthermore, TRAF5 lacking a RING finger domain acts as a dominant negative mutant to suppress CD40-mediated induction of CD23 expression in the WEHI231 mouse premature B cell line, suggesting that TRAF5 mediates CD40 signaling (Ishida et al., 1996b). To elucidate the role of TRAF5 in human and compare the structure of mouse and human TRAF5, we have moleculary cloned a cDNA for human TRAF5 and characterized its product.
2. Experimental and discussion 2.1. Structure of human TRAF5
* Corresponding author. Tel.: +81 3 54495302; Fax: +81 3 54495413; e-mail: [email protected] Abbreviations: CAT, chloramphenicol acetyltransferase; GST, glutathione-S transferase; NFkB, nuclear factor kB; TNFR, tumor necrosis factor receptor; TRAF, tumor necrosis factor receptor-associated factor. 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 6 16 - 1
From 1.2×106 clones of a human Barkitt’s lymphoma Daudi cDNA library, nine independent clones were hybridized with 32P-labeled mouse TRAF5 cDNA ( Ishida et al., 1996b). Since restriction mapping analysis revealed that clone 5 included the sequences of the other
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Fig. 1. Structure of the human TRAF5 protein. (A) Homology between the human and mouse TRAF5 protein. The RING finger, Zn fingers, coiled-coil and TRAF-C domains are indicated by arrows. The amino acids sequences were compared using ‘Gap’, as implemented in the University of Wisconsin GCG program (Devereux et al., 1984). Identity and similarity of amino acids are indicated by vertical lines and dots, respectively. A full-length mouse TRAF5 cDNA was used as a probe to screen a Burkitt’s B cell lymphoma line Daudi cDNA library in lgt 11 (Clontech). After hybridization, the filters were washed with 1×SSC/0.1% (w/v) SDS at 50°C for 30 min. (B) Diagram of the domain structure of the human TRAF5 protein. Identities and similarities (parentheses) of entire molecule and those of each domain between the human and mouse TRAF5 protein are shown.
eight clones, cDNA clone 5 was subjected to further analysis. The complete nucleotide sequence has been deposited in the GenBank database (Accession No.
AB000509). An open reading frame encoding a predicted protein of 557 aa (calculated molecular weight of 64 236) was found. The encoded protein has typical
S-i. Mizushima et al. / Gene 207 (1998) 135–140
Fig. 2. Northern blot analysis of the human TRAF5 mRNA. Twelve micrograms of poly(A)+ RNA extracted from human B cell lines Daudi and Raji, were separated by 1% formaldehyde denaturing agarose gel and transferred onto nylon membrane (Hybond N, Amersham) as described (Ishida et al., 1995). The filter was incubated with 32P-labeled DNA fragment corresponding to the first 500 bp of human TRAF5 cDNA. The filter was finally washed with 0.5×SSC/0.2% SDS at 65°C for 30 min.
characteristics previously shown in the TRAF family of proteins: it has the RING finger, Zn finger, coiled-coil and TRAF-C domains (Rothe et al., 1994). The alignment of amino acid sequence of mouse TRAF5 with that of the encoded protein is shown in Fig. 1A. Overall identity is 79.8%, which is less than that between mouse and human TRAF3 (95.0% identity) (Hu et al., 1994; Cheng et al., 1995; Mosialos et al., 1995; Sato et al., 1995). However, identities of the RING finger and that of the TRAF-C domain are 92.5% and 91.6%, respectively ( Fig. 1B). Thus, we conclude that the cDNA cloned could encode the human TRAF5 homolog. Identities of the Zn finger and coiled-coil domain are 69.3% and 72.7%, respectively, which are less conserved than the RING finger and TRAF-C domain. The length of human TRAF5 cDNA obtained was 3993 bp, including a long 3∞-untranslated region of approx. 2300 bp. Northern blotting of poly(A)+ RNA from human B cell lines, Daudi and Raji, revealed that the human TRAF5 mRNA could be 4.2 or 7.5 kb (Fig. 2). Thus, the TRAF5 cDNA obtained could be derived from 4.2 kb mRNA. Whether the longer transcript contains additional untranslated sequences or encodes a TRAF5-related protein is not known. 2.2. Chromosomal mapping of the human TRAF5 gene Chromosomal localization of the cDNA probe for the human TRAF5 gene was performed by fluorescence in situ hybridization (FISH ) as described previously (Hirai et al., 1996). In total, 63 metaphase spreads were
137
Fig. 3. Chromosomal localization of the human TRAF5 gene. Partial metaphase showing the hybridization signals of the human TRAF5 cDNA on chromosome 1 at band q32.3-q41.1 (arrow). The biotinylated cDNA probe for human TRAF5 was hybridized to R-banded chromosomes prepared from PHA-stimulated lymphocytes of normal donors. After overnight hybridization at 37°C, the slides were washed in 50% formamide/2×SSC at 42°C for 10 min, followed by a wash in 1×SSC at room temperature for 15 min. Hybridization signals were amplified using rabbit antibiotin ( ENZO), fluorescein-labeled goat anti-rabbit IgG ( ENZO) and Cy2-labeled donkey anti-goat IgG (Amersham). The chromosomes were counterstained with propidium iodide.
analyzed. Thirty-five specific hybridization signals were localized to chromosome 1 at band q32.3-q41.1, with peak distribution at 1q32.3 (Fig. 3). No other hybridization sites were found. From these results, the TRAF5 gene was assigned to 1q32.3-q41.1. Although several human disorders including van der Woude syndrome (van der Woude, 1954), cardiomyopathy (Durand et al., 1995), Ripping muscle disease 1 ( Torbergsen, 1975) and Usher syndrome 2A ( Kimberling et al., 1995), are mapped to this locus, the relationship of the TRAF5 gene with these disorders is not known. 2.3. Expression of human TRAF5 mRNA in various tissues To analyze the expression level of the TRAF5 mRNA in various human tissues, quantitative RT–PCR was performed, since the amount of TRAF5 mRNA in human tissue is not abundant enough to be measured by Northern blotting. The TRAF5 fragment was amplified using the oligo-dT-primed cDNA pools synthesized from poly(A)+ RNA of various human tissues (Clontech, Palo Alto, CA) as templates ( Fig. 4A). To characterize the quality of cDNA pools used for templates, the b-actin fragment was also amplified ( Fig. 4B).
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Fig. 4. Analysis of TRAF5 mRNA expression in human tissues. One nanogram of the oligo-dT-primed cDNA pool synthesized from poly(A)+ RNA of various human tissues (Clontech) was used for PCR reaction (30 s denaturation at 94°C, 30 s annealing at 55°C, 1 min elongation at 72°C, 30 cycles). The TRAF5 fragment (1074 bp) was amplified by a sense primer (nucleotide 750–769) and an antisense primer (nucleotide 1804–1823) (A). The b-actin fragment (838 bp) was amplified from 0.1 mg of cDNA by a 5∞ primer (nucleotide 294–325) and a 3∞ primer (nucleotide 1100–1131) purchased from Clontech (B). The reaction mixture was analyzed on a 1.5% agarose gel. l phage DNA digested with EcoT14 I was used as a molecular weight marker (M.W.).
The human TRAF5 mRNA is easily detected in various tissues except brain. It is highly expressed in adrenal gland and thyroid gland as well as spleen, suggesting that TRAF5 could mediate signals triggered by some receptors which play a role in the specific function of these organs. Additional bands amplified from leukocyte cDNA could be alternate spliced products, since similar pattern was observed when we changed the 5∞-primer for amplification (data not shown).
2.4. Binding of human TRAF5 to various members of the TNF receptor superfamily The mouse TRAF5 protein could be involved in the signal derived from CD40, CD30 and lymphotoxin-b receptor (Ishida et al., 1996b; Aizawa et al., 1997; Nakano et al., 1996). To examine whether human TRAF5 could also mediate these signals, binding of human TRAF5 to the cytoplamic tail of CD40, CD30 and the lymphotoxin-b receptor was analyzed. The fusion protein of human TRAF5 with the maltose binding protien (MBP) was expressed in bacteria and was purified by passing through an amylose resin column. The MBP–TRAF5 was mixed with the fusion protein of glutathione-S-transferase (GST ) with the cytoplasmic tail of indicated receptors immobilized to agarose beads, and the amount of TRAF5 bound to each receptor was analyzed by Western blotting using anti-TRAF5 antibody (Fig. 5). TRAF5 bound to the lymphotoxin-b receptor more efficiently than to CD40 and CD30.
2.5. Human TRAF5 overexpression activates NFkB It has been demonstrated that previously identified TRAF family proteins except TRAF3 and TRAF1, mediate signals linked to NFkB activation (Rothe et al., 1995; Nakano et al., 1996; Ishida et al., 1996a,b). To examine the possible role of human TRAF5 in NFkB activation, transient transfection experiments were performed to determine whether human TRAF5 expression might lead to activation of transcription from a kB-sitedependent reporter gene. The kB-site-dependent reporter construct ([kB] TK-CAT ) (Inoue et al., 1991) was cot6 ransfected with a human TRAF5 expression vector (pME-FLAG-TRAF5) into human 293T cells. To confirm the specificity of transcription, the same reporter construct carrying mutant kB sites ([kBM ] TK-CAT ) 6 was transfected under the same conditions as the wild type reporter construct. The human TRAF5 protein activates kB-site-dependent transcription in a dosedependent manner ( Fig. 6), indicating that human TRAF5 could mediate the signal linked to NFkB activation. It has been shown that signal emanating from CD40 (Berberich et al., 1994), CD30 (Biswas et al., 1995) or the lymphotoxin-b receptor (Nakano et al., 1996) is linked to NFkB activation. Furthermore, human TRAF5 binds to the cytoplasmic tail of CD40, CD30 and the lymphotoxin-b receptor, as demonstrated in this paper. Thus, our data strongly suggest that human TRAF5 could mediate signals emanating from the various members of the TNFR superfamily including CD40, CD30 and the lymphotoxin-b receptor. During the preparation of this manuscript, Nakano et al. (1997)
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Fig. 6. NFkB activation by human TRAF5 in 293T cells. 293T cells (106) were transfected with 1 mg of [kB] TK-CAT (open circles) or 6 [kBM ] TK-CAT (closed circles), 1 mg of pbactin–bgal, the indicated 6 amounts of pME-FLAG-hTRAF5 and enough pME18S control plasmid to give 10 mg of total DNA by the calcium phosphate method. CAT assays were performed as described (Inoue et al., 1991). The percentage values of conversion were calculated from the results which were in the linear range of CAT activity (<60%). Experiments were performed three times and the representative result was shown.
Fig. 5. Binding of the human TRAF5 protein to the cytoplasmic tail of CD40, CD30 and lymphotoxin-b receptor. (A) Binding of human TRAF5 to the cytoplasmic tail of various receptors. The full-length human TRAF5 protein was expressed as fusion protein with the maltose binding protein (MBP) in bacteria. The MBP–TRAF5 protein recovered in the soluble fraction was purified by passing through amylose resin column. The purified MBP–TRAF5 was incubated in the presence (+) or absence (−) of the fusion protein of GST with the cytoplasmic tail of indicated receptors attached to beads. After washing, the amount of TRAF5 bound was analyzed by Western blotting using anti-TRAF5 antibody (sc-6195, Santa Cruz). An asterisk denotes background bands derived from the GST–LTbR fraction. (B) Analysis of GST fusion proteins. Each GST fusion protein used was analyzed on 10% acrylamide–SDS gel. Dots indicate the full-length GST or GST fusion proteins.
reported the molecular cloning of the human TRAF5 cDNA including chromosomal aassignment. However, we have shown here that human TRAF5 binds to various members of the TNFR family with a different affinity using the purified human TRAF5 protein and receptors.
3. Conclusions (1) A human cDNA encoding the TRAF5 protein was isolated. (2) The human TRAF5 gene is localized on chromosome 1q32.3-q41.1. (3) Human TRAF5 is expressed in various tissues.
(4) Human TRAF5 binds more efficiently to the cytoplasmic tail of lymphotoxin-b receptor than to that of CD40 and CD30. (5) Human TRAF5 mediates signal linked to NFkB activation.
Acknowledgement This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan, a Grant-in-Aid from the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists and a Grant-in-Aid for AIDS Research from the Japan Health Sciences Foundation.
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