Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1421–1426, February 1999 Cell Biology Specificities of CD40 signaling: Involvement of TRAF2 in CD40-induced NF-kB activation and intercellular adhesion molecule-1 up-regulation HO H. LEE*, PAUL W. DEMPSEY*, THOMAS P. PARKS†‡,XIAOQING ZHU*, DAVID BALTIMORE§, AND GENHONG CHENG*¶ *Department of Microbiology and Molecular Genetics, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California, Los Angeles, CA 90095; †Department of Inflammatory Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877; and §California Institute of Technology, Pasadena, CA 91125 Contributed by David Baltimore, December 22, 1998 ABSTRACT Several tumor necrosis factor receptor- not TRAFs 1, 3, and 4, in transient transfection experiments associated factor (TRAF) family proteins including TRAF2, activates both the NF-kB and stress-activated protein kinase TRAF3, TRAF5, and TRAF6, as well as Jak3, have been (SAPK) signal transduction pathways (7, 8, 11, 13, 16, 17). implicated as potential mediators of CD40 signaling. An To better understand the contributions of the different extensive in vitro binding study indicated that TRAF2 and CD40ct-interacting adapter molecules to specific CD40 sig- TRAF3 bind to the CD40 cytoplasmic tail (CD40ct) with much naling pathways and CD40-mediated biology, we initiated our higher affinity than TRAF5 and TRAF6 and that TRAF2 and studies by examining the relative binding affinities of TRAFs TRAF3 bind to different residues of the CD40ct. Using CD40 2, 3, 5, and 6 and Jak3 with the CD40ct. It was found that the mutants incapable of binding TRAF2, TRAF3, or Jak3, we binding affinities of TRAFs 2 and 3 for the CD40ct are much found that the TRAF2-binding site of the CD40ct is critical for greater than those of TRAFs 5 and 6. Using human CD40 NF-kB and stress-activated protein kinase activation, as well mutant constructs that specifically abolish binding of TRAF2, as the up-regulation of the intercellular adhesion molecule-1 TRAF3, or Jak3 to the CD40ct, we also found that the (ICAM-1) gene, whereas binding of TRAF3 and Jak3 is TRAF2-binding site in the CD40ct is required for CD40- dispensable for all of these functions. Overexpression of a mediated NF-kB and SAPK activation. Finally, we demon- k a dominantly active I B strongly inhibited CD40-induced strate that CD40-mediated up-regulation of ICAM-1, a cell- k NF- B activation, ICAM-1 promoter activity, and cell-surface adhesion molecule important for cell–cell interactions and ICAM-1 up-regulation. These studies suggest a potential inflammation, is mediated through NF-kB signaling by signal transduction pathway from the CD40 receptor to the TRAF2. transcriptional activation of the ICAM-1 gene. MATERIALS AND METHODS CD40 is a 50-kDa glycoprotein, a member of the tumor necrosis factor receptor (TNFR) superfamily, and is expressed Plasmids and Expression Vectors. Full-length wild-type on a variety of cells including B cells. Studies with X-linked human CD40 and its mutant derivatives were generated with hyper-IgM syndrome, a genetic defect in the corresponding PCR end primers and internal primers with appropriate CD40L gene that is expressed predominantly by CD41 T mutations and cloned into the BamHI and SalI sites of the helper cells, and studies with mice lacking either the CD40 or pBABEpuro vector (18, 19). Glutathione S-transferase (GST) CD40L gene, have demonstrated that interaction between fusion proteins all were constructed in the pGex2T prokaryotic CD40 and CD40L during T and B cell contact is essential for gene fusion expression vector (Pharmacia). The construction all events in T cell-dependent antigen responses, including of GST-C17, C10N, C10C, and the full-length GST-mCD40ct germinal center formation, Ig isotype switching, antibody has been described previously (19). Constructs used for the affinity maturation, memory B cell formation, and T cell alanine-scanning mutagenesis were cloned similarly. Expres- activation (1–4). In cell culture systems, stimulation of CD40 sion was confirmed by Coomassie staining. TRAF constructs receptor with either anti-CD40 antibodies or CD40L rescues B were generated by PCR and cloned into the BamHI and NotI cells from apoptosis, promotes B cell proliferation, and induces sites of the PEBB-Flu-tagged mammalian expression vector. Ig isotype switching in the presence of cytokines (2). The TRAF2 and TRAF3 templates were derived from con- The pleiotropic biological roles mediated by CD40 raise an structs used previously (19). pME-TRAF5 template used for important issue regarding how a single receptor generates PCR was a gift from Jun-ichiro Inoue (University of Tokyo). intracellular signals capable of mediating such diverse biolog- The TRAF6 PCR product, generated from a cDNA library, ical phenomena. Studies using yeast two-hybrid screening and was digested with BglII and NotI and cloned into the BamHI in vitro binding assays have led to the identification of TNFR- and NotI sites of the PEBB-Flu vector. The pGL 1.3, pGL associated factor (TRAF) family proteins 2, 3, 5, and 6 as k k 1.3 B-, and the ( B)3-IFN-LUC luciferase reporters have been proteins associated with CD40 (5–9). In addition, Janus kinase described previously (20, 21). The pBABEpuro CD40L con- 3 (Jak3) also has been reported recently to associate with the struct has also been described previously (19). CD40 cytoplasmic tail (CD40ct) (10). The TRAF family consists of six known family members, TRAFs 1–6 (1, 5–9, Abbreviations: TRAF, tumor necrosis factor receptor-associated fac- 11–15), which are capable of interacting with various TNF and tor; Jak, Janus kinase; CD40ct, CD40 cytoplasmic tail; SAPK, stress- non-TNF receptors. Overexpression of TRAFs 2, 5, and 6, but activated protein kinase; JNK, c-Jun N-terminal kinase; ICAM-1, intercellular adhesion molecule 1; IkB-a, inhibitor of NF-kBa; GST, The publication costs of this article were defrayed in part by page charge glutathione S-transferase; CD40L, CD40 ligand (gp39). ‡Present address: Cellegy Pharmaceuticals, Inc., San Carlos, CA payment. This article must therefore be hereby marked ‘‘advertisement’’ in 94070. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. e-mail: genhongc@ PNAS is available online at www.pnas.org. microbio.ucla.edu. 1421 Downloaded by guest on September 30, 2021 1422 Cell Biology: Lee et al. Proc. Natl. Acad. Sci. USA 96 (1999) Cell Culture and Transfection. 293T cells and BOSC pack- aging cells were maintained in DMEM (GIBCOyBRL) sup- plemented with 10% fetal bovine serum (FBS) and were transfected by standard calcium phosphate methods. A549 human lung carcinoma cells, WEHI 231 murine B cell lym- phoma cells, BI-141 murine T cell hybridoma cell lines, and Daudi human Burkitt’s lymphoma B cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillinystreptomycin. Transient transfections with reporter constructs were performed with the Superfect Transfection Reagent (Qiagen, Chatsworth, CA). Harvested samples were assessed for luciferase activity by measurement with Promega luminol substrate. Infections of the BI-141 and WEHI 231 cells FIG. 1. Characterization of the relative binding affinities of TRAFs 2, 3, 5, and 6 for the CD40 cytoplasmic tail and determination of the were performed by the use of BOSC packaging cells as minimal binding regions within the CD40 C17 region. (A) TRAFs 2 described previously (18, 22). For electroporation, Daudi cells y m and 3 bind to the CD40ct with much greater affinity than TRAFs 5 and were electroporated at 250 V 975 F and selected with 1 6. The same amount of glutathione beads bound with the GST-mouse mgyml of active G418 (Mediatech, Washington, DC). CD40ct fusion protein was incubated with extracts from 293T cells In Vitro Binding Assays, Electrophoretic Mobility-Shift transfected with pEBB-Flu TRAF2, pEBB-Flu TRAF3, pEBB-Flu Assays, and in Vitro Kinase SAPK Assays. GST pull-down TRAF5, or pEBB-Flu TRAF6. The beads were washed, and bound assays were performed as described previously (19). For proteins were detected by Western blot analysis with anti-Flu anti- gel-shift and SAPK assays, cells were treated as indicated in bodies (Fig. 1A Upper). Equal amounts of extracts (5% of input used the text. Nuclear extracts were prepared and used in binding for GST pull-down) were analyzed by Western blot analysis with anti-Flu antibodies to ensure similar levels of expression of the reactions with a g-P32-labeled major histocompatibility com- k different Flu-tagged TRAFs (Fig. 1A Lower). (B) The minimum plex II NF- B specific hairpin oligonucleotide (20) using binding regions of TRAF2 and TRAF3 within the C17 region are previously published protocols (23). Endogenous SAPK ac- different. Lysates from 293T cells expressing Flu-TRAF2 or Flu- tivity was assessed by immunoprecipitation with anti-c-Jun TRAF3 were incubated with GST, GST-C17, GST-C10N, or GST- N-terminal kinase 1(JNK1) polyclonal antibodies (Santa Cruz C10C fusion proteins immobilized on glutathione beads. The bound Biotechnology) using previously published protocols (24). proteins then were analyzed by Western blot analysis with anti-Flu Fluorescence-Activated Cell Sorter (FACS) Analysis. To antibodies. assess expression of the human CD40 constructs, BI-141 infectants and parental cells were stained with fluorescein (C17) corresponding to amino acid residues 230–246 of the isothiocyanate (FITC)-conjugated murine anti-human CD40 CD40ct that could bind to TRAF3 as strongly as the entire mAb (clone 5C3; PharMingen) and analyzed by FACScan flow CD40ct (19). This C17 peptide also was capable of binding to cytometer. To assess ICAM-1 induction, WEHI231 or Daudi TRAF2 as strongly as the full-length CD40ct (Fig. 1B). This cells were stimulated for 24 hr with 1 mgyml of G28.5 or with C17 fragment was dissected further into two overlapping soluble CD40L.