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The Journal of Immunology, 1999, 162: 4720-4730.
Copyright © 1999 by The American Association of Immunologists

An 11-Amino Acid Sequence in the Cytoplasmic Domain of CD40 Is Sufficient for Activation of c-Jun N-Terminal Kinase, Activation of MAPKAP Kinase-2, Phosphorylation of I{kappa}B{alpha}, and Protection of WEHI-231 Cells from Anti-IgM-Induced Growth Arrest1

Claire L. Sutherland, Danielle L. Krebs and Michael R. Gold2

Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35–45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of I{kappa}B{alpha} phosphorylation and degradation. Amino acids 35–45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-{kappa}B, JNK, and p38 MAPK.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD40 is a receptor on B cells that regulates proliferation, survival, Ig class switching, and memory cell formation (1). The ligand for CD40 (CD40L)3 is expressed on activated CD4+ T cells (2). The essential role of the CD40/CD40L interaction in the development of humoral immunity is illustrated by the X-linked hyperIgM syndrome. B lymphocytes from patients with this immunodeficiency disease fail to undergo Ig class switching and do not form germinal centers (3, 4). Similarly, mice lacking CD40 or CD40L are unable to generate secondary humoral immune responses to T cell-dependent Ags (5, 6).

Engagement of CD40 by CD40L or by anti-CD40 Abs activates multiple signaling pathways, including the kinase cascade that activates NF-{kappa}B, the JAK3/STAT3 pathway, the phosphatidylinositol 3-kinase pathway, and the kinase cascades that lead to activation of the ERK, JNK, and p38 mitogen-activated protein kinases (MAPKs) (7, 8, 9, 10, 11, 12, 13). The roles of individual signaling pathways in mediating the effects of CD40 on B cells for the most part remain to be elucidated.

MAPKs are key signaling intermediates that have been implicated in both mitogenic and apoptotic responses to receptor signaling (14). Upon activation, MAPKs translocate to the nucleus where they phosphorylate and activate transcription factors. The three families of MAPKs, the ERK, JNK, and p38 MAPKs, each phosphorylate and activate a different set of transcription factors. The ERKs phosphorylate ETS domain-containing transcription factors such as Elk-1; JNK phosphorylates c-Jun and ATF-2 (activating transcription factor-2) and p38 MAPK phosphorylates ATF-2, MEF2C, and CHOP (14, 15, 16, 17). The p38 MAPK also phosphorylates and activates MAPKAP kinase-2 (18, 19), a serine/threonine kinase whose targets include the heat shock protein hsp25 and the CREB transcription factor (20).

We have previously shown that CD40 activates the JNK and p38 MAPKs as well as MAPKAP kinase-2 in WEHI-231 B lymphoma cells (12). The mechanism by which CD40 activates these kinases is not completely understood. JNK and p38 MAPK are activated by dual specificity kinases called MAPK kinases (MKKs), which phosphorylate both threonine and tyrosine residues in a conserved threonine-X-tyrosine activation motif (21). The MKKs that phosphorylate JNK and p38 MAPK are activated by upstream kinases that are regulated by the Rac and Cdc42 GTPases (22, 23, 24). Several MKKs can phosphorylate both JNK and p38 MAPK, and many stimuli activate both of these MAPKs (25), suggesting that activation of JNK and p38 MAPK reflects the bifurcation of a single pathway. However, some MKKs preferentially activate only JNK (26, 27) or only p38 MAPK (28), and certain stimuli can activate p38 MAPK without the concomitant activation of JNK (29, 30). This raises the possibility that CD40 could use distinct pathways to activate JNK and p38 MAPK.

CD40 is a member of the TNF receptor (TNF-R) superfamily and has no intrinsic enzymatic activity. This suggests that CD40 interacts with adapter proteins that couple it to signaling pathways. Indeed, four members of the TNF-R-associated factor (TRAF) family of adapter proteins, TRAF2, TRAF3, TRAF5, and TRAF6, can bind to the cytoplasmic domain of CD40 (31, 32, 33, 34). When overexpressed in fibroblasts, TRAF2, TRAF5, and TRAF6 can activate both JNK and NF-{kappa}B (32, 33, 34, 35, 36, 37, 38, 39). The ability of these TRAF proteins to activate the p38 MAPK/MAPKAP kinase-2 pathway has not been examined. In addition to the TRAF proteins, two other proteins that associate with CD40 may also link CD40 to signaling pathways. A novel 23-kDa transmembrane protein associates with the extracellular domain of CD40 (40), while the JAK3 tyrosine kinase has been reported to bind to the cytoplasmic domain of human CD40 (9).

To determine which of these CD40-associated proteins might mediate activation of JNK and p38 MAPK as well as activation of NF-{kappa}B and prosurvival pathways in B cells, our approach was to map the regions of the CD40 cytoplasmic domain that are responsible for activating these signaling pathways. The cytoplasmic domain of murine CD40 contains 74 amino acids, while that of human CD40 contains 62 amino acids. Amino acids 31–62 (numbering from the inside the plasma membrane) of the murine and human CD40 cytoplasmic tails are completely identical (41, 42). In vitro studies have shown that amino acids 35–51 of the CD40 cytoplasmic domain contain sequences required for binding TRAF2 and TRAF3 (43, 44, 45). TRAF5 appears to bind to the same site (33). In contrast, TRAF6, which can also activate NF-{kappa}B, JNK, and perhaps ERK, binds to residues 15–23 of the human CD40 cytoplasmic domain (34, 45, 46), which is homologous to amino acids 19–28 of the murine CD40 cytoplasmic domain. Like TRAF6, JAK3 has been reported to bind to the membrane-proximal region of the human CD40 cytoplasmic domain. While the JAK3 binding site has been mapped to amino acids 5–14 of the human CD40 cytoplasmic domain (9), it has not been shown that JAK3 binds to murine CD40.

We have used a gain-of-function approach to identify the minimal regions of the CD40 cytoplasmic domain that can activate the JNK, p38 MAPK, NF-{kappa}B, and prosurvival pathways in B cells. We expressed in WEHI-231 cells chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to progressively smaller portions of the murine CD40 cytoplasmic domain. We found that an 11-amino acid linear sequence corresponding to amino acids 35–45 of the murine CD40 cytoplasmic tail was sufficient for maximal activation of the JNK and the p38 MAPK/MAPKAP kinase-2 pathways. Amino acids 35–45 of the CD40 cytoplasmic tail were also sufficient for activation of NF-{kappa}B and for protection of WEHI-231 cells from anti-IgM-induced growth arrest. These results suggest that amino acids 35–45 of the CD40 cytoplasmic domain constitute a minimal TRAF2/3/5 binding motif and are consistent with the idea that TRAF2, TRAF3, or TRAF5 couples CD40 to the JNK and p38 MAPK pathways, to NF-{kappa}B activation, and to prosurvival pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and other reagents

The hybridomas producing the OKT8 and 51.1 anti-human CD8{alpha} mAbs were obtained from the American Type Culture Collection (Manassas, VA). The 51.1 mAb was biotinylated using sulfo-NHS biotin (Pierce, Rockford, IL). The hybridoma producing the 1C10 anti-murine CD40 mAb (47) was a gift from Dr. M. Howard (DNAX Research Institute, Palo Alto, CA). Anti-mouse IgG-FITC was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Polyclonal rabbit Abs against JNK1 (Ab C-17) and I{kappa}B{alpha} were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The sheep anti-MAPKAP kinase-2 mAb was purchased from Upstate Biotechnology (Lake Placid, NY). Abs specific for I{kappa}B{alpha} phosphorylated at serine 32 were obtained from New England Biolabs (Beverly, MA). Horseradish peroxidase-conjugated protein A and the enhanced chemiluminescence detection system were obtained from Amersham (Oakville, Canada). Avidin, protein A-Sepharose, protein G-Sepharose, and glutathione-Sepharose were purchased from Sigma (St. Louis, MO). Glutathione-S-transferase (GST) fusion proteins containing amino acids 1–79 of c-Jun were expressed in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Bacteria containing the plasmid encoding GST-c-Jun1–79(1–79) were a gift from Dr. J. Hambleton (University of California, San Francisco). Recombinant murine hsp25 was obtained from StressGen Biotechnologies (Victoria, Canada).

Construction of CD8{alpha}/CD40 chimeric receptors

A plasmid containing cDNA encoding human CD8{alpha} in which a BglII site had been inserted after the fourth codon of the cytoplasmic domain (48) was a gift from Dr. A. Weiss (University of California, San Francisco). The CD8 cDNA was excised from this vector and subcloned into the pLXSN retroviral expression vector (49). The cDNAs encoding the full-length cytoplasmic domain of murine CD40 (amino acids 1–74), a region corresponding to amino acids 26–63, and a region corresponding to amino acids 26–53 were produced by RT-PCR using WEHI-231 B cell mRNA as a template. The primers used added a BglII site at the 5' end of the amplified cDNAs and a stop codon followed by a BglII site at the 3' end (Table IGo). The smaller CD40 segments were created by annealing together synthetic oligonucleotides that contained the relevant CD40 sequences as well as a BglII site at the 5' end and a stop codon followed by a BglII site at the 3' end (Table IIGo). The CD40 cDNA fragments were digested with BglII and ligated into pLXSN-CD8{alpha} at the BglII site. The sequence of each CD8{alpha}/CD40 chimeric cDNA was confirmed by DNA sequencing using a primer corresponding to codons 177–183 of the CD8 sense strand (5'-CTG GAC TTC GCC TGT GAT ATC-3').


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  		Table I. Primers used for PCR amplification of portions of the CD40 cytoplasmic domain1

 

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  		Table II. Oligonucleotides used to generate murine CD40 cytoplasmic domain fragments1

 
Expression of chimeric CD8{alpha}/CD40 receptors

The CD8{alpha}/CD40 constructs in the pLXSN retroviral expression vector were transfected into the BOSC23 packaging cell line (50) (a gift from Dr. W. Pear, Massachusetts Institute of Technology, Cambridge, MA) by the calcium phosphate method. BOSC23 cell supernatants containing the pLXSN vectors packaged into retroviruses were collected 2 days later and used to infect the WEHI-231 murine B lymphoma cell line. After 40 h, infected cells were selected by culturing the cells in medium containing 1.8 mg/ml of G418 (Life Technologies, Grand Island, NY). G418-resistant clones were screened for expression of human CD8{alpha} by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). The cells were stained with the OKT8 anti-CD8{alpha} mAb followed by goat anti-mouse IgG-FITC, both at a final concentration of 30 µg/ml.

Cell stimulation and preparation of cell lysates

WEHI-231 cells were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-ME. The cells were resuspended to 107/ml in modified HEPES-buffered saline (25 mM sodium HEPES (pH 7.2), 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 1 mg/ml glucose, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM 2-ME), warmed to 37°C, and stimulated either with a biotinylated anti-human CD8{alpha} mAb (51.1-biotin) and avidin or with the 1C10 anti-CD40 mAb. Reactions were stopped by adding ice-cold PBS containing 1 mM Na3VO4 and then centrifuging the cells for 3 min at 3000 rpm in the cold. Cell pellets were washed once, without resuspending, with 1 ml of ice-cold PBS/Na3VO4 and then solubilized in one of the following buffers: buffer A (20 mM Tris-HCl (pH 8), 137 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, and 10 mM ß-glycerophosphate), buffer B (20 mM Tris-HCl (pH 7.4), 1% Triton-X 100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM ß-glycerophosphate, 1 mM Na3VO4, 2 mM Na4P2O7, 1 mM PMSF, and 10 µg/ml leupeptin), or buffer C (20 mM Tris (pH 8), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1% Triton X-100, 1 mM Na3VO4, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF). After 10 min on ice, detergent-insoluble material was removed by centrifugation. Protein concentrations were determined using the bicinchoninic acid assay (Pierce).

JNK in vitro kinase assay

Following stimulation, 107 cells were lysed in 350 µl of buffer A. Cell lysates were precleared for 1 h at 4°C with 10 µl of protein A-Sepharose, then mixed with 0.5 µg of anti-JNK1 Ab for 90 min at 4°C. Immune complexes were collected by adding 10 µl of protein A-Sepharose and mixing for an additional hour. In vitro kinase assays using GST-c-Jun1–79(1–79) as substrate were conducted as described previously (12). 32P incorporation into GST-c-Jun1–79(1–79) was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

MAPKAP kinase-2 in vitro kinase assay

Cell lysates (500 µg protein in buffer B) were precleared for 1 h at 4°C with 10 µl of protein G-Sepharose, then mixed with 2 µg of sheep anti-MAPKAP kinase-2 Ab for 90 min at 4°C. Immune complexes were collected on 10 µl of protein G-Sepharose for 1 h, and in vitro kinase assays were performed as described previously (12). 32P incorporation into hsp25 was quantitated using a phosphorimager.

I{kappa}B{alpha} phosphorylation and degradation

Cell lysates (40 µg of protein in buffer C) were separated on 12% low bis-acrylamide (12% acrylamide and 0.1% bis-acrylamide, final concentrations) SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked overnight with 5% (w/v) nonfat dry milk in TBS (10 mM Tris-HCl (pH 7.5) and 150 mM NaCl). The filters were washed for 10 min with TBS/0.05% Tween-20 (TBST) and then incubated overnight in the cold with the anti-phospho-I{kappa}B{alpha} Ab diluted 1/1000 in TBST. After washing the filters for 10 min with TBST, the filters were incubated for 1 h at room temperature with protein A-horseradish peroxidase diluted 1/5000 in TBST. The membranes were washed extensively with TBST, and immunoreactive bands were visualized by enhanced chemiluminescence detection. To reprobe the blots, bound Abs were eluted by incubating the blots for 15 min with TBS, pH 2. The membranes were blocked as described above and then incubated with the I{kappa}B{alpha} Ab (diluted 1/1000 in TBST) for 3 h at room temperature. Immunoreactive bands were visualized as described for the anti-phospho-I{kappa}B{alpha} Ab.

Proliferation assays

WEHI-231 cells (1 x 104 cells/well) were cultured in triplicate in 200 µl of culture medium containing various stimuli. After 40 h at 37°C, 1 µCi of [3H]thymidine (Amersham) was added to each well, and the cells were harvested 4 h later. The incorporation of [3H]thymidine into DNA was determined by liquid scintillation counting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CD8{alpha}/CD40 chimeric receptor proteins

To identify signaling motifs in the CD40 cytoplasmic domain, we constructed a set of chimeric CD8{alpha}/CD40 receptors in which segments of the murine CD40 cytoplasmic domain were fused onto the C-terminus of a truncated human CD8{alpha} protein consisting of the extracellular domain, the transmembrane region, and the first four cytoplasmic amino acids of CD8{alpha} (Fig. 1Go). Chimeric receptors were constructed containing the full-length CD40 cytoplasmic domain (amino acids 1–74 of the murine CD40, counting from the inside face of the plasma membrane), the membrane-proximal portion of the CD40 cytoplasmic domain (amino acids 1–25), or amino acids 26–63, which is the homology box region that is nearly identical in the murine and human CD40 proteins. The homology box region of the CD40 cytoplasmic domain was also subdivided into smaller regions in chimeric receptors that contained amino acids 26–53, 35–53, 26–44, 35–45, 45–63, or 43–53 of the CD40 cytoplasmic domain. Finally, we constructed a chimeric receptor containing residues 35–53 of the CD40 tail in which the threonine residue at position 40 was changed to an alanine. We were interested in determining whether this threonine was required for CD40-induced survival as well as activation of JNK, MAPKAP kinase-2, and NF-{kappa}B, since changing this threonine residue to an alanine in human CD40 abrogates several responses to CD40 engagement, including up-regulation of CD23, B7.1, and Fas (51, 52, 53, 54). These 10 chimeric receptors were stably expressed in WEHI-231 cells, as was the truncated CD8{alpha} protein (CD8/). For each receptor expressed, WEHI-231 clones were screened for CD8{alpha} expression, and clones with similar levels of expression were selected for further study (Fig. 2Go).



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  		FIGURE 1. Schematic representation of the CD8{alpha}/CD40 chimeric receptors. The CD8/ protein contains the extracellular and transmembrane domains of CD8{alpha} as well as the first four amino acids of the CD8 cytoplamic domain. For the chimeric receptors, various portions of the murine CD40 cytoplamic domain were fused to the C terminus of CD8/. The amino acid sequence of the CD40 cytoplasmic domain is shown, and the residues are numbered starting at the inner face of the membrane. The numbers in brackets indicate which portions of the CD40 cytoplamic domain have been fused to CD8{alpha} for each chimeric receptor. In the CD8/(35–53 T40A) chimeric receptor, the threonine residue at position 40 was changed to an alanine.

 


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  		FIGURE 2. Expression of CD8{alpha}/CD40 chimeric receptors in WEHI-231 cells. Untransfected parental WEHI-231 cells (solid lines) and two stable clones expressing each CD8/CD40 chimeric receptor (dotted lines) were stained with the human CD8{alpha}-specific OKT8 mAb followed by anti-mouse IgG-FITC. The numbers in brackets denote which amino acids of the CD40 cytoplasmic domain were present in each chimeric receptor. Note that the CD8/(35–53 T40A) chimeric receptor is referred to as CD8/(35–53 T-A) in this figure.

 
Mapping the portion of the CD40 cytoplasmic domain required for activating JNK and MAPKAP kinase-2

We have previously shown that in WEHI-231 cells CD40 strongly activates the JNK and p38 MAPKs as well as MAPKAP kinase-2, a downstream target of p38 MAPK (12). To identify proteins that may link CD40 to activation of these kinase signaling pathways, we used the chimeric CD8{alpha}/CD40 receptors to map the portion of the CD40 cytoplasmic domain responsible for activating the JNK and p38 MAPK/MAPKAP kinase-2 pathways. We chose to use MAPKAP kinase-2 activation as an indirect measure of p38 MAPK activation, since MAPKAP kinase-2 is usually activated to a greater extent than p38 MAPK (12). This gave us a larger range with which to quantitate the relative abilities of different regions of CD40 to activate the p38 MAPK/MAPKAP kinase-2 pathway. In WEHI-231 cells, CD40-stimulated activation of MAPKAP kinase-2 is entirely dependent on p38 MAPK activity. Fig. 3Go shows that a specific inhibitor of p38 MAPK, SB203580 (18), completely blocked the ability of CD40 to activate MAPKAP kinase-2.



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  		FIGURE 3. Activation of MAPKAP kinase-2 by CD40 is dependent on the p38 MAPK. WEHI-231 cells were pretreated with 10 µM of the p38 MAPK inhibitor SB203580 (SB) for 25 min before being stimulated with 5 µg/ml of the 1C10 anti-CD40 mAb for 15 min. Cell lysates were immunoprecipitated with an Ab to MAPKAP kinase-2, and in vitro kinase assays were performed using Hsp25 as a substrate.

 
We first determined whether a chimeric receptor containing the entire cytoplasmic domain of CD40 (amino acids 1–74) could activate JNK and MAPKAP kinase-2 when expressed in WEHI-231 B lymphoma cells. We found that clustering this CD8/1–74(1–74) chimeric receptor with a biotinylated anti-CD8 mAb (51.1-biotin) and avidin routinely caused a 15-fold increase in JNK activity (Fig. 4Go) and a 10-fold increase in MAPKAP kinase-2 activity (Fig. 5Go). This is similar to the magnitude of JNK activation and MAPKAP kinase-2 activation by treating parental WEHI-231 cells with the 1C10 anti-CD40 mAb or with soluble CD40 ligand (12) (data not shown). Thus, interactions mediated by the CD40 cytoplasmic domain are sufficient to fully activate JNK and MAPKAP kinase-2 in the absence of any interactions mediated by the extracellular or transmembrane domains of CD40.



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  		FIGURE 4. Amino acids 35–45 of the CD40 cytoplasmic domain constitute a signaling motif that is sufficient for the activation of JNK. WEHI-231 clones expressing the indicated CD8{alpha}/CD40 chimeric receptors were stimulated with 10 µg/ml biotinylated 51.1 (anti-CD8 mAb) and 10 µg/ml avidin for 15 min. Cell lysates were immunoprecipitated with the anti-JNK1 Ab, and in vitro kinase assays were performed using GST-c-Jun(1–79) as a substrate. The JNK activity induced by engaging CD8/(1–74), the chimeric receptor containing the full-length CD40 cytoplasmic domain, was assigned a value of 100%. The dashed line indicates the JNK activity from CD8/(1–74)-expressing cells that were not stimulated. The average value for this basal JNK activity was 7% of the JNK activity induced by engaging the CD8/(1–74) chimeric receptor; i.e., engaging CD8/(1–74) caused a 15-fold increase in JNK activity. The basal levels of JNK activity in clones expressing other chimeric receptors were very similar. Activation of JNK caused by engaging the other chimeric receptors is expressed as a percent of the JNK activation caused by engaging CD8/(1–74). The data represent the mean and standard deviation from a total of three or more independent expreiments using two different clones expressing that particular chimeric receptor. Avidin alone did not stimulate JNK activity (data not shown).

 


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  		FIGURE 5. Amino acids 35–45 of the CD40 cytoplasmic domain constitute a signaling motif that is sufficient for the activation of MAPKAP kinase-2. WEHI-231 clones expressing the indicated CD8{alpha}/CD40 chimeric receptors were stimulated with 10 µg/ml biotinylated 51.1 (anti-CD8 mAb) and 10 µg/ml avidin for 15 min. Cell lysates were immunoprecipitated with the anti-MAPKAP kinase-2 Ab, and in vitro kinase assays were performed using Hsp25 as a substrate. The MAPKAP kinase-2 activity induced by engaging CD8/(1–74), the chimeric receptor containing the full-length CD40 cytoplasmic domain, was assigned a value of 100%. The dashed line indicates the MAPKAP kinase-2 activity from cells expressing CD8/(1–74) which were not stimulated. The average value for this basal MAPKAP kinase-2 activity was 9.6% of the activity induced by engaging the CD8/(1–74) chimeric receptor; i.e., engaging CD8/(1–74) caused about a 10-fold increase in MAPKAP kinase-2 activity. The basal levels of MAPKAP kinase-2 activity in clones expressing other chimeric receptors were very similar. Activation of MAPKAP kinase-2 caused by engaging the other chimeric receptors is expressed as a percent of the MAPKAP kinase-2 activation caused by engaging CD8/(1–74). The data represent the mean and standard deviation from a total of three or more independent experiments using two different clones expressing that particular chimeric receptor. Avidin alone did not stimulate MAPKAP kinase-2 activity (data not shown).

 
Our next goal was to map the portion of the CD40 cytoplasmic domain responsible for activating JNK and MAPKAP kinase-2. Amino acids 26–63 of the cytoplasmic domains of murine and human CD40 are 92% identical, whereas the membrane-proximal region of CD40 diverges considerably between the mouse and human (41, 42). This suggested that the homology box region (amino acids 26–63) might contain the important CD40 signaling motifs. Consistent with this idea, a chimeric receptor containing the membrane-proximal 25 residues of the CD40 cytoplasmic domain (CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)) did not activate JNK (Fig. 4Go) or MAPKAP kinase-2 (Fig. 5Go). In contrast, the CD8/26–63(26–63) chimeric receptor, which contains the homology box region, activated both JNK (Fig. 4Go) and MAPKAP kinase-2 (Fig. 5Go) to a similar extent as the chimeric receptor containing the full-length CD40 cytoplasmic tail. These results show that residues 64–74 of the murine CD40 tail are not required for activation of the JNK and p38 pathways. Human CD40 terminates after residue 62, consistent with the idea that this C-terminal extension in murine CD40 is unlikely to carry out important signaling functions. When expressed in murine cells, human CD40 can mediate the same responses as the endogenous murine CD40 (53, 54, 55, 56).

To further delineate the region of CD40 responsible for activation of JNK and MAPKAP kinase-2, we constructed chimeric receptors containing progressively smaller portions of this CD40 homology box. We first tested whether the last 10 residues of the homology box were required for activating these kinases. We found that the CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor activated JNK and MAPKAP kinase-2 to similar extents as CD8/26–63(26–63) (Figs. 4Go and 5Go). Thus, the last 10 residues of the homology box (residues 54–63) are not needed for activating the JNK and p38 MAPK signaling pathways.

Our results thus far had indicated that the JNK and p38 MAPK activation motifs were contained within residues 26–53 of the CD40 cytoplasmic domain. In vitro studies with fusion proteins have shown that TRAF2 can bind to peptides corresponding to amino acids 35–51 of murine CD40 (43, 44). TRAF3 and TRAF5 appears to associate with an identical or overlapping region of CD40 (33, 45). When overexpressed in fibroblasts, TRAF2 and TRAF5 can activate JNK, while TRAF3 overexpression does not (35). Although the ability of TRAF proteins to activate the p38 MAPK/MAPKAP kinase-2 pathway has not been examined, expressing a dominant negative form of TRAF3 in the RAMOS human B cell line has been shown to selectively block CD40-induced activation of the p38 MAPK (57). Thus, TRAF2 or TRAF5 may couple CD40 to JNK, while TRAF3 couples CD40 to the p38 MAPK. To determine whether the proposed TRAF2/3/5 binding region of CD40 corresponds to the region capable of activating JNK and MAPKAP kinase-2, we made a chimeric receptor containing residues 35–53 of the CD40 cytoplasmic domain. We found that CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) strongly activated JNK (Fig. 4Go) and MAPKAP kinase-2 (Fig. 5Go), consistent with the idea that TRAF2, TRAF3, or TRAF5 might couple CD40 to activation of JNK and the p38 MAPK in B cells.

To further refine the CD40 signaling motif required for activation of JNK and MAPKAP kinase-2, we first used chimeric receptors containing either the N- or C-terminal portions of the region spanning amino acids 35–53 of the CD40 cytoplasmic domain. We found that CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44), a chimeric receptor containing the N-terminal half of this region could activate JNK and MAPKAP kinase-2 (Figs. 4Go and 5Go), while two chimeric receptors containing the C-terminal half of this region, CD8/(45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) and CD8/(43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), were incapable of activating JNK and MAPKAP kinase-2 (Figs. 4Go and 5Go). Since only CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) and CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) of this set of chimeric receptors could activate JNK and MAPKAP kinase-2, it indicated that residues other than 35–44 in the CD40 cytoplasmic domain are dispensable for CD40-induced activation of the JNK and p38 MAPK/MAPKAP kinase-2 pathways. To determine whether this region was sufficient for activation of these kinases, we constructed a chimeric receptor containing only residues 35–45 of the CD40 cytoplasmic domain. CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) was able to activate these kinases to the same extent as the chimeric receptor containing the full-length CD40 tail (Figs. 4Go and 5Go). Thus, residues 35–45 of murine CD40 constitute a JNK/p38 MAPK activation motif and may be the minimal TRAF2/3/5 binding site.

Residues 35–45 of the murine CD40 cytoplasmic domain mediate activation of the NF-{kappa}B pathway and protection from anti-IgM-induced growth arrest

CD40 engagement activates the NF-{kappa}B transcription factor (7, 8). NF-{kappa}B is retained in the cytosol in an inactive state, bound to the inhibitory I{kappa}B proteins (58). NF-{kappa}B activation occurs via phosphorylation of I{kappa}B{alpha} at serines 32 and 36 (59). This targets I{kappa}B{alpha} for degradation and allows NF-{kappa}B to translocate to the nucleus (58, 59, 60). When overexpressed in fibroblasts, TRAF2, TRAF5, and TRAF6 can all activate NF-{kappa}B (32, 33, 34, 35, 36). However, it is not clear whether all of these TRAF proteins can link CD40 to NF-{kappa}B activation in B cells. TRAF2 and TRAF5 bind to amino acids 35–51 of CD40 (33, 44), while TRAF6 binds to the membrane-proximal region of CD40 (34). To determine which regions of CD40 activate NF-{kappa}B in B cells, we tested the ability of our chimeric receptors to induce phosphorylation and degradation of I{kappa}B{alpha}.

Cross-linking the CD8/1–74(1–74) chimeric receptor caused a time-dependent increase in I{kappa}B{alpha} phosphorylation that was readily detectable after 1 min and was maximal by 2 min after receptor engagement (Fig. 6Go). Consistent with the phosphorylation kinetics, I{kappa}B{alpha} degradation was apparent within 2 min of CD8/1–74(1–74) engagement and was complete within 5 min. Similar results were observed when the 1C10 anti-CD40 mAb was used to engage the endogenous CD40 in parental WEHI-231 cells, although the kinetics of I{kappa}B{alpha} phosphorylation and degradation were slightly slower (Fig. 6Go). In contrast, ligation of CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) did not cause I{kappa}B{alpha} phosphorylation or degradation after 1–10 min (Fig. 6Go) or at 40 min (data not shown). Thus, our data suggest that the membrane-proximal region of the CD40 cytoplasmic tail is incapable by itself of causing significant activation of NF-{kappa}B in WEHI-231 cells. The CD8/26–63(26–63), CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), and CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) chimeric receptors all induced marked phosphorylation of I{kappa}B{alpha} within 1–2 min of engagement, and this was followed by degradation of I{kappa}B{alpha} (Fig. 6Go), suggesting that the NF-{kappa}B activation motif was contained within residues 35–44 of the CD40 cytoplasmic domain. Consistent with this idea, the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) chimeric receptor was capable of inducing I{kappa}B{alpha} phosphorylation and degradation. Since residues 35–45 of the CD40 cytoplasmic domain participate in the binding of TRAF2 and TRAF5 (45), our data suggest that TRAF2 and/or TRAF5 mediate CD40 activation of NF-{kappa}B in WEHI-231 cells.



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  		FIGURE 6. Residues 35–45 of the murine CD40 cytoplasmic domain are sufficient to induce I{kappa}B{alpha} phosphorylation and degradation. WEHI-231 cell clones expressing the indicated CD8{alpha}/CD40 chimeric receptors were stimulated with 10 µg/ml biotinylated 51.1 (anti-CD8 mAb) and 10 µg/ml avidin for 1–10 min. The parental WEHI-231 cells (lower right panel) were stimulated with 10 µg/ml of the 1C10 anti-CD40 mAb. In the upper panel of each pair, cell lysates were analyzed by immunoblotting with an Ab specific for the phosphrylated form of I{kappa}B{alpha} (P-I{kappa}B{alpha}). The filters were then stripped and reprobed with an anti-I{kappa}B{alpha} Ab (lower panels) to assess I{kappa}B{alpha} degradation. Two different WEHI-231 clones expressing each chimeric receptor were analyzed in at least three independent experiments. Representative results are shown. Note that the lower band in the anti-I{kappa}B{alpha} blots is an unidentified protein that reacts with the anti-I{kappa}B{alpha} Ab. The intensity of this band did not change upon receptor engagement. In some panels, this band was not completely resolved from the I{kappa}B{alpha} band. To facilitate the resolution of these two bands, short exposures were done for the I{kappa}B{alpha} blots. Thus, in some lanes there appears to be no I{kappa}B{alpha} present even though there is a substantial amount of phosphorylated I{kappa}B{alpha}. Longer exposures revealed the presence of small amounts of I{kappa}B{alpha} in these lanes.

 
While amino acids 35–45 of the CD40 cytoplasmic domain were sufficient to induce I{kappa}B{alpha} phosphorylation and degradation, additional sequences appear to be required for maximal activation of NF-{kappa}B by CD40. Figure 6Go shows that the I{kappa}B{alpha} phosphorylation caused by CD8/1–74(1–74), the chimeric receptor containing the full-length CD40 cytoplasmic domain, was significantly stronger than that caused by the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) chimeric receptor. CD8/1–74(1–74)-induced I{kappa}B{alpha} phosphorylation was also more rapid than that caused by CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). CD8/1–74(1–74) caused near maximal phosphorylation of I{kappa}B{alpha} after 1 min, while CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) did not induce significant I{kappa}B{alpha} phosphorylation until 2 min. Comparing the magnitude and kinetics of I{kappa}B{alpha} phosphorylation induced by the other chimeric receptors allowed us to determine which CD40 sequences were required for maximal I{kappa}B{alpha} phosphorylation. We found that the CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor could induce I{kappa}B{alpha} phosphorylation to the same extent and with the same rapid kinetics as CD81–74(1–74). Thus, residues 26–34 and/or 46–53 of the CD40 cytoplasmic domain contribute to the ability of CD40 to induce I{kappa}B{alpha} phosphorylation and degradation. Both these flanking sequences may be required for maximal I{kappa}B{alpha} phosphorylation and degradation, since CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) induced significantly stronger and more rapid I{kappa}B{alpha} phosphorylation than either CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) or CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53), both of which induced the slower and less robust response characteristic of CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45). Moreover, the CD8/(45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) and CD8/(43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptors were unable to activate NF-{kappa}B, suggesting that if the C-terminal flanking regions contribute to NF-{kappa}B activation, they do so by cooperating with residues 35–45 as opposed to independently binding activators of NF-{kappa}B.

Recent work by Sonenshein and colleagues has shown that activation of NF-{kappa}B is essential for CD40 to prevent BCR-induced growth arrest and apoptosis in WEHI-231 cells (61). We found that engaging the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) chimeric receptor with anti-CD8 Abs could completely protect WEHI-231 cells from anti-IgM-induced growth arrest (Fig. 7Go). This indicates that the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) chimeric receptor can activate NF-{kappa}B to a biologically significant extent even though it does not induce I{kappa}B{alpha} phosphorylation to the same extent as the chimeric receptor containing the full-length CD40 cytoplasmic domain.



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  		FIGURE 7. Residues 35–45 of the CD40 cytoplasmic tail are sufficient to protect WEHI-231 cells from anti-IgM-induced apoptosis. A WEHI-231 clone expressing the CD8/(35–45) chimeric receptor was cultured with or without 3 µg/ml anti-IgM Ab in the presence of medium alone, 10 µg/ml of the 1C10 anti-CD40 mAb, or 10 µg/ml each of biotinylated 51.1 (anti-CD8 mAb) and avidin. [3H]Thymidine was added after 40 h, and 4 h later the incorportation of [3H]thymidine into DNA was determined by liquid scintillation counting. All determinations were carried out in triplicate, and the mean and standard deviation for each data point are shown. This is one of three similar experiments performed with two different clones expressing CD8/(35–45).

 
Threonine 40 is essential for CD40 signaling

The threonine residue at position 40 of the human CD40 cytoplasmic region has previously been shown to be important for CD40 signaling (51, 52, 53, 54). We asked whether changing this residue in murine CD40 would affect its ability to signal. We found that this threonine to alanine mutation completely abolished the ability of the murine CD40 cytoplasmic domain to activate JNK and MAPKAP kinase-2. The CD8(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor was fully active, whereas the CD8/(35–45 T40A) chimeric receptor in which residue 40 was changed to an alanine did not activate JNK (Fig. 4Go) or MAPKAP kinase-2 (Fig. 5Go). The CD8/(35–45 T40A) chimeric receptor also did not induce I{kappa}B{alpha} phosphorylation or degradation (Fig. 6Go). Thus, threonine 40 is essential for murine CD40 to activate the JNK, p38 MAPK/MAPKAP kinase-2, and NF-{kappa}B signaling pathways. Presumably, this residue interacts with proteins that link CD40 to these signaling pathways. This threonine residue has been shown to be important for CD40 to bind TRAF2, TRAF3, and TRAF5 (31, 33, 45), again consistent with the idea that these TRAF proteins link CD40 to activation of JNK, p38 MAPK, and NF-{kappa}B.

The isolated TRAF6 binding site of CD40 is not sufficient for signaling in WEHI-231 cells

Our results show that amino acids 35–45 of the CD40 cytoplasmic domain contain a signaling motif that can mediate the activation of JNK and MAPKAP kinase-2 and induce the phosphorylation and degradation of I{kappa}B{alpha}. In contrast, chimeric receptors containing other regions of CD40 were unable to induce these signaling events. Most notably, both the CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) and CD8/(45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) chimeric receptors were incapable of activating the JNK and p38 MAPKs or inducing the phosphorylation and degradation of I{kappa}B{alpha} ( Figs. 4–6GoGoGo). The inability of the CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) chimeric receptor to signal was surprising, since it appeared to contain the minimal binding site for TRAF6, and TRAF6 has been shown to activate JNK and NF-{kappa}B when overexpressed in fibroblasts (34). However, more detailed mapping studies by Pullen et al. (45) have recently shown that the optimal TRAF6 binding site corresponds to amino acids 19–28 of the murine CD40 cytoplasmic domain. Since our CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) chimeric was missing key residues of the TRAF6 binding site, we constructed a new chimeric receptor that contained amino acids 15–30 of the murine CD40 cytoplasmic domain and expressed this chimeric receptor in WEHI-231 cells.

FACS analysis showed that the cell surface expression of the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor was lower than that of the other CD8 chimeric receptors we had expressed. Twelve WEHI-231 clones expressing the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor were analyzed by staining with anti-CD8 Abs. For the two clones expressing the highest levels of the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor, the mean fluorescence intensities of anti-CD8 staining were 38 and 52%, respectively, of that for a CD8/1–74(1–74)-expressing WEHI-231 clone that we had used in our previous experiments (Fig. 8Go). However, WEHI-231 clones expressing similar levels of the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor (i.e., 40–50% that of the CD8/1–74(1–74) clone) showed strong activation of JNK, MAPKAP kinase-2, and NF-{kappa}B in response to anti-CD8 Abs (data not shown). Thus, the level of cell surface expression of the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor should not be a limiting factor in its ability to initiate signals.



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  		FIGURE 8. Expression of CD8/(15–30) chimeric receptors. Two stable WEHI-231 clones expressing the CD8/(15–30) chimeric receptor as well as a CD8/(1–74)-expressing clone used in previous figures were stained with the human CD8{alpha}-specific OKT8 mAb followed by anti-mouse IgG-FITC. Of the 12 CD8/(15–30)-expressing clones analyzed, the two shown in this figure had the highest expression. The level of CD8/(15–30) expression on the surface of these two clones (mean fluorescence intensity) was 38% (clone 1) and 52% (clone 2) of the surface expression of CD8(1–74) on theCD8/(1–74) clone shown in the figure.

 
The CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor contains the TRAF6 binding site but not the TRAF2/3/5 binding site. In Fig. 9Go we analyzed the ability of this receptor to activate JNK and MAPKAP kinase-2 and to induce the phosphorylation and degradation of I{kappa}B{alpha}. Although overexpression of TRAF6 has been reported to activate JNK and NF-{kappa}B in fibroblasts (34, 35), we found that the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor caused little or no activation of JNK or MAPKAP kinase-2 in WEHI-231 cells (Fig. 9GoA) and did not induce I{kappa}B{alpha} phosphorylation or degradation (Fig. 9GoB). Although we cannot rule out the possibility that the cytoplasmic domain of the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor is improperly folded and unable to interact with TRAF6, the simplest interpretation of these data is that the TRAF6 binding motif of murine CD40 is not sufficient by itself to mediate these responses, at least in WEHI-231 cells.



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  		FIGURE 9. The CD8/(15–30) chimeric receptor causes little or no activation of JNK, MAPKAP kinase-2, or NF-{kappa}B. A, Two different WEHI-231 clones expressing CD8/(15–30) as well as WEHI-231 clones expressing either the truncated CD8{alpha} (CD8/) or the CD8/(1–74) chimeric receptor containing the full-length CD40 cytoplasmic domain were stimulated with 10 µg/ml biotinylated 51.1 (anti-CD8 mAb) and 10 µg/ml avidin for 15 min. Cell lysates were immunoprecipitated with the anti-JNK Ab (upper panel) or the anti-MAPKAP kinase 2 Ab (lower panel), and in vitro kinase assays were performed using GST-c-Jun as a substrate for JNK and Hsp25 as a substrate for MAPKAP kinase 2. The JNK or MAPKAP kinase-2 activity induced by engaging CD8/(1–74) was assigned a value of 100%. The data represent the mean and standard deviation from three independent experiments. B, The two WEHI-231 clones expressing CD8/(15–30) were stimulated with 10 µg/ml biotinylated 51.1 (anti-CD8 mAb) and 10 µg/ml avidin for 1–10 min. In the upper panel of each pair, cell lysates were analyzed by immunoblotting with an Ab specific for the phosphorylated form of I{kappa}B{alpha} (P-I{kappa}B{alpha}). The filters were then stripped and reprobed with an anti-I{kappa}B{alpha} Ab (lower panels) to assess I{kappa}B{alpha} degradation. Lysates of CD8/(1–74)-expressing cells were used as a positive control for I{kappa}B{alpha} phosphorylation.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we have used chimeric receptors containing different portions of the CD40 cytoplasmic domain to show that an 11-amino acid linear sequence corresponding to amino acids 35–45 of the CD40 cytoplasmic domain is sufficient for 1) activating JNK, 2) activating the p38 MAPK/MAPKAP kinase-2 pathway, and 3) inducing the phosphorylation and degradation of I{kappa}B{alpha}. Grafting these 11 amino acids from CD40 onto CD8{alpha} was also sufficient to create a chimeric receptor with the ability to fully protect WEHI-231 cells from anti-IgM-induced growth arrest. While previous studies involving the expression of truncated or mutated CD40 proteins have shown that amino acids 35–45 of the CD40 cytoplasmic domain are important for some CD40 functions, they could not rule out that other regions of CD40 were also required. In contrast, our findings demonstrate that this 11-amino acid region of CD40 constitutes an independent signaling motif. Our results also imply that interactions mediated by the extracellular and transmembrane domains of CD40 are not required for activation of JNK, MAPKAP kinase-2, or NF-{kappa}B by CD40 and that oligomerization of CD40 is sufficient to initiate these responses.

This is the first report directly identifying the region of CD40 that activates the JNK and p38 MAPK/MAPKAP kinase-2 pathways. We found that this same region of CD40, amino acids 35–45 of the cytoplasmic domain, was also sufficient for CD40 to induce the phosphorylation and degradation of I{kappa}B{alpha}. Previous studies had shown that amino acids 36–52 of the CD40 cytoplasmic domain are sufficient to activate NF-{kappa}B in 293 cells (43) and that amino acids 32–41 are necessary for CD40 to activate NF-{kappa}B in B cells (53). Taken together, these results indicate that amino acids 36–41 of CD40 (PVQETL) are critical for CD40 to activate NF-{kappa}B. This is consistent with findings that a PVQET motif is essential for the CD40-related EBV LMP1 protein to activate NF-{kappa}B (62). Further mutational analysis is required to determine whether the PVQET motif contained within amino acids 35–45 of the CD40 cytoplasmic domain is essential for CD40 to activate JNK and MAPKAP kinase-2. Nevertheless, we have clearly shown that residues 35–45 of the CD40 cytoplasmic domain contain a signaling motif that can activate JNK and MAPKAP kinase-2 to the same extent as the complete CD40 cytoplasmic domain.

Our results as well as those from previous studies (51, 52, 53, 54) indicate that the threonine residue at position 40 of the CD40 cytoplasmic domain is particularly important for CD40 signaling. We found that changing this threonine residue to an alanine abolished the ability of the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor to activate JNK, MAPKAP kinase-2, and NF-{kappa}B. Other studies have shown that changing this threonine residue to an alanine abrogates the ability of CD40 to activate NF-{kappa}B and to induce homotypic aggregation, Ab secretion, and up-regulation of B7.1, Fas, and CD23 (53, 54). Thus, either the PVQET motif or another overlapping signaling motif containing threonine 40 is responsible for the majority of CD40-induced signaling events including, as we have shown, activation of the JNK and the p38 MAPK/MAPKAP kinase-2 pathways.

Our results suggest that the activation of JNK, p38 MAPK, and NF-{kappa}B by CD40 as well as protection of WEHI-231 cells from anti-IgM-induced growth arrest are mediated by proteins that bind to residues 35–45 of the CD40 cytoplasmic domain. TRAF2, TRAF3, TRAF5, and TRAF6 can bind directly to the cytoplasmic domain of CD40 via their highly related C-terminal TRAF domains (31, 32, 33, 34). In vitro binding assays have shown that TRAF2 and TRAF3 can bind to fusion proteins or peptides containing amino acids 36–52 of murine CD40 (43, 44, 45), while TRAF5 binds to an identical or overlapping region of CD40 (33). The CD40 signaling motif we have identified, residues 35–45 of the CD40 cytoplasmic domain, may therefore contain the essential elements for binding TRAF2, TRAF3, and TRAF5. TRAF6, in contrast, binds to a membrane-proximal region of CD40 that includes residues 14–23 of human CD40 or residues 19–28 of murine CD40 (34, 45). Thus, our results are consistent with the idea that TRAF2, TRAF3, or TRAF5 mediates the ability of CD40 to activate NF-{kappa}B, JNK, and the p38 MAPK/MAPKAP kinase-2 pathway as well as the ability of CD40 to protect WEHI-231 cells from anti-IgM-induced growth arrest. Several lines of evidence support this conclusion. First, overexpression of TRAF2 or TRAF5 in fibroblasts results in activation of both NF-{kappa}B and JNK (32, 33, 35, 36, 37, 38, 39). Second, expressing a truncated (i.e., dominant negative) form of TRAF2 in B cells blocks the ability of CD40 to activate JNK (63), implicating the portion of CD40 that binds TRAF2, TRAF3, and TRAF5 in this response. Similarly, it has recently been shown that expressing a truncated dominant-negative form of TRAF3 in B cells blocks activation of p38 MAPK by CD40 (57). Finally, changing the threonine residue in the PVQET motif of human CD40 to an alanine not only prevents CD40 signaling but also abolishes the ability of CD40 to bind TRAF2, TRAF3, and TRAF5 (31, 33, 43). Although we cannot rule out the involvement of other proteins that bind to residues 35–45 of the CD40 cytoplasmic domain, these data all support the idea that residues 35–45 of CD40 constitute a minimal TRAF binding motif and that TRAF2, TRAF3, or TRAF5 couples CD40 to activation of JNK, p38 MAPK, and NF-{kappa}B.

Comparing the various chimeric receptors we constructed to the one containing the full-length CD40 cytoplasmic domain revealed that residues 35–45 of the CD40 cytoplasmic domain were sufficient for maximal activation of JNK and MAPKAP kinase-2 (Figs. 4Go and 5Go) but not for maximal activation of NF-{kappa}B (Fig. 6Go). CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) did not induce I{kappa}B{alpha} phosphorylation and degradation to the same extent or as rapidly as CD8/1–74(1–74), which contains the complete CD40 cytoplasmic domain. However, CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) could induce I{kappa}B{alpha} phosphorylation and degradation as rapidly and to the same extent as CD8/1–74(1–74), indicating that the regions flanking residues 35–45 (residues 26–34 and/or 46–53) contribute to the binding of proteins that activate NF-{kappa}B. Both residues 26–34 and residues 46–53 appear to be required for full activation of NF-{kappa}B, since neither the CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) nor CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor could induce the more robust and more rapid response that was stimulated by CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) or CD8/1–74(1–74). How do residues 26–34 and 46–53 contribute to the ability of CD40 to activate NF-{kappa}B? Although TRAF6 can activate NF-{kappa}B when overexpressed in fibroblasts, it is unlikely that CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) binds TRAF6. The TRAF6 binding site in murine CD40 corresponds to residues 19–28, and residue 19 is essential for TRAF6 binding (45). Similarly, the inability of the CD8/(43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) and CD8/(45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) chimeric receptors to induce I{kappa}B{alpha} phosphorylation and degradation indicates that the C-terminal flanking region does not by itself bind activators of NF-{kappa}B. Thus, a more likely explanation for the contribution of residues 26–34 and 46–53 to CD40-induced NF-{kappa}B activation is that these residues stabilize the binding of a protein to residues 35–45 either by directly interacting with this protein or by improving the ability of residues 35–45 to assume a conformation that is optimal for binding this protein that links CD40 to NF-{kappa}B. Interactions mediated by the regions flanking residues 35–45 of the CD40 cytoplasmic domain could improve the efficiency of TRAF binding. Consistent with this idea, Chaudhuri et al. showed that optimal binding of TRAF2 to CD40 requires residues 33–62 of the CD40 cytoplasmic domain (44). While this would explain the contribution of residues 46–53, further analysis is required to assess the contribution of residues 26–34 to the binding of TRAF proteins to CD40.

Why are residues 26–34 and 46–53 required for maximal activation of NF-{kappa}B by CD40 but not for full activation of JNK and MAPKAP kinase-2? One possibility is that the TRAF proteins that couple CD40 to NF-{kappa}B are different from the TRAF proteins that couple CD40 to JNK and p38 MAPK. The TRAF protein that links CD40 to NF-{kappa}B may need to interact with the regions flanking residues 35–45 to bind efficiently, while the TRAF proteins that link CD40 to JNK and p38 MAPK may not interact with these regions. Alternatively, the same TRAF proteins may link CD40 to NF-{kappa}B, JNK, and p38 MAPK, but greater amounts of these TRAF proteins may need to be bound to CD40 to cause maximal activation of NF-{kappa}B than to cause maximal activation of JNK and p38 MAPK. Interactions mediated by the regions flanking residues 35–45 of the CD40 cytoplasmic domain could improve the efficiency of TRAF binding, as is the case for TRAF2 binding (44).

While loss-of-function studies employing truncated CD40 proteins or CD40 with point mutations have shown that amino acids 35–45 of the CD40 cytoplasmic domain are important for some CD40 functions, our work shows for the first time that this 11-amino acid sequence is sufficient to mediate many CD40 signaling functions. This raises the question as to whether other regions of the CD40 cytoplasmic domain make significant contributions to CD40 signaling. In particular, TRAF6 binds to a distinct region of the CD40 cytoplasmic domain, residues 14–23 of human CD40 (which corresponds to residues 19–28 of murine CD40), and has been shown to activate both NF-{kappa}B and JNK when overexpressed in fibroblasts (34, 35). However, we found that the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor, which contains the TRAF6 binding site, was unable to activate NF-{kappa}B, JNK, or MAPKAP kinase-2 when expressed in WEHI-231 cells. Although WEHI-231 cells express TRAF6 mRNA (34), they may not express sufficient amounts of the TRAF6 protein to allow this region of CD40 to activate these signaling pathways. Alternatively, the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor may not be able to interact efficiently with TRAF6 either because it is misfolded or because additional sequences are required. While we cannot rule out that the cytoplasmic domain of CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) is misfolded, Pullen et al. showed that a peptide corresponding to residues 18–28 of murine CD40 can bind TRAF6 with high affinity in vitro (45). Thus, if folded properly, the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor should be able to bind TRAF6. Further work is required to determine the relative contribution of TRAF6 to the ability of CD40 to activate NF-{kappa}B, JNK, and p38 MAPK in B cells.

In addition to the CD8/(15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) chimeric receptor, which contains the TRAF6 binding site, the CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) and CD8/(43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) chimeric receptors did not activate JNK, MAPKAP kinase-2, or NF-{kappa}B to a significant extent. The simplest interpretation of these data is that these regions of CD40 cannot by themselves activate these signaling pathways. Recent work by Pullen et al. (45) using a set of overlapping 10- and 14-mer peptides covering the entire CD40 cytoplasmic domain indicated that human CD40 contains a single TRAF2/3/5 binding site within residues 30–40 of the cytoplasmic domain and a single TRAF6 binding within residues 15–23 (equivalent to residues 19–28 in murine CD40). This implies that the CD8/(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) and CD8/(43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63) chimeric receptors cannot bind the TRAF proteins that activate NF-{kappa}B, JNK, and p38 MAPK and that these regions of CD40, by themselves, do not mediate activation of these signaling pathways. While neither our experiments nor those of Pullen et al. can definitively rule out that these regions of CD40 are misfolded when expressed as peptides or when fused to the truncated CD8{alpha}, our observation that CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) activates JNK and MAPKAP kinase-2 to the same extent as CD8/1–74(1–74) supports the idea that other regions of CD40 do not contribute significantly to the activation of these MAPK signaling pathways. Similarly, the ability of the CD8/(26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptor to induce I{kappa}B{alpha} phosphorylation as well as CD8/1–74(1–74) suggests that residues 1–25 of the CD40 cytoplasmic domain do not by themselves make a significant contribution to NF-{kappa}B activation.

Our studies show that residues 35–45 of the CD40 cytoplasmic domain contain a major signaling motif that is able to mediate full activation of JNK and p38 MAPK as well as substantial activation of NF-{kappa}B. Work by Hostager et al. (53) together with recent experiments we have performed, indicate that there is a second signaling motif in the CD40 cytoplasmic domain that partially overlaps the NF-{kappa}B/JNK/p38 MAPK activation motif contained within residues 35–45 of the CD40 cytoplasmic domain. Hostager et al. showed that residues 41–62 of the CD40 cytoplasmic domain as well as the threonine at position 40 are required for CD40 to induce expression of the cell surface markers CD23, Fas, and B7.1 in the M12.4.1 murine B cell line (53). Recently, we have shown that the CD8/1–74(1–74) chimeric receptor could induce expression of CD23 in M12.4.1 cells, but that the CD8/(35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) and CD8/(43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) chimeric receptors could not (data not shown). This indicates that induction of CD23 expression is mediated by a CD40 signaling motif that is not entirely contained within residues 35–45 of the CD40 cytoplasmic domain. Thus, the CD40 cytoplasmic domain appears to contain two overlapping, but distinct, signaling motifs. The first motif, which we have defined in this report, is contained within residues 35–45 and mediates activation of NF-{kappa}B, JNK, and p38 MAPK. The second signaling motif requires the threonine at position 40 as well as other amino acids contained within residues 41–62 and is responsible for up-regulation of CD23 and other cell surface markers. The definition of this second CD40 signaling motif as well as the identification of adapter proteins that bind differentially to the two motifs are the next steps in elucidating the molecular basis of CD40 signaling.


   Acknowledgments
 
We thank Yvonne Yang for analyzing CD23 expression in M12.4.1 cells, and Marilyn Kehry, Debbie Law, Douglas Carlow, and James Weiler for helpful discussions.


   Footnotes
 
1 This work was supported by grants (to M.R.G.) from the Arthritis Society of Canada and the Medical Research Council of Canada and a Medical Research Council Scholarship (to M.R.G.). Back

2 Address correspondence and reprint requests to Dr. Michael R. Gold, Department of Microbiology and Immunology, University of British Columbia, 6174 University Blvd., Vancouver, British Columbia, Canada V6T 1Z3. E-mail address: Back

3 Abbreviations used in this paper: CD40L, CD40 ligand; JAK, Janus kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; MAPKAP kinase-2, mitogen-activated protein kinase-activated protein kinase-2; MKK, MAP kinase kinase; TNF-R, TNF receptor; TRAF, TNF-R-associated factor; GST, glutathione S-transferase. Back

Received for publication March 23, 1998. Accepted for publication January 21, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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