Figure 1.
Figure 1. The TRAF2/TRAF3 binding domain of CD40 mediates CD154-induced death signals. (A) Graphical representation of CD40 and its TRAF-binding domains. A double Q234E235 → AA mutation, yielding CD40mT6, selectively abolishes the interaction of TRAF6 with CD40, whereas a T254 → A mutation (CD40mT2/T3) inhibits TRAF2 and TRAF3 but not TRAF6 binding to CD40. CD40mT2/T3/T6 combines the aforementioned mutations and perturbs the binding of all TRAFs (Davies et al., 2005b). WT, wild type. (B) The TRAF6-interacting domain of CD40 transduces ERK and Akt signaling. Lysates from CD154-stimulated HeLa/CD40 and HeLa/CD40mT6 cells were analyzed for the expression of phosphorylated, active ERK and Akt, or the total proteins. (C) The TRAF2/TRAF3-interacting domain of CD40 mediates CD154-induced death signals. HeLa clones expressing the WT or mutated CD40 sequences described in A were stimulated with CD154 for 12 h before assessment of apoptosis. Mean values of percentage apoptotic cells from at least three independent experiments are shown with the exception of HeLa/CD40mT6 clone 10, where two determinations were performed. (D and E) The TRAF2/TRAF3-binding domain of CD40 mediates cell death via caspase-8 activation. HeLa/CD40mT6 clone 21 cells were stimulated with CD154 for the indicated time points, and lysates were analyzed for the expression of caspase-8, caspase-3, or β-actin (D). Inhibitors of caspase-8 and -3 but not caspase-9 protect HeLa/CD40mT6 cells from CD154-induced apoptosis assessed by cell death ELISA. Data are expressed as fold increase (±SD; n = 4) in apoptosis induced by CD154 relative to untreated cultures, which was given the arbitrary value of 1. (F) RT-PCR showing up-regulation of TNF mRNA after treatment of HeLa/CD40mT6 cells with CD154. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (G) Early CD154-mediated death signals are independent of autocrine TNF production. HeLa/CD40mT6 cells were exposed to 0.5 µg/ml neutralizing anti-TNF mAb and then treated as described in C before assessment of apoptosis. Error bars indicate SD.

The TRAF2/TRAF3 binding domain of CD40 mediates CD154-induced death signals. (A) Graphical representation of CD40 and its TRAF-binding domains. A double Q234E235 → AA mutation, yielding CD40mT6, selectively abolishes the interaction of TRAF6 with CD40, whereas a T254 → A mutation (CD40mT2/T3) inhibits TRAF2 and TRAF3 but not TRAF6 binding to CD40. CD40mT2/T3/T6 combines the aforementioned mutations and perturbs the binding of all TRAFs (Davies et al., 2005b). WT, wild type. (B) The TRAF6-interacting domain of CD40 transduces ERK and Akt signaling. Lysates from CD154-stimulated HeLa/CD40 and HeLa/CD40mT6 cells were analyzed for the expression of phosphorylated, active ERK and Akt, or the total proteins. (C) The TRAF2/TRAF3-interacting domain of CD40 mediates CD154-induced death signals. HeLa clones expressing the WT or mutated CD40 sequences described in A were stimulated with CD154 for 12 h before assessment of apoptosis. Mean values of percentage apoptotic cells from at least three independent experiments are shown with the exception of HeLa/CD40mT6 clone 10, where two determinations were performed. (D and E) The TRAF2/TRAF3-binding domain of CD40 mediates cell death via caspase-8 activation. HeLa/CD40mT6 clone 21 cells were stimulated with CD154 for the indicated time points, and lysates were analyzed for the expression of caspase-8, caspase-3, or β-actin (D). Inhibitors of caspase-8 and -3 but not caspase-9 protect HeLa/CD40mT6 cells from CD154-induced apoptosis assessed by cell death ELISA. Data are expressed as fold increase (±SD; n = 4) in apoptosis induced by CD154 relative to untreated cultures, which was given the arbitrary value of 1. (F) RT-PCR showing up-regulation of TNF mRNA after treatment of HeLa/CD40mT6 cells with CD154. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (G) Early CD154-mediated death signals are independent of autocrine TNF production. HeLa/CD40mT6 cells were exposed to 0.5 µg/ml neutralizing anti-TNF mAb and then treated as described in C before assessment of apoptosis. Error bars indicate SD.

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