Abstract
FADD is a key adaptor modulating several signaling pathways such as apoptosis induced by Fas (CD95) and tumor necrosis factor receptor 1, and cell proliferation induced by mitogens. Whereas mutations in Fas disrupt its binding to FADD and cause autoimmune lymphoproliferative (lpr) syndromes, a FADD deficiency blocks embryonic development in mice. To delineate the multifunction of FADD in vivo, we performed functional reconstitution analysis by introducing wild type and mutant FADD into FADD-/- cells or FADD-/- mice lacking the endogenous FADD. An lpr-like FADD mutant, V121N, was reported previously as being defective in Fas binding in vitro. However, we found that in mice V121N can bind to Fas and is functional in signaling apoptosis. Unexpectedly, this lpr-like mutant FADD failed to support mouse development, indicating that the death domain of FADD has an additional function required for embryogenesis, which is independent of that required for receptor-induced apoptosis. Further mutagenesis was targeted at charged residues in the FADD death domain, presumably mediating electrostatic interactions with Fas. We showed that the target binding and apoptosis signaling functions of FADD were not affected when mutations were introduced to a majority of the charged residues. In one exception, replacing arginine 117 with an uncharged residue disrupted target binding and apoptosis signaling, but restoring the positive charge at position 117 failed to reconstitute the FADD function. Therefore, in vivo target binding of FADD involves an additional mechanism distinct from electrostatic interaction.
Highlights
Fas-induced apoptosis is essential for maintaining lymphoid homeostasis and suppressing autoimmunity [21]
Because mice lacking each of the FADD-dependent death receptors develop normally [26, 33,34,35], it is possible that mutations disabling the cell death function of FADD would still allow a partial or complete embryogenesis
FADD-deficient cells are resistant to cell death induced by Fas, tumor necrosis factor (TNF)-R1, and TRAIL-R (24, 25, 29 –32)
Summary
Genomic Reconstitution of FADDϪ/Ϫ Mice—The 12-kb EcoRV genomic DNA containing the promoter region, two coding exons, and the intron of the mouse FADD gene was subcloned from a 23-kb genomic DNA inserted in a cosmid clone [12]. Stable FADDϪ/Ϫ MEFs infected with the wild type or mutant FADDcontaining viruses were detached from culture plates by trypsinization and were resuspended in 5 ml of DMEM containing 10% fetal calf serum. Agonistic monoclonal anti-Fas antibodies (Jo2, Pharmingen) were added (10 g), followed by a 15-min incubation at 37 °C These treated cells were washed with ice-cold PBS and lysed for 1 h at 4 °C in a lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 10 mM -glycerophosphate, 1 mM sodium vanadate, 0.1 mM NaF, 1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Roche Applied Science). After a 10-h incubation at 37 °C (about 70% confluence), cells
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