Dissecting Fas signaling with an altered-specificity death-domain mutant: requirement of FADD binding for apoptosis but not Jun N-terminal kinase activation

Abstract

Fas is a cell surface death receptor that regulates peripheral tolerance and lymphoid homeostasis. In many pathologic conditions, ectopic Fas activation mediates tissue destruction. Several proteins that can bind to the cytoplasmic death domain of Fas have been implicated in Fas signal transduction. Here we show that FADD, which couples Fas to pro-caspase-8, and, Daxx, which couples Fas to the Jun N-terminal kinase pathway, bind independently to the Fas death domain. We have isolated a death domain mutant, termed FasDelta, that selectively binds Daxx but not FADD. In tranfected tissue culture cells, FasDelta activated Jun N-terminal kinase normally but was impaired in cell death induction. These results suggest that FADD and Daxx activate two independent pathways downstream of Fas and confirm the essential role of FADD binding in apoptosis induction.

Figure 1

Independent binding of DaxxC and FADD(80–205) to Fas. (A) Binding of in vitro-translated [³⁵S]DaxxC and FADD(80–205) to GST-FasIC. [³⁵S]DaxxC (5 μl) and [³⁵S]FADD(80–205) (0.1 μl) IVT reactions were added in all lanes. Lanes 2, 3, and 4 contained 5, 10, or 20 μl of IVT nonradioactive DaxxC; lanes 5, 6, and 7 contained 0.5, 2, or 5 μg of recombinant purified hFADD(95–208). (B) Lack of cooperative binding of DaxxC and FADD(80–205) to GST-FasIC. Upper, [³⁵S]FADD(80–205) IVT reaction (0.1 μl in lanes 1 and 4; 0.3 μl in lanes 2 and 5; 1.0 μl in lanes 3 and 6) were incubated with GST-FasIC in the absence (lanes 1–3) or presence (lanes 4–6) of 20 μl of nonradioactive IVT DaxxC. Lower, [³⁵S]DaxxC IVT reaction (2 μl in lanes 1 and 4; 5 μl in lanes 2 and 5; 10 μl in lanes 3 and 6) were incubated with GST-FasIC in the absence (lanes 1–3) or presence (lanes 4–6) of 5 μg of recombinant purified hFADD(95–208). Positions of molecular weight standards (in kDa) are shown at right.

Figure 2

Identification of FasΔ. (A) Schematic representation of fusion proteins encompassing the cytoplasmic domain of murine Fas (41). Boundaries of the death domain and FasΔ deletion are indicated. (B) Binding of in vitro translated [³⁵S]DaxxC and FADD(80–205) to GST-fusion proteins. [³⁵S]DaxxC (10 μl) and [³⁵S]FADD(80–205) (0.2 μl) IVT reactions were added to all lanes. Positions of MW standards (in kDa) are shown at right (Upper). Coomassie blue-stained GST fusion proteins from the same gel is shown (Lower).

Figure 3

(A) Expression of Myc-Fas and Myc-FasΔ. Human embryonic kidney 293 cells were transfected with the indicated plasmids, immunoprecipitated with anti-Myc antibody conjugated to agarose beads, and immunoblotted with anti-Myc antibody. (B) JNK activation by Myc-Fas and Myc-FasΔ. Flag-tagged JNK1 (Flag-JNK) and the indicated plasmids were cotransfected into 293 cells; 24 hours after transfection, cells were treated with 0.5 μg/ml Jo2 antibody for 30 min and assayed for JNK kinase activity. Upper, Phosphorylation of GST-cJun. Lower, Expression of Flag-JNK. The data shown are representative of three independent assays.

Figure 4

Apoptosis by Fas and FasΔ. HeLa cells were transfected with pEBB, pEBB-Myc-Fas, or pEBB-Myc-FasΔ plasmids with 0.5 μg of pCMV-lacZ. Jo2 antibody was added 16 hr later. The cells were stained with 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-gal) 24 hours (A) or 48 hours (B) after transfection, and blue cells were analyzed for apoptotic morphology. (C) Synergistic killing of MycFasΔ and ASK1. The indicated plasmids and pCMV-lacZ were cotransfected in HeLa cells; amount of transfected DNA were equalized by adding vector DNA. The cells were stained with X-gal 24 hr after transfection and analyzed for apoptotic morphology as described in Materials and Methods.