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SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane.

Siegel RM, Muppidi JR, Sarker M, Lobito A, Jen M, Martin D, Straus SE, Lenardo MJ - J. Cell Biol. (2004)

Bottom Line: Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex.We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling.Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. rsiegel@nih.gov

ABSTRACT
Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

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ALPS-associated Fas mutations block SPOTS formation by distinct mechanisms. (A) GST pull-down assay with the indicated mutant Fas DD proteins or GST alone, and 35S-labeled FADD, was performed as described previously (Martin et al., 1999). Vertical black lines divide lanes taken from the same blot. (B) Signaling complex formation and caspase-8 processing in EBV lines from selected Fas mutant ALPS patients and zVAD-fmk treated normal donor-derived cells. EBV cell lines were stimulated with 1 μg/ml of APO-1 anti-Fas mAb for 10 min or the times indicated, and the DISC was immunoprecipitated as described previously (Martin et al., 1999). Caspase-8 cleavage in cell lysates are shown in the bottom panel. Arrowheads denote protein fragments of interest and open circles denote background bands. Vertical black lines divide lanes taken from the same blot. (C) DEVDase effector caspase assay of lysates from EBV-transformed cell lines or ALPS patient EBV cell lines heterozygous for the indicated Fas mutations. Cells were treated for 1 h with 1 μg/ml of APO-1 anti-Fas mAb. Assays were performed as described previously (Martin et al., 1999). Values are the fluorescence of treated minus control for each cell line in arbitrary fluorescence units. Error bars represent the SEM for triplicate measurements.
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fig7: ALPS-associated Fas mutations block SPOTS formation by distinct mechanisms. (A) GST pull-down assay with the indicated mutant Fas DD proteins or GST alone, and 35S-labeled FADD, was performed as described previously (Martin et al., 1999). Vertical black lines divide lanes taken from the same blot. (B) Signaling complex formation and caspase-8 processing in EBV lines from selected Fas mutant ALPS patients and zVAD-fmk treated normal donor-derived cells. EBV cell lines were stimulated with 1 μg/ml of APO-1 anti-Fas mAb for 10 min or the times indicated, and the DISC was immunoprecipitated as described previously (Martin et al., 1999). Caspase-8 cleavage in cell lysates are shown in the bottom panel. Arrowheads denote protein fragments of interest and open circles denote background bands. Vertical black lines divide lanes taken from the same blot. (C) DEVDase effector caspase assay of lysates from EBV-transformed cell lines or ALPS patient EBV cell lines heterozygous for the indicated Fas mutations. Cells were treated for 1 h with 1 μg/ml of APO-1 anti-Fas mAb. Assays were performed as described previously (Martin et al., 1999). Values are the fluorescence of treated minus control for each cell line in arbitrary fluorescence units. Error bars represent the SEM for triplicate measurements.

Mentions: Most ALPS-associated Fas mutations in the DD fail to bind to FADD, resulting in dominant inhibition of DISC formation (Martin et al., 1999; Vaishnaw et al., 1999). However, unlike all other previously described Fas DD mutants, the Fas T225K mutation from ALPS kindred #27 retained the ability to interact with FADD in GST pull-down and coimmunoprecipitation assays, although FADD binding was weaker than for wild-type Fas (Fig. 7 A and not depicted). Mutation of the same amino acid to proline in the T225P mutant from another ALPS family completely abrogated FADD binding and globally perturbed DD folding (Fig. 7 A; Martin et al., 1999). However, the Fas T225K DD retained a normal overall structure (unpublished data). Analysis of DISC formation in patient-derived cell lines harboring the T225K mutant showed normal recruitment of FADD and procaspase-8 after anti-Fas stimulation, whereas cells from ALPS patients harboring the non-FADD binding R234Q mutation or other Fas DD mutations demonstrated severe defects in DISC formation (Fig. 7 B; Martin et al., 1999). Despite normal DISC formation, cells harboring the T225K mutation produced no detectable fully processed caspase-8 in cell lysates even after prolonged Fas stimulation (Fig. 7 B). This pattern was similar to wild-type cells treated with anti-Fas in the presence of zVAD-fmk (Fig. 7 B). Apoptosis, as measured by effector caspase activation, was impaired in cell lines derived from patients heterozygous for the Fas T225K mutation, but was slightly higher than that in cell lines from patients harboring non FADD-binding DD mutations (Fig. 7 C). This finding is consistent with data showing that FasT225K is defective when transfected alone and is a weak dominant-negative inhibitor of apoptosis triggered by wild-type Fas (Jackson et al., 1999).


SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane.

Siegel RM, Muppidi JR, Sarker M, Lobito A, Jen M, Martin D, Straus SE, Lenardo MJ - J. Cell Biol. (2004)

ALPS-associated Fas mutations block SPOTS formation by distinct mechanisms. (A) GST pull-down assay with the indicated mutant Fas DD proteins or GST alone, and 35S-labeled FADD, was performed as described previously (Martin et al., 1999). Vertical black lines divide lanes taken from the same blot. (B) Signaling complex formation and caspase-8 processing in EBV lines from selected Fas mutant ALPS patients and zVAD-fmk treated normal donor-derived cells. EBV cell lines were stimulated with 1 μg/ml of APO-1 anti-Fas mAb for 10 min or the times indicated, and the DISC was immunoprecipitated as described previously (Martin et al., 1999). Caspase-8 cleavage in cell lysates are shown in the bottom panel. Arrowheads denote protein fragments of interest and open circles denote background bands. Vertical black lines divide lanes taken from the same blot. (C) DEVDase effector caspase assay of lysates from EBV-transformed cell lines or ALPS patient EBV cell lines heterozygous for the indicated Fas mutations. Cells were treated for 1 h with 1 μg/ml of APO-1 anti-Fas mAb. Assays were performed as described previously (Martin et al., 1999). Values are the fluorescence of treated minus control for each cell line in arbitrary fluorescence units. Error bars represent the SEM for triplicate measurements.
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fig7: ALPS-associated Fas mutations block SPOTS formation by distinct mechanisms. (A) GST pull-down assay with the indicated mutant Fas DD proteins or GST alone, and 35S-labeled FADD, was performed as described previously (Martin et al., 1999). Vertical black lines divide lanes taken from the same blot. (B) Signaling complex formation and caspase-8 processing in EBV lines from selected Fas mutant ALPS patients and zVAD-fmk treated normal donor-derived cells. EBV cell lines were stimulated with 1 μg/ml of APO-1 anti-Fas mAb for 10 min or the times indicated, and the DISC was immunoprecipitated as described previously (Martin et al., 1999). Caspase-8 cleavage in cell lysates are shown in the bottom panel. Arrowheads denote protein fragments of interest and open circles denote background bands. Vertical black lines divide lanes taken from the same blot. (C) DEVDase effector caspase assay of lysates from EBV-transformed cell lines or ALPS patient EBV cell lines heterozygous for the indicated Fas mutations. Cells were treated for 1 h with 1 μg/ml of APO-1 anti-Fas mAb. Assays were performed as described previously (Martin et al., 1999). Values are the fluorescence of treated minus control for each cell line in arbitrary fluorescence units. Error bars represent the SEM for triplicate measurements.
Mentions: Most ALPS-associated Fas mutations in the DD fail to bind to FADD, resulting in dominant inhibition of DISC formation (Martin et al., 1999; Vaishnaw et al., 1999). However, unlike all other previously described Fas DD mutants, the Fas T225K mutation from ALPS kindred #27 retained the ability to interact with FADD in GST pull-down and coimmunoprecipitation assays, although FADD binding was weaker than for wild-type Fas (Fig. 7 A and not depicted). Mutation of the same amino acid to proline in the T225P mutant from another ALPS family completely abrogated FADD binding and globally perturbed DD folding (Fig. 7 A; Martin et al., 1999). However, the Fas T225K DD retained a normal overall structure (unpublished data). Analysis of DISC formation in patient-derived cell lines harboring the T225K mutant showed normal recruitment of FADD and procaspase-8 after anti-Fas stimulation, whereas cells from ALPS patients harboring the non-FADD binding R234Q mutation or other Fas DD mutations demonstrated severe defects in DISC formation (Fig. 7 B; Martin et al., 1999). Despite normal DISC formation, cells harboring the T225K mutation produced no detectable fully processed caspase-8 in cell lysates even after prolonged Fas stimulation (Fig. 7 B). This pattern was similar to wild-type cells treated with anti-Fas in the presence of zVAD-fmk (Fig. 7 B). Apoptosis, as measured by effector caspase activation, was impaired in cell lines derived from patients heterozygous for the Fas T225K mutation, but was slightly higher than that in cell lines from patients harboring non FADD-binding DD mutations (Fig. 7 C). This finding is consistent with data showing that FasT225K is defective when transfected alone and is a weak dominant-negative inhibitor of apoptosis triggered by wild-type Fas (Jackson et al., 1999).

Bottom Line: Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex.We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling.Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. rsiegel@nih.gov

ABSTRACT
Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.

Show MeSH
Related in: MedlinePlus