doi: 10.1093/emboj/cdf530.
Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1
Affiliations
- PMID: 12356728
- PMCID: PMC129052
- DOI: 10.1093/emboj/cdf530
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Jafrac2 is an IAP antagonist that promotes cell death by liberating Dronc from DIAP1
EMBO J.
.
Abstract
Members of the Inhibitor of Apoptosis Protein (IAP) family are essential for cell survival in Drosophila and appear to neutralize the cell death machinery by binding to and ubiquitylating pro-apoptotic caspases. Cell death is triggered when "Reaper-like" proteins bind to IAPs and liberate caspases from IAPs. We have identified the thioredoxin peroxidase Jafrac2 as an IAP-interacting protein in Drosophila cells that harbours a conserved N-terminal IAP-binding motif. In healthy cells, Jafrac2 resides in the endoplasmic reticulum but is rapidly released into the cytosol following induction of apoptosis. Mature Jafrac2 interacts genetically and biochemically with DIAP1 and promotes cell death in tissue culture cells and the Drosophila developing eye. In common with Rpr, Jafrac2-mediated cell death is contingent on DIAP1 binding because mutations that abolish the Jafrac2-DIAP1 interaction suppress the eye phenotype caused by Jafrac2 expression. We show that Jafrac2 displaces Dronc from DIAP1 by competing with Dronc for the binding of DIAP1, consistent with the idea that Jafrac2 triggers cell death by liberating Dronc from DIAP1-mediated inhibition.
Figures
Fig. 1. Jafrac2 is an IAP-binding protein. (A) DIAP2 (3) and DIAP1 (4) but not an unrelated control protein (5) specifically co-purified a protein with an apparent molecular weight of 26 kDa (Jafrac2). Purified material was resolved by SDS–PAGE and visualized by silver staining. Molecular mass markers in kDa are shown (1). (2) is an empty lane. (B) Bar diagram representing the structure of Jafrac2 and the location of the AKP motif. ERS designates the ER targeting sequence. Cysteine residues important for the thioredoxin peroxidase activity of Jafrac2 are indicated. Lower panel: alignment of the N-terminal sequences of Rpr, Grim and Hid (left) and Jafrac2, Smac/DIABLO and HtrA2/Omi (right). Proteins were grouped based on the maturation by a methionine amino peptidase (left) and by proteolytic cleavage (right). Identical residues are shown in black, residues identical in two out of the three sequences are indicated in light grey, while residues with ≥60% similarity are shown in dark grey. (C) Co-immunoprecipitation of endogenous Jafrac2 with endogenous DIAP1 from S2 cells. Jafrac2 was co-immunoprecipitated by anti-full-length (3) and anti-RING DIAP1 (5) antibodies, but not the pre-immune serum (2 and 4). (D) The BIR2 of DIAP1 is required for Jafrac2 binding. S2 cells were transiently transfected with the indicated combinations of constructs encoding Jafrac2-V5 and wild-type or mutant DIAP1–GST. DIAP1–GST was purified from cell lysates using gluthathione beads and associated Jafrac2 was detected by immunoblot analysis with anti-V5 antibodies (top panel). Effective DIAP1 purification was determined by immunoblotting the eluate with an anti-DIAP1 RING antibody (second panel). Expression of the indicated constructs was confirmed by immunoblotting with the indicated antibodies (third and fourth panels). An asterisk denotes endogenous DIAP1.
Fig. 1. Jafrac2 is an IAP-binding protein. (A) DIAP2 (3) and DIAP1 (4) but not an unrelated control protein (5) specifically co-purified a protein with an apparent molecular weight of 26 kDa (Jafrac2). Purified material was resolved by SDS–PAGE and visualized by silver staining. Molecular mass markers in kDa are shown (1). (2) is an empty lane. (B) Bar diagram representing the structure of Jafrac2 and the location of the AKP motif. ERS designates the ER targeting sequence. Cysteine residues important for the thioredoxin peroxidase activity of Jafrac2 are indicated. Lower panel: alignment of the N-terminal sequences of Rpr, Grim and Hid (left) and Jafrac2, Smac/DIABLO and HtrA2/Omi (right). Proteins were grouped based on the maturation by a methionine amino peptidase (left) and by proteolytic cleavage (right). Identical residues are shown in black, residues identical in two out of the three sequences are indicated in light grey, while residues with ≥60% similarity are shown in dark grey. (C) Co-immunoprecipitation of endogenous Jafrac2 with endogenous DIAP1 from S2 cells. Jafrac2 was co-immunoprecipitated by anti-full-length (3) and anti-RING DIAP1 (5) antibodies, but not the pre-immune serum (2 and 4). (D) The BIR2 of DIAP1 is required for Jafrac2 binding. S2 cells were transiently transfected with the indicated combinations of constructs encoding Jafrac2-V5 and wild-type or mutant DIAP1–GST. DIAP1–GST was purified from cell lysates using gluthathione beads and associated Jafrac2 was detected by immunoblot analysis with anti-V5 antibodies (top panel). Effective DIAP1 purification was determined by immunoblotting the eluate with an anti-DIAP1 RING antibody (second panel). Expression of the indicated constructs was confirmed by immunoblotting with the indicated antibodies (third and fourth panels). An asterisk denotes endogenous DIAP1.
Fig. 1. Jafrac2 is an IAP-binding protein. (A) DIAP2 (3) and DIAP1 (4) but not an unrelated control protein (5) specifically co-purified a protein with an apparent molecular weight of 26 kDa (Jafrac2). Purified material was resolved by SDS–PAGE and visualized by silver staining. Molecular mass markers in kDa are shown (1). (2) is an empty lane. (B) Bar diagram representing the structure of Jafrac2 and the location of the AKP motif. ERS designates the ER targeting sequence. Cysteine residues important for the thioredoxin peroxidase activity of Jafrac2 are indicated. Lower panel: alignment of the N-terminal sequences of Rpr, Grim and Hid (left) and Jafrac2, Smac/DIABLO and HtrA2/Omi (right). Proteins were grouped based on the maturation by a methionine amino peptidase (left) and by proteolytic cleavage (right). Identical residues are shown in black, residues identical in two out of the three sequences are indicated in light grey, while residues with ≥60% similarity are shown in dark grey. (C) Co-immunoprecipitation of endogenous Jafrac2 with endogenous DIAP1 from S2 cells. Jafrac2 was co-immunoprecipitated by anti-full-length (3) and anti-RING DIAP1 (5) antibodies, but not the pre-immune serum (2 and 4). (D) The BIR2 of DIAP1 is required for Jafrac2 binding. S2 cells were transiently transfected with the indicated combinations of constructs encoding Jafrac2-V5 and wild-type or mutant DIAP1–GST. DIAP1–GST was purified from cell lysates using gluthathione beads and associated Jafrac2 was detected by immunoblot analysis with anti-V5 antibodies (top panel). Effective DIAP1 purification was determined by immunoblotting the eluate with an anti-DIAP1 RING antibody (second panel). Expression of the indicated constructs was confirmed by immunoblotting with the indicated antibodies (third and fourth panels). An asterisk denotes endogenous DIAP1.
Fig. 1. Jafrac2 is an IAP-binding protein. (A) DIAP2 (3) and DIAP1 (4) but not an unrelated control protein (5) specifically co-purified a protein with an apparent molecular weight of 26 kDa (Jafrac2). Purified material was resolved by SDS–PAGE and visualized by silver staining. Molecular mass markers in kDa are shown (1). (2) is an empty lane. (B) Bar diagram representing the structure of Jafrac2 and the location of the AKP motif. ERS designates the ER targeting sequence. Cysteine residues important for the thioredoxin peroxidase activity of Jafrac2 are indicated. Lower panel: alignment of the N-terminal sequences of Rpr, Grim and Hid (left) and Jafrac2, Smac/DIABLO and HtrA2/Omi (right). Proteins were grouped based on the maturation by a methionine amino peptidase (left) and by proteolytic cleavage (right). Identical residues are shown in black, residues identical in two out of the three sequences are indicated in light grey, while residues with ≥60% similarity are shown in dark grey. (C) Co-immunoprecipitation of endogenous Jafrac2 with endogenous DIAP1 from S2 cells. Jafrac2 was co-immunoprecipitated by anti-full-length (3) and anti-RING DIAP1 (5) antibodies, but not the pre-immune serum (2 and 4). (D) The BIR2 of DIAP1 is required for Jafrac2 binding. S2 cells were transiently transfected with the indicated combinations of constructs encoding Jafrac2-V5 and wild-type or mutant DIAP1–GST. DIAP1–GST was purified from cell lysates using gluthathione beads and associated Jafrac2 was detected by immunoblot analysis with anti-V5 antibodies (top panel). Effective DIAP1 purification was determined by immunoblotting the eluate with an anti-DIAP1 RING antibody (second panel). Expression of the indicated constructs was confirmed by immunoblotting with the indicated antibodies (third and fourth panels). An asterisk denotes endogenous DIAP1.
Fig. 2. Subcellular localization of Jafrac2. (A) Endogenous Jafrac2 resides in the ER. Immunofluorescence confocal microscopy of SR2 cells stained with an anti-Jafrac2 antibody (red) and GFP–Spitz (green). Right panels represent merged micrographs. (B) The N-terminal 17 Aa of Jafrac2 are required for targeting Jafrac2 to the ER. Confocal micrographs of SR2 cells transfected with ΔN1–17-Jafrac2 lacking the N-terminal signal sequence and stained with anti-V5 (red) and anti-DIAP1 antibodies (green). (C) Confocal micrographs of NIH 3T3 cells transfected with Jafrac2 (red) and the ER-marker ECFP-ER (green). (D) Jafrac2 is an intracellular protein. Endogenous Jafrac2 is present exclusively in the cellular but absent from the medium fraction. (E) Jafrac2 is released from the ER in UV-mediated apoptosis. Normal and UV-treated S2 cells were lysed in a buffer containing 0.025% digitonin and subfractionated into membrane and cytosolic fractions. The cytosolic fractions were examined by immunoblot analysis with the indicated antibodies. (F) Jafrac2 is released from the ER by the ER stress-inducing agents brefeldin A or tunicamycin. Normal and treated NIH 3T3 cells stably expressing Jafrac2-V5 were analysed as in (E). At 3–4 h post-UV (G) or -brefeldin A treatment (H) of NIH 3T3, the majority of Jafrac2 (red) is no longer co-localized with ECFP-ER (green), as seen by overlaying the green and red images (yellow).
Fig. 2. Subcellular localization of Jafrac2. (A) Endogenous Jafrac2 resides in the ER. Immunofluorescence confocal microscopy of SR2 cells stained with an anti-Jafrac2 antibody (red) and GFP–Spitz (green). Right panels represent merged micrographs. (B) The N-terminal 17 Aa of Jafrac2 are required for targeting Jafrac2 to the ER. Confocal micrographs of SR2 cells transfected with ΔN1–17-Jafrac2 lacking the N-terminal signal sequence and stained with anti-V5 (red) and anti-DIAP1 antibodies (green). (C) Confocal micrographs of NIH 3T3 cells transfected with Jafrac2 (red) and the ER-marker ECFP-ER (green). (D) Jafrac2 is an intracellular protein. Endogenous Jafrac2 is present exclusively in the cellular but absent from the medium fraction. (E) Jafrac2 is released from the ER in UV-mediated apoptosis. Normal and UV-treated S2 cells were lysed in a buffer containing 0.025% digitonin and subfractionated into membrane and cytosolic fractions. The cytosolic fractions were examined by immunoblot analysis with the indicated antibodies. (F) Jafrac2 is released from the ER by the ER stress-inducing agents brefeldin A or tunicamycin. Normal and treated NIH 3T3 cells stably expressing Jafrac2-V5 were analysed as in (E). At 3–4 h post-UV (G) or -brefeldin A treatment (H) of NIH 3T3, the majority of Jafrac2 (red) is no longer co-localized with ECFP-ER (green), as seen by overlaying the green and red images (yellow).
Fig. 2. Subcellular localization of Jafrac2. (A) Endogenous Jafrac2 resides in the ER. Immunofluorescence confocal microscopy of SR2 cells stained with an anti-Jafrac2 antibody (red) and GFP–Spitz (green). Right panels represent merged micrographs. (B) The N-terminal 17 Aa of Jafrac2 are required for targeting Jafrac2 to the ER. Confocal micrographs of SR2 cells transfected with ΔN1–17-Jafrac2 lacking the N-terminal signal sequence and stained with anti-V5 (red) and anti-DIAP1 antibodies (green). (C) Confocal micrographs of NIH 3T3 cells transfected with Jafrac2 (red) and the ER-marker ECFP-ER (green). (D) Jafrac2 is an intracellular protein. Endogenous Jafrac2 is present exclusively in the cellular but absent from the medium fraction. (E) Jafrac2 is released from the ER in UV-mediated apoptosis. Normal and UV-treated S2 cells were lysed in a buffer containing 0.025% digitonin and subfractionated into membrane and cytosolic fractions. The cytosolic fractions were examined by immunoblot analysis with the indicated antibodies. (F) Jafrac2 is released from the ER by the ER stress-inducing agents brefeldin A or tunicamycin. Normal and treated NIH 3T3 cells stably expressing Jafrac2-V5 were analysed as in (E). At 3–4 h post-UV (G) or -brefeldin A treatment (H) of NIH 3T3, the majority of Jafrac2 (red) is no longer co-localized with ECFP-ER (green), as seen by overlaying the green and red images (yellow).
Fig. 2. Subcellular localization of Jafrac2. (A) Endogenous Jafrac2 resides in the ER. Immunofluorescence confocal microscopy of SR2 cells stained with an anti-Jafrac2 antibody (red) and GFP–Spitz (green). Right panels represent merged micrographs. (B) The N-terminal 17 Aa of Jafrac2 are required for targeting Jafrac2 to the ER. Confocal micrographs of SR2 cells transfected with ΔN1–17-Jafrac2 lacking the N-terminal signal sequence and stained with anti-V5 (red) and anti-DIAP1 antibodies (green). (C) Confocal micrographs of NIH 3T3 cells transfected with Jafrac2 (red) and the ER-marker ECFP-ER (green). (D) Jafrac2 is an intracellular protein. Endogenous Jafrac2 is present exclusively in the cellular but absent from the medium fraction. (E) Jafrac2 is released from the ER in UV-mediated apoptosis. Normal and UV-treated S2 cells were lysed in a buffer containing 0.025% digitonin and subfractionated into membrane and cytosolic fractions. The cytosolic fractions were examined by immunoblot analysis with the indicated antibodies. (F) Jafrac2 is released from the ER by the ER stress-inducing agents brefeldin A or tunicamycin. Normal and treated NIH 3T3 cells stably expressing Jafrac2-V5 were analysed as in (E). At 3–4 h post-UV (G) or -brefeldin A treatment (H) of NIH 3T3, the majority of Jafrac2 (red) is no longer co-localized with ECFP-ER (green), as seen by overlaying the green and red images (yellow).
Fig. 3. DIAP1 binding is mediated by the N-terminal IBMs of Jafrac2 and Rpr. (A) AKP- but not KP-Jafrac2 interacts with DIAP1. Co-purification of Jafrac2 with DIAP1 from cellular extracts. In this and subsequent figures, expression and purification of the indicated constructs was determined as in Figure 1D. (B) The thioredoxin peroxidase activity of Jafrac2 is not required for DIAP1 binding. Co-purification of wild-type and Jafrac2 mutants with DIAP1 from cellular extracts. (C) AKP-Jafrac2 directly interacts with DIAP1. In vitro co-purification of recombinant AKP- and KP-Jafrac2 with recombinant DIAP1 protein is shown. First and second panel: immunoblot analysis of Jafrac2- and DIAP1-specific antibodies. Third panel: Coomassie-stained gel of recombinant AKP- and KP-Jafrac2.
Fig. 3. DIAP1 binding is mediated by the N-terminal IBMs of Jafrac2 and Rpr. (A) AKP- but not KP-Jafrac2 interacts with DIAP1. Co-purification of Jafrac2 with DIAP1 from cellular extracts. In this and subsequent figures, expression and purification of the indicated constructs was determined as in Figure 1D. (B) The thioredoxin peroxidase activity of Jafrac2 is not required for DIAP1 binding. Co-purification of wild-type and Jafrac2 mutants with DIAP1 from cellular extracts. (C) AKP-Jafrac2 directly interacts with DIAP1. In vitro co-purification of recombinant AKP- and KP-Jafrac2 with recombinant DIAP1 protein is shown. First and second panel: immunoblot analysis of Jafrac2- and DIAP1-specific antibodies. Third panel: Coomassie-stained gel of recombinant AKP- and KP-Jafrac2.
Fig. 3. DIAP1 binding is mediated by the N-terminal IBMs of Jafrac2 and Rpr. (A) AKP- but not KP-Jafrac2 interacts with DIAP1. Co-purification of Jafrac2 with DIAP1 from cellular extracts. In this and subsequent figures, expression and purification of the indicated constructs was determined as in Figure 1D. (B) The thioredoxin peroxidase activity of Jafrac2 is not required for DIAP1 binding. Co-purification of wild-type and Jafrac2 mutants with DIAP1 from cellular extracts. (C) AKP-Jafrac2 directly interacts with DIAP1. In vitro co-purification of recombinant AKP- and KP-Jafrac2 with recombinant DIAP1 protein is shown. First and second panel: immunoblot analysis of Jafrac2- and DIAP1-specific antibodies. Third panel: Coomassie-stained gel of recombinant AKP- and KP-Jafrac2.
Fig. 4. Induced expression of AKP- but not KP-Jafrac2 causes induction of apoptosis in S2 cells. Similarly, AVA- and AKP-Rpr but not VA- and KP-Rpr promote cell death. The indicated Ub fusion constructs were co-transfected with a lacZ reporter plasmid, and 24 h post-transfection cells from each well were divided into two dishes to avoid variations in transfection efficiencies. Expression of the indicated constructs was induced by copper sulfate and cells were examined for β-galactosidase (β-gal) activity. (A–D) and (I–L), untreated cells; (E–H) and (M–O), cells treated with copper sulfate (induced state).
Fig. 5. Jafrac2 competes with Dronc for the binding of DIAP1. The ability of DIAP1 to bind to Dronc is significantly impaired in the presence of AKP- but not KP-Jafrac2. Co-purification of a catalytically inactive Dronc mutant (Dronc C>A) with DIAP1–GST in the presence of KP- or AKP-Jafrac2 from cellular extracts. Expression and purification of the indicated constructs was determined as in Figure 1D.
Fig. 6. Ectopic expression of AKP-Jafrac2, AVA- and AKP-Rpr in the developing eye causes eye ablations. (B) GMR-akp-jafrac2/+ transgenic flies display a spotted eye phenotype that is reminiscent to the eye phenotype caused by Dronc expression in the eye. (A) GMR-gal4/UAS-pro-droncW). (C) GMR-ava-rpr/+ and (D) GMR-akp-rpr/+ flies show eyes of reduced size. (E) Control flies (Canton S). (F) GMR-kp-jafrac2/+, (G) GMR-va-rpr/+ and (H) GMR-akp-rpr/+ transgenic flies show no detectable effects on eye development.
Fig. 7. The eye ablation phenotype caused by ectopic expression of Jafrac2 is contingent on DIAP1 binding and is suppressed by co-expression of DIAP1 and p35. The eye phenotypes of GMR-akp-jafrac2/+, GMR-ava-rpr/+ and GMR-akp-rpr/+ transgenic flies on their own (A, F and K), or in combination with th4 (B, G and L), GMR-diap1 (C, H and M), GMR-p35 (D, I and N) or Df(3L)AC1 [indicated as Df(Dronc); E, J and O] are shown. The th4 diap1 mutation suppresses AKP-Jafrac2 but enhances AVA- and AKP-Rpr induced eye phenotypes. Co-expression of DIAP1 and p35 rescues cell death induced by Jafrac2 and Rpr. Flies with a chromosomal deletion that removes the dronc locus (Df(3L)AC1) display a suppressed Jafrac2 and Rpr eye phenotype.
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