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. 2007 Dec;27(23):8152-63.
doi: 10.1128/MCB.00227-07. Epub 2007 Aug 27.

Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1

Go Fujino  1 , Takuya Noguchi, Atsushi Matsuzawa, Shota Yamauchi, Masao Saitoh, Kohsuke Takeda, Hidenori Ichijo
Affiliations

Thioredoxin and TRAF family proteins regulate reactive oxygen species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1

Go Fujino et al. Mol Cell Biol. 2007 Dec.

Abstract

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, plays pivotal roles in reactive oxygen species (ROS)-induced cellular responses. In resting cells, endogenous ASK1 constitutively forms a homo-oligomerized but still inactive high-molecular-mass complex including thioredoxin (Trx), which we designated the ASK1 signalosome. Upon ROS stimulation, the ASK1 signalosome unbinds from Trx and forms a fully activated higher-molecular-mass complex, in part by recruitment of tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF6. However, the precise mechanisms by which Trx inhibits and TRAF2 and TRAF6 activate ASK1 have not been elucidated fully. Here we demonstrate that the N-terminal homophilic interaction of ASK1 through the N-terminal coiled-coil domain is required for ROS-dependent activation of ASK1. Trx inhibited this interaction of ASK1, which was, however, enhanced by expression of TRAF2 or TRAF6 or by treatment of cells with H2O2. Furthermore, the H2O2-induced interaction was reduced by double knockdown of TRAF2 and TRAF6. These findings demonstrate that Trx, TRAF2, and TRAF6 regulate ASK1 activity by modulating N-terminal homophilic interaction of ASK1 VSports手机版. .

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Figures

FIG. 1.
FIG. 1.
Identification of Trx-binding region of ASK1. (A) Schematic representation of mouse ASK1WT and its deletion mutants, with amino acid numbers indicated. The NCC and CCC domains are indicated. (B) Interaction of ASK1 with Trx in yeast. A reporter plasmid encoding β-galactosidase and a prey plasmid encoding Trx or Trx-CS fused to the transcriptional activation domain were cotransformed into yeast strain EGY188 with bait plasmids encoding N-terminal fragments corresponding to aa 1 to 277, 1 to 244, and 46 to 277 of ASK1 fused to the DNA-binding domain. Each transformant was patched onto an indicator plate. Galactose-dependent blue spots indicate positive interactions. (C) Interaction of ASK1 with endogenous Trx in HEK293A cells. HEK293A cells were transfected with HA-ASK1WT, HA-ASK1Δ47-120, HA-ASK1Δ244-277, and HA-ASK1Δ277. Cell lysates were immunoprecipitated with anti-Trx or anti-IgG1 antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Trx antibodies. (D) Dissociation of Trx from ASK1 in response to H2O2. HEK293A cells were transfected with HA-ASK1WT. After 24 h, cells were treated with the indicated concentrations of H2O2 for 20 min. Cell lysates were immunoprecipitated with anti-Trx or anti-IgG1 antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. P-ASK1 represents the activated form of ASK1. (E) Dissociation of Trx from ASK1-NT277 in response to H2O2. HEK293A cells were transfected with HA-ASK1-NT277. After 24 h, cells were treated with the indicated concentrations of H2O2 for 20 min. Cell lysates were immunoprecipitated with anti-Trx or anti-IgG1 antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Trx antibodies. IP, immunoprecipitation; IB, immunoblotting.
FIG. 2.
FIG. 2.
Identification of TRAF2- and TRAF6-binding region of ASK1. (A) Schematic representation of ASK1WT and its deletion mutants. (B) Interaction of ASK1 with TRAF2 in HEK293A cells. HEK293A cells were cotransfected with Flag-TRAF2 and either ASK1WT or its deletion mutants, as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Flag antibodies. (C) Interaction of ASK1 with TRAF6 in HEK293A cells. HEK293A cells were cotransfected with Flag-TRAF6 and either ASK1WT or its deletion mutants, as indicated. Samples were analyzed as described for panel B. (D) Schematic representation of mouse ASK1WT, ASK1-NT, and deletion mutants, with amino acid numbers indicated. (E) TRAF2 preferentially interacts with aa 384 to 655 of ASK1. HEK293A cells were cotransfected with Flag-TRAF2 and either ASK1-NT or its deletion mutants, as indicated. Samples were analyzed as described for panel B. (F) TRAF6 preferentially interacts with aa 384 to 655 of ASK1. HEK293A cells were cotransfected with Flag-TRAF6 and either ASK1-NT or its deletion mutants, as indicated. Samples were analyzed as described for panel B. IP, immunoprecipitation; IB, immunoblotting.
FIG. 3.
FIG. 3.
Trx-binding-region truncated mutants of ASK1 strongly interact with TRAF2 and TRAF6. (A) Schematic representation of mouse ASK1WT and its N-terminal deletion mutants, with amino acid numbers indicated. (B) ASK1Δ277 and ASK1Δ384 strongly interact with TRAF2. HEK293A cells were cotransfected with Flag-TRAF2 and either ASK1WT, ASK1Δ277, or ASK1Δ384. Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Flag antibodies. (C) ASK1Δ277 and ASK1Δ384 strongly interact with TRAF6. HEK293A cells were cotransfected with Flag-TRAF6 and either ASK1WT, ASK1Δ277, or ASK1Δ384. Samples were analyzed as described for panel B. (D) Interaction of ASK1 mutants with endogenous Trx in HEK293A cells. HEK293A cells were transfected with HA-ASK1WT, HA-ASK1Δ277, and HA-ASK1Δ384. Cell lysates were immunoprecipitated with anti-Trx or anti-IgG1 antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Trx antibodies. IP, immunoprecipitation; IB, immunoblotting.
FIG. 4.
FIG. 4.
The NCC domain is required for ROS-induced activation of ASK1. (A) Expression of Trx inhibits ASK1 activity but does not inhibit ASK1Δ277 activity. HEK293A cells were transfected with the indicated combinations of HA-ASK1WT, HA-ASK1Δ277, and Flag-Trx. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. P-ASK1 represents the activated form of ASK1. (B) ASK1Δ277 exhibits high basal activity. HEK293A cells were transfected with various amounts of HA-ASK1WT, HA-ASK1Δ277, and HA-ASK1Δ384. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. P-ASK1 and P-JNK represent the activated forms of ASK1 and JNK, respectively. Relative activities for ASK1WT, ASK1Δ277, and ASK1Δ384 in lanes 4, 8, and 13, respectively, are shown in the graph. Values are the means ± standard errors (SE) for three independent experiments. (C) Schematic representation of mouse ASK1WT and its NCC domain-deleted mutant, with amino acid numbers indicated. (D) H2O2-induced activation of ASK1WT and ASK1ΔNCC. HEK293A cells were transfected with HA-ASK1WT or HA-ASK1ΔNCC. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. P-ASK1 represents the activated form of ASK1. Amounts of activated ASK1 (P-ASK1WT or P-ASK1ΔNCC) relative to total protein (HA-ASK1WT or HA-ASK1ΔNCC) were calculated and are shown as x-fold increases by considering the value for the cells treated without H2O2 as 1.00. Values are the means ± SE for three independent experiments. (E) ASK1 induces caspase-3-like activity in HEK293A cells. HEK293A cells were transfected with HA-ASK1WT, HA-ASK1Δ277, HA-ASK1Δ384, or pcDNA3. After 48 h, cells were lysed and caspase-3-like activity in lysates was measured, using DEVD-AFC as a cleaved substrate. Activity is shown as the x-fold increase relative to the value for the extract from pcDNA3-transfected cells. Values are the means ± SE for three independent experiments (upper panel). Cell lysates were subjected to SDS-PAGE followed by immunoblotting with anti-HA antibody (lower panel). (F) ASK1Δ277 exhibits the highest activity among the N-terminal deletion mutants. HEK293A cells were transfected with various amounts of HA-ASK1Δ277, HA-ASK1Δ384, and HA-ASK1ΔN. Cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. IB, immunoblotting; ns, nonspecific.
FIG. 5.
FIG. 5.
Homophilic interaction of ASK1 through the NCC domain. (A) Schematic representation of mouse ASK1WT and its C-terminal deletion mutants, with amino acid numbers indicated. (B) The NCC domain contributes to homophilic interaction of ASK1. HEK293A cells were transfected with the indicated combinations of Flag- or HA-tagged ASK1-NT277 and ASK1-NT384. Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Flag antibodies. (C) Homophilic interaction of ASK1 through the N-terminal region is much weaker than that through the C-terminal region. HEK293A cells were transfected with the indicated combinations of Flag- or HA-tagged ASK1WT, ASK1ΔC, and ASK1-NT384. Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Flag antibodies. IP, immunoprecipitation; IB, immunoblotting; ns, nonspecific.
FIG. 6.
FIG. 6.
Trx inhibits homophilic interaction of ASK1 through the N-terminal region. (A) Expression of Trx has no effect on homophilic interaction of ASK1WT. HEK293A cells were transfected with the indicated combinations of Flag-ASK1WT, HA-ASK1WT, and Flag-Trx. Cell lysates were immunoprecipitated with anti-HA antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. The amounts of activated ASK1 (P-ASK1) relative to total protein (ASK1) were calculated and are shown as x-fold increases by considering the value for cells expressing HA-ASK1WT and Flag-ASK1WT but not Flag-Trx (lane 3) as 1.00. (B) Expression of Trx inhibits homophilic interaction of ASK1ΔC. HEK293A cells were transfected with the indicated combinations of Flag-ASK1ΔC, HA-ASK1ΔC, and Flag-Trx. Cell lysates were immunoprecipitated with anti-HA antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (C) TRAF2 and TRAF6 are required for H2O2-induced homophilic interaction of ASK1ΔC. HEK293A cells were transfected with the indicated siRNAs. After 24 h, cells were transfected with the indicated combinations of Flag-ASK1ΔC and HA-ASK1ΔC. After another 24 h, cells were treated with 5.0 mM H2O2 for 20 min. Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. (D) Dissociation of Trx from ASK1ΔC in response to H2O2. HEK293A cells were transfected with HA-ASK1ΔC. After 24 h, cells were treated with 1.0 mM H2O2 for 20 min. Cell lysates were immunoprecipitated with anti-Trx or anti-IgG1 antibody. Immunoprecipitates and aliquots of each lysate were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Trx antibodies. (E) Expression of TRAF2 promotes homophilic interaction of ASK1ΔC. HEK293A cells were transfected with the indicated combinations of Flag-ASK1ΔC, HA-ASK1ΔC, and Flag-TRAF2. Samples were analyzed as described for panel B. (F) Expression of TRAF6 promotes homophilic interaction of ASK1ΔC. HEK293A cells were transfected with the indicated combinations of Flag-ASK1ΔC, HA-ASK1ΔC, and Flag-TRAF6. Samples were analyzed as described for panel B. IP, immunoprecipitation; IB, immunoblotting.
FIG. 7.
FIG. 7.
Predicted model of mechanisms of regulation of Trx-mediated ASK1 activation. In resting cells, Trx binds to the Trx-binding domain of ASK1, inhibiting N-terminal homophilic interaction. Upon ROS stimulation, oxidized Trx dissociates from ASK1, with reciprocal recruitment of TRAF2 and TRAF6 to ASK1, promoting N-terminal homophilic interaction. This interaction contributes to effective autophosphorylation and activation of ASK1.
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