Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin

Abstract

The stress-activated protein kinases (SAPKs, also called c-Jun NH(2)-terminal kinases) and the p38s, two mitogen-activated protein kinase (MAPK) subgroups activated by cytokines of the tumor necrosis factor (TNF) family, are pivotal to the de novo gene expression elicited as part of the inflammatory response. Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAP3K) that activates both the SAPKs and p38s in vivo. Here we show that TNF receptor (TNFR) associated factor 2 (TRAF2), an adapter protein that couples TNFRs to the SAPKs and p38s, can activate ASK1 in vivo and can interact in vivo with the amino- and carboxyl-terminal noncatalytic domains of the ASK1 polypeptide. Expression of the amino-terminal noncatalytic domain of ASK1 can inhibit TNF and TRAF2 activation of SAPK. TNF can stimulate the production of reactive oxygen species (ROS), and the redox-sensing enzyme thioredoxin (Trx) is an endogenous inhibitor of ASK1. We also show that expression of TRAF2 fosters the production of ROS in transfected cells. We demonstrate that Trx significantly inhibits TRAF2 activation of SAPK and blocks the ASK1-TRAF2 interaction in a reaction reversed by oxidants. Finally, the mechanism of ASK1 activation involves, in part, homo-oligomerization. We show that expression of ASK1 with TRAF2 enhances in vivo ASK1 homo-oligomerization in a manner dependent, in part, upon the TRAF2 RING effector domain and the generation of ROS. Thus, activation of ASK1 by TNF requires the ROS-mediated dissociation of Trx possibly followed by the binding of TRAF2 and consequent ASK1 homo-oligomerization.

FIG. 1

Activation of ASK1 upon coexpression with TRAF2: requirement for the TRAF2 RING finger domain. 293 cells were transiently transfected with pcDNA-HA-ASK1 (0.1 μg/dish) and either GST (pEBG vector), pEBG (GST)-TRAF2, or TRAF2-ΔRING (5 μg/dish) as indicated. ASK1 was immunoprecipitated and assayed in vitro for autophosphorylation and phosphorylation of the ASK1 substrate GST-SEK1[K129R]. Crude cell extracts were subjected to SDS-PAGE and immunoblotting with anti-HA to determine expression. Expression of the TRAF2 constructs was judged by isolating GST constructs on GSH agarose and subjecting the isolates to SDS-PAGE and immunoblotting with anti-GST.

FIG. 2

In vivo interaction between ASK1 and TRAF2. (A) The binding of ASK1 and TRAF2 involves the amino-terminal (aa 1 to 460) and carboxyl-terminal (aa 937 to 1375) noncatalytic domains of ASK1. 293 cells were transfected with GST (pEBG vector) or pEBG (GST)-TRAF2 plus pcDNA-HA-ASK1, pMT3-HA-ASK1[1-460], pMT3-HA-ASK1[667-936], or pMT3-HA-ASK1[937-1375], as indicated. Cells were transfected with 5 μg of TRAF2 construct. The levels of ASK1 plasmid used are indicated. TRAF2 was isolated on GSH agarose and subjected to SDS-PAGE and immunoblotting with anti-HA to detect associated HA-ASK1 constructs. Alternatively, GSH isolates were probed with anti-GST to gauge expression of TRAF2. Likewise, anti-HA immunoprecipitates were subjected to SDS-PAGE and immunoblotting with anti-HA to determine expression of the HA-ASK1 constructs (ASK1[937-1375] consistently comigrates with a species that nonspecifically reacts with anti-HA). IP, immunoprecipitate; IB, immunoblot. (B) The TRAF2 TRAF domains are necessary and sufficient to mediate the ASK1-TRAF2 interaction. Assays were performed as above except that the indicated GST-TRAF2 truncation constructs (in pEBG) were employed. (C) The free TRAF domain of TRAF2 can interact in vivo with either ASK1[1-460] or ASK1[937-1375]. Coimmunoprecipitations were performed as above except that the indicated HA-ASK1 and GST-TRAF2 constructs were employed. IP, immunoprecipitate; IB, immunoblot.

FIG. 3

Dose-dependent inhibition of TNF and TRAF2 activation of SAPK upon expression of ASK1[1-460]. (A) Inhibition of TNF recruitment of the SAPKs by ASK1[1-460]. 293 cells were transfected with GST-SAPK (p54α1 isoform, 1 μg/plate) along with the indicated levels of HA-ASK1[1-460]. Plasmid levels were balanced with empty HA vector (pMT3). Cells were then treated with vehicle or 100 ng of TNF per ml for 5 min, as indicated. SAPK was isolated on GSH beads and assayed for phosphorylation of c-Jun as described (top). Anti-HA immunoprecipitates or GSH agarose pulldowns were probed, respectively, with anti-HA or GST to determine expression of the transfected constructs (bottom). (B) Inhibition of TRAF2 activation of the SAPKs by ASK1[1-460]. Experiments were performed as above except that half of the cells were transfected with FLAG-TRAF2 (3 μg/dish), and the cells were left untreated.

FIG. 4

TNF stimulation of the association of endogenous ASK1 and TRAF2 is ROS-dependent: reversal with Nac. Overexpression of TRAF2 against a background of low Trx blocks the ASK1-Trx interaction. (A) The TNF-dependent association of endogenous ASK1 and TRAF2 is reversed by free radical scavengers. L929 cells were treated with TNF and Nac as indicated. Endogenous TRAF2 was immunoprecipitated and immunoblotted with anti-ASK1 to detect endogenous ASK1 associated with TRAF2. Crude lysates were blotted with anti-ASK1 and TRAF2, as indicated, in order to monitor the levels of the endogenous proteins present in each assay sample. (B) Excess TRAF2 reverses the ASK1-Trx interaction. 293 cells were transfected with the indicated HA-ASK1 (0.3 μg), FLAG-Trx (1 μg) constructs (in pcDNA3), and either vector or increasing levels of Myc-TRAF2 (0.1, 0.2, or 0.4 μg of pcDNA3). FLAG-Trx was immunoprecipitated and subjected to SDS-PAGE and immunoblotting with anti-FLAG. IP, immunoprecipitate; IB, immunoblot.

FIG. 5

Inhibition of the ASK1-TRAF2 interaction by Trx under conditions of a relative excess of Trx: reversal with oxidant (hydrogen peroxide [H₂O₂]. (A) Under conditions of comparatively low TRAF2 expression, Trx inhibits TRAF2 activation of SAPK. 293 cells were transfected with pMT3 (HA)-SAPK-p46β1 (1 μg/dish) and either vector pEBG-(GST)-TRAF2 (1 μg/dish) or pCMV-Myc-Trx (5 μg/dish), as indicated. SAPK was immunoprecipitated from cell extracts and assayed in immune complexes as indicated. TRAF2 was isolated on GSH beads and subjected to SDS-PAGE and immunoblotting with anti-HA to detect bound ASK1. (B) Under conditions of lower TRAF2 expression, Trx inhibits the ASK1-TRAF2 interaction. 293 cells were transfected as indicated with pcDNA3-HA-ASK1 (1 μg/dish), pEBG-(GST)-TRAF2 (1 μg/dish), and pCMV5-Myc-Trx (5 μg/dish). GSH agarose isolates of TRAF2 were probed with anti-HA to detect bound ASK1. GSH isolates of TRAF2, as well as the Myc and HA immunoprecipitates, were also probed with the cognate antibody to monitor expression of the transfected constructs. (C) Expression of Trx does not inhibit the interaction between TRAF2 and GCK under conditions wherein the ASK1-TRAF2 interaction is disrupted. 293 cells were transfected with GST-TRAF2 and HA-ASK1 or the TRAF2 binding domain of GCK (GST-GCK-CTD) (42) and FLAG-TRAF2 plus either vector or Myc-Trx. GST polypeptides were isolated on GSH agarose and probed with anti-FLAG (GCK-TRAF2 interaction) or anti-HA (TRAF2-ASK1 interaction). For all panels, expression of the transfected constructs was determined by subjecting GSH agarose, anti-HA, anti-FLAG, or anti-Myc isolates to SDS-PAGE and immunoblotting with the cognate antibody. (D) TRAF6 association with ASK1 is also reversed by excess Trx. 293 cells were transfected with FLAG-TRAF6, HA-ASK1, and Myc-Trx. TRAF6 was immunoprecipitated with anti-FLAG and probed with anti-HA to detect bound ASK1. HA and FLAG immunoprecipitates were also immunoblotted with the corresponding antibodies in order to judge expression of the relevant constructs. IP, immunoprecipitation; IB, immunoblot.

FIG. 6

Oxidant reverses Trx inhibition of the ASK1-TRAF2 interaction. 293 cells were transfected with HA-ASK1 (1 μg/dish), GST-TRAF2 (1 μg/dish), and Myc-Trx (5 μg/dish) as indicated. Cells were then treated with water or 10 mM H₂O₂ as indicated. GST-TRAF2 was isolated on GSH beads and subjected to SDS-PAGE and immunoblotting with anti-HA to detect associated HA-ASK1.

FIG. 7

TRAF2-dependent enhancement of ASK1 oligomerization. (A) TRAF2 enhancement of ASK1 oligomerization is dependent in part on the TRAF2 RING domain. 293 cells were transfected with Myc-ASK1 and HA-ASK1 plus either vector FLAG-TRAF2 or FLAG-TRAF2ΔRING. Myc-ASK1 immunoprecipitates were probed with anti-HA to detect formation of ASK1 oligomers. HA, Myc, and FLAG immunoprecipitates were probed with the cognate antibodies indicated to judge expression of the transfected constructs. (B) TRAF2-dependent oligomerization of ASK1 is dependent on ROS. 293 cells were transfected with Myc-ASK1 and HA-ASK1 plus either vector or untagged TRAF2. A portion of the TRAF2-transfected cells was treated with Nac (5 mM, 16 h) as indicated. After SDS-PAGE, Myc-ASK1 immunoprecipitates were probed with anti-HA to detect formation of ASK1 oligomers. HA and Myc immunoprecipitates were probed with the cognate antibodies to judge expression of the transfected constructs. Crude extracts were probed with anti-TRAF2 to detect expression of TRAF2. Anti-TRAF2 immunoprecipitates were probed with anti-HA to detect the formation of the ASK1-TRAF2 complex.

FIG. 8

Model for the regulation of ASK1 by TNF. The parallel TRAF2 → GCK/GCKR → MEKK1 pathway is indicated for comparison. See text for details.