Oncogene (2006) 25, 5787–5800 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Human glutathione S- P1-1 interacts with TRAF2and regulates TRAF2–ASK1 signals

YWu1, Y Fan1, B Xue2, L Luo2, J Shen1, S Zhang1, Y Jiang3 and Z Yin1

1Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, JiangSu, People’s Republic of China; 2State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, People’s Republic of China and 3Key Laboratory of Functional of Guangdong Province, Department of Pathophysiology, Southern Medical University, Guangzhou, People’s Republic of China

Human glutathione S-transferase P1-1 (GSTP1-1) is an Introduction ubiquitously expressed that plays an important role in the detoxification and xenobiotics . It -a (TNF-a) is a pleiotropic has been shown that GSTP1-1 interacts with c-Jun NH2- cytokine that elicits a wide spectrum of physiological terminal kinase (JNK) and suppresses its activity. Here, and pathogenic events including cell proliferation, we report a novel function of GSTP1-1 in regulating differentiation and (Arch and Thompson, tumor necrosis factor-a (TNF-a)-triggered signaling. The 1999; Locksley et al., 2001). TNF-a triggers the present experiments showed that GSTP1-1 physically activation of the factors, such as activator associated with tumor necrosis factor receptor-associated protein 1 (AP-1) and nuclear factor-kB (NF-kB), factor 2(TRAF2) in vivo and in vitro. Overexpression of through stimulating cascades including GSTP1-1 inhibited TRAF2-induced activation of both c-Jun NH2-terminal kinases (JNKs)/stress-activated JNK and p38 but not of nuclear factor-jB (NF-jB). protein kinases (SAPKs) and IkB kinases pathways Glutathione S-transferase P1-1 also attenuated TRAF2- (Feldmann and Maini, 2001). These cellular responses to enhanced apoptosis signal-regulating kinase 1 (ASK1) TNF-a is signaled through two different cell surface and inhibited TRAF2–ASK1- receptors, p55 TNF-R1 and p75 TNF-R2 (Tartaglia and induced cell apoptosis by suppressing the interaction of Goeddel, 1992). TNF-a induces homotypic aggregation TRAF2and ASK1. Conversely, silencing of GSTP1-1 of these receptors, resulting in the recruitment of a expression through RNA interference (RNAi) resulted in number of adaptor to the cytoplasmic amino- increase of TNF-a-dependent TRAF2–ASK1 association terminal domains of the clustered receptors and con- followed by hyper-activation of ASK1 and JNK. A mutant sequent initiation of (Smith et al., GSTP1-1 lacking TRAF domain-binding motif exhibited 1994; Ashkenazi and Dixit, 1998; Wajant et al., 2003). a significant decline of capacity to bind TRAF2and block Tumor necrosis factor receptor-associated factor 2 TRAF2–ASK1 signaling compared with the wild type of (TRAF2) is a prototypical member of the TRAF family, GSTP1-1. Moreover, the glutathione-conjugating activity which act as important signal transducers for the TNF of GSTP1-1 was not involved in the regulation of TRAF2 receptor (TNF-R) and the interleukin-1 receptor/Toll- signaling. These findings indicate that GSTP1-1 plays an like receptor superfamily members. To date, seven important regulatory role in TNF-a-induced signaling by members of the TRAF family have been identified forming ligand-binding interactions with TRAF2, which (TRAF1–7) (Arch et al., 1998; Baud et al., 1999; Bradley provides a new insight for analysing the protective effects and Pober, 2001; Chung et al., 2002; Xu et al., 2004). of GSTP1-1 in tumor cells. TRAF2 mediates TNF-a-induced activation of AP-1 Oncogene (2006) 25, 5787–5800. doi:10.1038/sj.onc.1209576; and NF-kB by directly associating with TNF-R2 (Rothe published online 24 April 2006 et al., 1994, 1995) and/or recruiting to TNF-R1 through interacting with TNF-R1-associated death domain Keywords: glutathione S-transferase P1-1; TNF-a; protein (TRADD) (Hsu et al., 1995, 1996a, b). It has TRAF2; ASK1; apoptosis been well demonstrated that TRAF2 is crucial for downstream signaling events in response to TNF-a,and controls or death (Xia and Chen, 2005). Previous overexpression experiments revealed that TRAF2 plays an important role in TNF-a-triggered Correspondence:Dr Z Yin, Jiangsu Province Key Laboratory for activation of JNK and NF-kB signaling pathways Molecular and Medical Biotechnology, College of Life Sciences, (Takeuchi et al., 1996; Natoli et al., 1997; Reinhard Nanjing Normal University, 122 Ninghai Road, Nanjing, JiangSu et al., 1997; Song et al., 1997). The lack of TRAF2 or the 210097, People’s Republic of China. E-mail:[email protected] expression of a dominant-negative form of TRAF2 only Received 5 December 2005; revised 21 February 2006; accepted 8 March led to a modest defect in TNF-a-induced NF-kB 2006; published online 24 April 2006 activation, but resulted in a severe reduction of JNK GSTP1-1 modulates TNF-a signaling YWuet al 5788 activation (Lee et al., 1997; Yeh et al., 1997). However, activity. GSTP1-1 functions as an endogenous inhibitor the precise mechanism by which TRAF2 is capable of of JNK, which interact with the C-terminal of that mediating these signaling pathways remains largely kinase (Adler et al., 1999; Yin et al., 2000; Wang et al., exclusive. 2001). GSTM1-1 (GSTm), another member of GST Mitogen-activated protein kinases (MAPKs) cascades superfamily, has been identified as a negative regulator are pivotal components in TRAF2-elicited signal path- of ASK1 and MEKK1 (Cho et al., 2001; Dorion et al., ways (Ichijo 1999; Davis 2000; Chang and Karin, 2001). 2002; Ryoo et al., 2004). Our previous study also Two major MAPKs , JNK and p38, are known to be showed that GSTP1-1 repressed ROS-induced ASK1 activated by TRAF2 and act as the major primary activation in a dose-dependent manner (Yin et al., activators of AP-1. TRAF2 interacts directly or 2001). indirectly with several MAPK kinase kinases For more than a decade, GSTP1-1 has generated (MAPKKKs) such as apoptosis signal-regulating kinase interest as a neoplastic marker based on its elevated 1 (ASK1), germinal center kinase and MEK kinase 1 expression in many tumor tissues relative to matched (MEKK1), and thereby activates MAPKs cascades normal tissue (Shea et al., 1988; Moscow et al., 1989). (Nishitoh et al., 1998; Yuasa et al., 1998; Baud et al., Recently, elevated expression of GSTP1-1 has been 1999; Hoeflich et al., 1999). implicated in resistance to apoptosis initiated by a ASK1 is a MAPKKK that activates both MKK4/7– variety of stimuli (Voehringer et al., 2000; Cumming JNK and MKK3/6–p38 signaling pathways. TNF-a and et al., 2001). However, little is known about the effects some oxidative stressors have been reported to activate of GSTP1-1 on TNF-a-triggered MAPKs activation and ASK1 and then to induce cell apoptosis (Ichijo et al., apoptotic cell death, particularly it is still unclear 1997; Gotoh and Copper, 1998; Liu et al., 2000). whether and how GSTP1-1 regulates TNF-a-activated Depending on its TRAF domain and RING-finger ASK1–JNK signaling pathway. Here, we show that motif, TRAF2 could enhance ASK1 homo-oligomeriza- GSTP1-1 blocks TRAF2–ASK1 interaction and tion and autophosphorylation at Thr845 by direct TRAF2-induced ASK1 activation via binding the protein–protein interaction with ASK1. It has been TRAF domain of TRAF2, and consequently suppresses documented that ASK1 is specifically required for TNF- TNF-a- and TRAF2–ASK1-triggered cell death. These a- and TRAF2-induced sustained JNK and p38 activa- data revealed a novel function of GSTP1-1 in modulat- tion. TNF-a or TRAF2 overexpression stimulates ing TNF-a/TRAF2-elicited ASK1 activation. production of reactive oxygen species (ROS) that in turn activates ASK1–MKKs–JNK/p38 signal pathways through eliciting TRAF2–ASK1 interaction in target cells (Nishitoh et al., 1998; Hoeflich et al., 1999; Results Liu et al., 2000; Tobiume et al., 2002). Some redox- GSTP1-1 inhibits TNF-a-induced ASK1–JNK cascade sensing proteins including thioredoxin (Trx), heat-shock activation protein 72 (HSP72) and 90 () and glutathione In order to determine whether TNF-a-induced ASK1– S-transferase (GST) Mu (GSTM1-1) (Saitoh et al., 1998; JNK cascade activation could be regulated by GSTP1-1, Cho et al., 2001; Park et al., 2002; Zhang et al., 2005) human cervical carcinoma HeLa cells were transiently could bind ASK1 and inhibit its activity. TRAF2- transfected with hemagglutinin (HA)-tagged GSTP1-1 induced ASK1 activation is likely to be owing to prior or empty vector, followed by treatment with TNF-a dissociation of Trx, a major endogenous inhibitor of ASK1, from ASK1. These findings imply that the free (50 ng/ml) for 30 min. The endogenous phosphorylated form of JNKs, MKK4 and ASK1 were determined by radical scavengers play a crucial role in regulating immunoblotting using phospho-specific antibodies TRAF2–ASK1 signals (Saitoh et al., 1998; Liu et al., 2000). against JNK/SAPK (Thr183/Tyr185), MKK4 (Ser257/ Thr261) and ASK1 (Thr845), respectively. The results The GSTs are identified as a multigene family of showed that TNF-a-activated ASK1–JNK cascade was isozymes that catalyse the nucleophilic attack of the markedly reduced in GSTP1-1 overexpressed cells sulfur atom of glutathione (GSH) on electrophilic (Figure 1). The activation of p38, another important groups of molecules (Tew, 1994). On the basis MAPK, induced by TNF-a was also impaired by of the amino-acid sequence, the mammalian GSTs are GSTP1-1- overexpression, but TNF-a-induced degrada- divided into six classes: a, m, o, p, y and z (Townsend and Tew, 2003a). Among this family of isozymes, tion of cellular IkBa was not affected (Figure 1). Similar results have been obtained from human glutathione S-transferase P1-1 (GSTP1-1, GSTp) is the U937 cells (data not shown). These data suggested most prevalent isozyme in mammalian cells. Previous studies show that GSTP1-1 is determinant in cellular that, in addition to binding JNK and suppressing its kinase activity, GSTP1-1 might also target on response to oxidative stress and protects tumor cells upstream molecule(s) involved in TNF-a-activated from apoptosis elicited by a variety of cytotoxic agents, MAPK signaling pathways. such as H2O2, UV, cisplatin and arsenic trioxide (Gate and Tew, 2001; Gilot et al., 2002; Townsend et al., 2002; Lu et al., 2004; Zhou et al., 2004). More recently, GSTP1-1 physically interacts with TRAF2 however, it has been shown that GSTs appear to Glutathione S- have been shown to act as regulate MAPKs via their non-catalytic, ligand-binding endogenous negative regulators of the JNK signaling

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5789 immunopellets were analysed by immunoblotting with anti-GSTP1-1 monoclonal antibody. The results showed that GSTP1-1 associated with JNK1 in unstimulated cells, but the binding of GSTP1-1-JNK1 decreased in response to TNF-a stimulus and returned to the basal level thereafter. A weak interaction between GSTP1-1 and p38a, which was not affected by TNF-a, was also observed (Figure 2a, top two panels, lanes 1–8). However, the bindings of GSTP1-1 with MKK4 or MKK6, the two upstream MAPKKs of JNKs and p38, were not detected in the same experimental conditions (Figure 2a, top two panels, lanes 9–16). Previous studies have revealed that GSTM1-1, another member of GST superfamily, could associate with ASK1 and MEKK1 directly and inhibit their kinase activities. As these two kinases are the most important MAPKKKs in the TNF-a-activated signaling cascades, we speculated that GSTP1-1 might also target on them directly as GSTM1-1. Unfortunately, no obvious interaction between GSTP1-1 and ASK1 or MEKK1 was found in our experiments (Figure 2a, middle two panels, lanes 1–8). We then focused on the effects of GSTP1-1 on upstream molecule(s) of ASK1. Two TRAF family proteins, TRAF2 and TRAF6, have been demonstrated to bind to and thereby activate ASK1. -knockout experiments showed that TRAF2 was essential for TNF-a-stimulated ASK1– JNK activation (Lee et al., 1997; Nishitoh et al., 1998; Noguchi et al., 2005), and TRAF6–ASK1 axis is selectively required for lipopolysaccharide (LPS)-in- duced activation of p38 (Matsuzawa et al., 2005). To explore the possibility that TRAF2 or TRAF6 was Figure 1 GSTP1-1 inhibits TNF-a-induced ASK1–JNK pathway related with the inhibitory effect of GSTP1-1 on TNF-a activation. HeLa cells were transiently transfected with hemagglu- triggered ASK1–JNK signaling cascade, endogenous tinin (HA)-GSTP1-1 (0.3 and 1.0 mg) or empty expression vectors. After 30 h, the transfected cells were treated with ( þ ) TRAF2 and TRAF6 in HeLa cells were immunopreci- or without (À) TNF-a (50 ng/ml) for 30 min. Cell lysates were pitated with respective antibodies. The results from subjected to immunoblot (IB) analysis with anti-phospho- immunoblot analysis using anti-GSTP1-1 antibody of JNK (Thr183/Tyr185), anti-phospho-MKK4 (Ser257/Thr261), the TRAF2 or TRAF6 immunoprecipitates revealed anti-phospho-ASK1 (Thr845), anti-IkB, anti-GSTP1-1 and control antibodies as indicated. of kinases was deter- that GSTP1-1 specifically associated with TRAF2, but mined. Relative kinases phosphorylation fold is shown as mean not TRAF6 (Figure 2a, middle two panels, lane 9–16). of duplicates from three independent experiments, with untreated The interaction of GSTP1-1 with TRAF2 was next as 1.0. investigated in human embryonic kidney (HEK) 293 cells under overexpression conditions. HEK293 cells were co-transfected with HA-tagged GSTP1-1 and Flag- tagged TRAF2 expression vectors or empty vector, pathways through binding different kinases, which is respectively, and then the cell lysates were subjected to revealed by the effects of GSTM1-1 on ASK1 and immunoprecipitation with anti-Flag M2 affinity gels. MEKK1, whereas GSTP1-1 on JNK (Adler et al., 1999; The results of immunoblot analysis using anti-HA Cho et al., 2001; Ryoo et al., 2004). Our previous studies antibody showed that GSTP1-1 significantly associated indicated that overexpression of GSTP1-1 suppresses with TRAF2 (Figure 2b). Further reciprocal experi- ROS-induced activation of ASK1 and MKK7 as well as ments that were carried out by immunoprecipitating JNK activation (Yin et al., 2000, 2001). Furthermore, as HA-GSTP1-1 from transfected HEK293 cell lysates showed in Figure 1, GSTP1-1 could also attenuate using anti-HA antibody also demonstrated that Flag- TNF-a-stimulated activation of p38 and JNK upstream TRAF2 but not Flag-TRAF6 specifically associated kinases, such as ASK1 and MKK4. with GSTP1-1 (Figure 2c). Moreover, in vitro pull-down Therefore, we next probed whether GSTP1-1 could assay using bacteria expressed recombinant His-GSTP1- directly interact with the upstream molecules of MAPKs 1 and the HEK293 cell lysates containing Flag-tagged to exert its inhibitory effects in TNF-a-triggered signals. TRAF2 or TRAF6 showed the same result (Figure 2d). HeLa cells were treated with TNF-a or left untreated, Taken together, these results strongly demonstrated and then the endogenous JNK1 and p38a were that GSTP1-1 physically associated with TRAF2 in immunoprecipitated with respective antibodies. The mammalian cells.

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5790

Figure 2 GSTP1-1 physically interacts with TRAF2 in vivo and in vitro.(a) Lysates from HeLa cells treated with TNF-a (50 ng/ml) were subjected to immunoprecipitation (IP) using indicated antibodies (anti-JNK1, anti-p38a, anti-MKK4, anti-MKK6, anti-ASK1, anti-MEK kinase 1, anti-TRAF2 and anti-TRAF6, 0.5 mg each) or control normal immunoglobulin G (IgG). The precipitates were analysed by immunoblotting with GSTP1-1 monoclonal antibody and corresponding antibodies. The total amounts of GSTP1-1 and glyceraldehyde-3- dehydrogenase (GAPDH) in the whole-cell lysates were determined by immunoblot analysis (bottom panel). (b) HEK293 cells were cotransfected with hemagglutinin (HA)-tagged GSTP1-1 (1 mg) and Flag-tagged TRAF2 (0.5 mg) or empty expression vectors as indicated. After 36 h, the cell lysates (500 mg) were immunoprecipitated with EzviewTM red anti-Flag M2 affinity gel (5 ml), followed by immunoblot analysis with anti-HA antibody or anti-TRAF2 antibody, and the whole-cell lysates were also immunoanalysed with anti-HA antibody. (c) HEK293 cells were co-transfected with HA-GSTP1-1 (0.5 mg, lanes 2 and 3), Flag- TRAF2 (1 mg, lane 2) and Flag-TRAF6 (1 mg, lane 3) as indicated. The whole-cell lysates were subjected to immunoprecipitation using anti-HA antibody and analysed by immunoblotting with anti-Flag antibody to verify the coprecipitated Flag-TRAF2 or Flag-TRAF6. The presence of overexpressed exogenous proteins in the same lysates was verified by immunoblotting. (d) Lysates (1 mg) of HEK293 cells transfected with Flag-tagged TRAF2 (upper panel) or TRAF6 (lower panel) were incubated with His-GSTP1-1 immobilized on Ni2 þ –IDA–agarose beads in the lysis buffer at 41C for 8 h. Immunoblotting of the bound proteins with anti-Flag antibody was shown. One representative experiment is shown in each panel.

Effects of TNF-a and glutathione on the interaction of TNF-a treatment up to 10 h and not affected by the TRAF2 with GSTP1-1 presence of cycloheximide (CHX), a protein synthesis In order to further analyse the effects of GSTP1-1 and inhibitor (Figure 3b). Therefore, these results indicated TRAF2 interaction on TNF-a-triggered signal cascades, that TNF-a stimulation could change the interaction of we determined whether TNF-a stimulus could alter the GSTP1-1 and TRAF2 without altering GSTP1-1 binding of GSTP1-1 and TRAF2 that existed in protein level. unstressed cells. HeLa cells were treated with TNF-a As GSTP1-1 is an that catalysed the (50 ng/ml) for different time intervals as indicated and conjugation of reduced glutathione to a variety of subjected to co-immunoprecipitation and immunoblot- electrophiles, we also tested whether the intracellular ting assay. The results showed that the amount of glutathione concentration could influence the interac- endogenous TRAF2–GSTP1-1 complex decreased to tion between GSTP1-1 and TRAF2 by treating the minimum 45 min after TNF-a treatment, and then HeLa cells with N-acetyl-L-cysteine (NAC), an ami- returned to the basal level at 90 min (Figure 3a), whereas nothiol and synthetic precursor of intracellular glu- the GSTP1-1 protein level did not change within this tathione, or buthionine-(S,R)-sulfoximine (BSO), an period. In fact, GSTP1-1 level was constant under the inhibitor of GSH synthesis. The data showed that the

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5791 association of GSTP1-1 and TRAF2 was independent of 2002). TRAF2 is an adaptor protein with several cellular glutathione level in resting cells or TNF-a- domains (Figure 4a) including the RING-finger and treated cells (Figure 3c and d). zinc-finger motifs for mediating downstream signaling events and the TRAF domain for interacting with upstream receptors and other signaling molecules, such The TRAF domain of TRAF2 mediates GSTP1-1 binding as ASK1 (Nishitoh et al., 1998; Hoeflich et al., 1999; Liu As TRAF2 has no intrinsic catalytic activity, protein– et al., 2000). To determine which domain of TRAF2 was protein interactions are essential for regulating TRAF2- responsible for the association with GSTP1-1, different mediated cellular signaling pathways (Chung et al., Flag-tagged TRAF2 deletion mutants and HA-tagged GSTP1-1 were co-transfected into HEK293 cells. Immunoblot analysis of co-immunoprecipitation assay using anti-Flag M2 affinity gels showed that the TRAF domain amino acids (aa 272–501) but not the RING- finger (aa 1–87) or zinc-finger motif (aa 87–271) of TRAF2 was required for the interaction of TRAF2 with GSTP1-1 (Figure 4b).

GSTP1-1 inhibits TRAF2-activated JNK and p38, but not NF-kB Above data indicated that GSTP1-1 associated with TRAF2 and played a regulatory role in TNF-a- activated signaling cascades. As TRAF2 mediates TNF-a-induced downstream signal pathways including JNK and NF-kB, the role of GSTP1-1 in regulating

Figure 3 TRAF2 and GSTP1-1 interaction can be altered by TNF-a and is independent of intracellular glutathione level. (a) Lysates (1 mg) from HeLa cells treated with TNF-a (50 ng/ml) Figure 4 Mapping of TRAF2 domains required for GSTP1-1 for indicated periods were immunoprecipitated with anti-TRAF2 binding. (a) Schematic diagram of wild-type TRAF2 and its monoclonal antibody (0.5 mg each). The immunopellets were deletion mutants. Their abilities to interact with GSTP1-1 are analysed by immunoblot analysis with anti-GSTP1-1 antibody. shown. ‘ þ ’ represents binding of GSTP1-1 with the respective (b) HeLa cells were treated with TNF-a (50 ng/ml) for indicated TRAF proteins and ‘À’ represents lacking of such interaction in time in presence of cycloheximide (CHX, 1 mg/ml) or not. The this assay. (b) Interaction of GSTP1-1 with respective TRAF2 protein levels of GSTP1-1 and GAPDH were determined by deletion mutants. HA-tagged GSTP1-1 was transiently co-trans- immunoblot analysis. (c, d) HeLa cells were incubated with 5 mM fected into HEK293 cells with expression plasmids for Flag-tagged N-acetyl-L-cysteine (NAC) for 5 h or 50 mM buthionine-(S,R)- wild-type and deletion mutant TRAF2. After 36 h, cell lysates sulfoximine (BSO) for 16 h followed by treatment of TNF-a (50 ng/ (1 mg) were subjected to immunoprecipitation with EzviewTM red ml) for 30 min. As described in (a), equal amounts of cell lysates anti-Flag M2 affinity gel (10 ml). Co-precipitated HA-GSTP1-1 was were immunoprecipitated and immunoblot analysed (c) and detected by immunoblot analysis with anti-HA antibody. Expres- intracellular glutathione level was measured as described in sion of GSTP1-1 and TRAF2 wild-type and deletion proteins was Materials and methods (d). Values shown are averages determined by immunoblotting with anti-HA or anti-Flag anti- (mean7s.d.) of three independent experiments. bodies.

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5792 TRAF2-mediated JNK and NF-kB activation were antibodies as indicated. Immunoblot analysis using addressed in transfection studies using Jun2-luciferase phospho-specific antibodies revealed that forced expres- or NF-kB-luciferase reporters. HeLa cells were co- sion of TRAF2 obviously activated JNK1, p38a and transfected with reporter constructs and expression MKK7, and this activation was significantly reduced by vectors as indicated (Figure 5a and b). At 36 h after the co-expression of GSTP1-1 (Figure 5c–e). These data transfection, the cells were treated with TNF-a for 4 h or verified the effects of GSTP1-1 previously described in left untreated, and the luciferase activities were assayed. Figure 1, and strongly suggested that GSTP1-1 inhibited Without TNF-a, forced expression of TRAF2 alone was both TNF-a- and TRAF2-mediated MAPKs but not sufficient to stimulate JNK and NF-kB activation in NF-kB activation via interacted with TRAF2. transfected cells. Overexpression of GSTP1-1 sup- pressed TRAF2 or TNF-a-mediated c-Jun activity in a GSTP1-1 regulates TRAF2-induced ASK1 activation dose-dependent manner, but did not alter the expression It has been well documented that TNF-a-induced of the NF-kB-luciferase reporter in either the presence TRAF2–ASK1 complex is required for ASK1 activation or absence of TNF-a (Figure 5a). In comparison, (Nishitoh et al., 1998; Liu et al., 2000; Noguchi et al., GSTP1-1 did not obviously inhibit the interleukin-1-b 2005). We and another group also found that GSTP1-1 (IL-1b) and TRAF6-mediated activation of either c-Jun inhibited the enzymatic activity of ASK1 in vivo (Yin or NF-kB in the same conditions (Figure 5b). et al., 2001) but not in vitro (Cho et al., 2001). In the We next observed the effects of GSTP1-1 on present study, GSTP1-1 was found to interact directly regulating TRAF2-elicited downstream kinases activa- with TRAF2 but not ASK1 (Figure 2), and repressed tion. HeLa cells were transiently cotransfected with TNF-a-induced ASK1 activation (Figure 1). Further- expression vectors encoding TRAF2 and/or GSTP1-1 more, both GSTP1-1 and ASK1 were associated with together with JNK1, p38a or MKK7. Exogenous the TRAF domain of TRAF2. All of these findings kinases were immunoprecipitated with corresponding suggested a possible mechanism that, through binding to

Figure 5 GSTP1-1 suppresses TRAF2-induced activation of JNK and p38, but not of NF-kB. (a) Expression vectors for TRAF2 and GSTP1-1 were transfected into HeLa cells together with 5 Â Jun2-Luc or 2 Â NF-kB-Luc reporter plasmid as indicated. After 36 h, cells were left untreated (white) or treated with TNF-a (50 ng/ml) for 4 h (black), and then the luciferase activities were measured using a luciferase assay system and values were normalized based on b-gal activities. Values shown are averages (mean7s.d.) of one representative experiment in which each transfection was performed in triplicate. (b) The transfection experiments were carried out as in (a), expect that TRAF6 plasmid was co-transfected together in lieu of TRAF2, then the cells were treated with (gray) or without (white) interleukin-1-b (10 ng/ml) for 6 h. Relative luciferase activities were determined as described in (a). (c–e) HeLa cells were co- transfected with the indicated combinations of expressing vectors encoding GSTP1-1 (1.5 mg), TRAF2 (1.5 mg), and HA-tagged JNK1 (2 mg) (c), Flag-tagged p38a (2 mg) (d), or Xpress-tagged MKK7 (3 mg) (e). Cells were harvested 36 h after transfection and lysates were subjected to immunoprecipitation with anti-HA, anti-Flag or anti-Xpress antibody. The immunopellets were analysed by immunoblotting with anti-phospho-JNK (Thr183/Tyr185) (c), anti-phospho-p38 (Thr180/Tyr182) (d), or anti-phospho-MKK7 (Ser271/Thr275) (e) antibody, respectively. The overexpressed exogenous proteins were detected using corresponding antibodies. Relative kinases phosphorylation fold is shown as mean of duplicates from three independent experiments.

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5793 TRAF2, GSTP1-1 blocked TRAF2–ASK1 complex HeLa cells were transfected with either siNS (Figure 7b, information, and subsequent ASK1 activation. lanes 1–12) or siGSTP1-1 (Figure 7b, lanes 13–18). After Therefore, the effect of GSTP1-1 on the TRAF2- 36 h, the cells were transfected again with the same induced ASK1 autophosphorylation and kinase cataly- amount of siRNA together with HA-tagged GSTP1-1 tic activity was first investigated in overexpression expression vector (Figure 7b, lanes 7–12) or empty conditions. HEK293 cells were co-transfected with the vector (Figure 7b, lanes 1–6 and 13–18). At 30 h indicated combinations of expression vectors producing following the second transfection, the cells were treated HA-ASK1 (0.1 mg), Flag-TRAF2 (2 mg) and Xpress- with TNF-a (50 ng/ml) for the indicated times. Endo- GSTP1-1 (1 mg), and then HA-ASK1 was immunopre- genous TRAF2 of different samples were immunopre- cipitated. Immunoblot analysis using phospho-specific cipitated with anti-TRAF2 antibody, followed by antibody against ASK1 (Thr845) showed that ASK1 immunoblot analysis. TRAF2 interacted with ASK1 at phosphorylation at Thr845 stimulated by TRAF2 was apparently inhibited by co-expression of GSTP1-1 (Figure 6a). As expected, the kinase catalytic activity of HA-ASK1 (measured using an in vitro kinase assay with His-MKK7 as a substrate) induced by forced expressed TRAF2 was inhibited by GSTP1-1 in a dose- dependent manner (Figure 6b). In addition, co-immu- noprecipitation study showed that overexpression of GSTP1-1 blocked the physical association of exogenous expressed TRAF2 with ASK1 (Figure 6c). Similar to that in HeLa cells (Figure 2), we also failed to find the GSTP1-1–ASK1 complex in co-transfected HEK293 cells. To confirm that GSTP1-1 counteract ASK1 activa- tion during TNF-a stimulation through inhibition of endogenous TRAF2–ASK1 interaction, GSTP1-1 pro- tein expression was silenced using HP validated Small interfering RNA (siRNA) targeted against human GSTP1-1 (siGSTP1-1) (Qiagen GmbH, Hilden, Ger- many). The successful knockdown of the GSTP1-1 gene was exhibited by the specific reduction of the GSTP1-1 expression in siGSTP1-1 but not RNAiFectTM alone or non-silencing siRNA (siNS)-transfected HeLa cells (Figure 7a). We thus tested the effect of GSTP1-1 silencing on TNF-a-induced ASK1 and JNK activation.

Figure 6 GSTP1-1 inhibits TRAF2-induced ASK1 catalytic activity. (a) HEK293 cells were co-transfected with the indicated combinations of expression vectors producing HA-ASK1 (0.1 mg), Flag-TRAF2 (2 mg) and Xpress-GSTP1-1 (1 mg). The cell lysates (1 mg) were immunoprecipitated with anti-HA antibody (0.5 mg), and the immunopellets were detected by immunoblot analysis with anti-phospho-ASK1 (Thr845) antibody. (b) HEK293 cells were transiently co-transfected with HA-tagged ASK1 (1 mg), Flag- tagged TRAF2 (1.5 mg) and Xpress-tagged GSTP1-1 (0.5, 1.0 and 1.5 mg) as indicated. After 36 h, the HA-tagged ASK1 were immunoprecipitated with anti-HA antibody (0.5 mg) from 200 mg of whole-cell lysates, and their kinase activities (KA) were assessed using His-MKK7 (K149M) as substrate. Equal amount of HA- tagged ASK1 used in each assay were confirmed by immunoblot- ting. Data shown are the representative of three independent experiments. The fold activation relative to that of cells co- transfected with an empty vector and an expression vector encoding HA-tagged ASK1 is the average of the three experiments. (c) HEK293 cells were co-transfected with Xpress-tagged GSTP1-1 (1 mg), Flag-tagged TRAF2 (1.5 mg) and HA-tagged ASK1 (1 mg) as indicated. After 36 h, the cells lysates (600 mg) were immunopreci- pitated with EzviewTM red anti-Flag M2 affinity gel or anti-Xpress antibody (0.5 mg), and the immunopellets were analysed by immunoblot analysis with anti-HA antibodies. Overexpressed ASK1 and GSTP1-1 were detected using anti-HA and anti-Xpress antibodies, respectively. One representative experiment is shown in each panel.

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5794 15 min after TNF-a stimulating in control cells In comparing the regulatory effects of the different (Figure 7b, panel 1, lanes 1–6). Overexpression of GSTP1-1 proteins on TRAF2-induced ASK1–JNK GSTP1-1 repressed the amount of TNF-a-induced cascade using luciferase reporter system, we found that TRAF2–ASK1 complex (Figure 7b, panel 1, lanes the transcriptional activity of c-Jun was dramatically 7–12). In comparison, marked enhancement of suppressed by GSTP1-1 (WT) and GSTP1-1(Y7F), but TRAF2–ASK1 interaction was observed in GSTP1-1 was only slightly inhibited by GSTP1-1 (TWAL) knockdown cells (Figure 7b, panel 1, lanes 13–18). As (Figure 8d). Furthermore, the GSTP1-1 (TWAL) the consequence of TRAF2 and ASK1 association, revealed no ability to block TRAF2-induced ASK1 ASK1 and JNK activation also dramatically increased catalytic activity compared with GSTP1-1 (WT) and and sustained by the GSTP1-1-knockdown (Figure 7b, GSTP1-1 (Y7F) (Figure 8e). panel 4 and 5, lanes 13–18). However, the interaction Forced expression of high levels of TRAF2 and ASK1 between TRAF2 and MEKK1, another important could make the HEK293 cells shrink, detach and float, MAPKKK (Baud et al., 1999), was not affected by the which, together with the results of previous study GSTP1-1 expression level (Figure 7b, panel 7). In the (Hoeflich et al., 1999), suggested that TRAF2 not only above experiments, the amounts of ASK1, MEKK1 as mediated cell survival pathways but also led to well as glyceraldehyde-3-phosphate dehydrogenase by interacting with ASK1. The (GAPDH) proteins used as the control were comparable result from flow cytometry showed that TRAF2 and in the different samples. ASK1 co-expression elicited apoptotic cell death and As TRAF2–ASK1 axis is essential for mediating such cell apoptosis were inhibited by co-expression TNF-a and oxidative stresses stimulated apoptotic cell GSTP1-1 (WT) or GSTP1-1 (Y7F) but only partially death (Tobiume et al., 2001; Noguchi et al., 2005), we suppressed by GSTP1-1 (TWAL) (Figure 8f), corre- also determined the effect of GSTP1-1 on TNF-a- sponding to the effects of GSTP1-1 and its mutants on induced HeLa cell apoptosis by microscopy (Figure 7c) inhibition of TRAF2-induced ASK1 kinase activity and flow cytometry using propidium iodide (PI) staining (Figure 8e). These results showed that the TRAF2- (Figure 7d). Exposure of GSTP1-1-knockdown HeLa binding function rather than GSH-conjugating catalytic cells to TNF-a plus CHX markedly enhanced apoptotic activity of GSTP1-1 is critical for its regulatory effects cell death. On the other hand, overexpression of on TRAF2–ASK1 signaling cascade. GSTP1-1 alleviated apoptosis of transfected HeLa cells to the same extent as ASK1 (K709R), a catalytically inactive form of ASK1 (Figure 7c and d). Moreover, pretreating the cells with NAC reversed the enhance- Discussion ment of TNF-a-induced cell death in GSTP1-1 knock- down cells. These results indicated that cellular GSTP1- Glutathione S-transferases are members of a phase II- 1 indeed played an important role in regulating TNF-a- metabolizing superfamily, which play an triggered TRAF2–ASK1 association and was a survival important role in detoxification of ROS and main- factor for protecting cells against TRAF2–ASK1 axis- tenance of the cellular redox state. Although GSTs were dependent apoptosis. early investigated because of their enzymatic activity involved in the conjugation of GSH to a wide range of A TRAF2-binding motif, TWQE from GSTP1-1, is electrophilic metabolites and effects on protecting tumor responsive for the interaction of TRAF2 with GSTP1-1 cells against toxic drugs and oxidative stress, they were Structural studies of TRAF2-binding proteins revealed a then discovered as ‘ligandins’ owing to their abilities to major consensus motif, (P/S/A/T)x(Q/E)E, and a minor interact covalently and non-covalently with various consensus motif, PxQxxD (Park et al., 1999; Ye et al., compounds that were not substrates for enzymatic 1999; Ye and Wu, 2000). In the GSTP1-1 sequence, a activity, including steroids, thyroid hormones, bilirubin, potent TRAF2-binding motif TWQE (aa 38–41) was prostaglandins and bile acid (Townsend and Tew, found. To evaluate the importance of this motif for 2003a, b). As the ligand-binding characters of GSTs TRAF2- binding, we generated a mutant of GSTP1-1, were confirmed, the regulatory effects of GSTs on stress- GSTP1-1 (TWAL), in which the Gln-40 and Glu-41 that activated cellular signal pathway were revealed in did not affect the enzyme activity of GSTP1-1 in vitro various experiments. (data not shown) were mutated with Ala and Leu, Recent reports showed that GSTs functioned to respectively (Figure 8a). GSTP1-1 (TWAL) mutant sequester MAPKs and acted as negative regulators, displayed an obvious decreased affinity to bind TRAF2 but the mechanisms of these regulations were different. in immunoprecipitation experiments, which indicated Although both GSTP1-1 and GSTM1-1 protect cells that the TRAF2-binding motif of GSTP1-1 is impor- from death elicited by various stresses (H2O2,UV, tant for GSTP1-1–TRAF2 association. In comparison, serum depletion, etc.) through inhibiting JNK signal- another GSTP1-1 mutant, GSTP1-1 (Y7F), whose transduction pathway, GSTP1-1 associated with JNK catalytic activity was blocked by replacing a tyrosine to keep its basal activity low in non-stressed cells with phenylalanine in the seventh amino-terminal (Adler et al., 1999), whereas GSTM1-1 interacted with position acted as effective as GSTP1-1 (WT) in binding ASK1 and MEKK1 directly and repressed ASK1- to TRAF2 (Figure 8b). In vitro pull-down assay also and MEKK1-dependent apoptotic cell death (Cho showed the similar results (Figure 8c). et al., 2001; Ryoo et al., 2004). GSTP1-1 inhibits

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5795

Figure 7 Effects of GSTP1-1 on TNF-a-induced interaction between TRAF2 and ASK1. (a) Characterization of hGSTP1-1 small interfering RNA (siRNA). HeLa cells were transfected with either RNAiFectTM transfection reagent alone (left lane), 100 nM non- silencing siRNA (middle lane) or hGSTP1-1-specific siRNA (right lane) as described in Materials and methods. Cell lysates (20 mg) were subjected to immunoblotting using monoclonal anti-GSTP1 antibody to determine the efficiency of GSTP1-1 suppression (upper panel) and GAPDH expression (bottom panel). (b) HeLa cells were transfected with either control siRNA (lanes 1–12) or GSTP1-1 siRNA (lanes 13–18). After 36 h, the cells were transfected again with the same amount of siRNA together with HA tagged GSTP1-1 expression vector (lanes 7–12) or empty vector (lanes 1–6 and 13–18). At 36 h after the second transfection, the cells were treated with TNF-a (50 ng/ml) for the indicated periods. Cell lysates were subjected to immunoblot analysis with anti-phospho-JNK (Thr183/ Tyr185) and anti-phospho-ASK1 (Thr845) antibodies. The same lysates were immunoprecipitated with anti-TRAF2 antibody followed by immunoblot analysed with anti-ASK1 or anti-MEK kinase 1 antibodies. The efficiency of GSTP1-1 overexpression (lanes 7–12) and suppression (lanes 13–18) was determined using anti-GSTP1-1 antibody. (c) The transfected HeLa cells were treated with TNF-a (50 ng/ml) and cycloheximide (CHX, 1 mg/ml) for 12 h. Phase-contrast photography of transfected HeLa cells treated with or without TNF-a/CHX as indicated. (d) The transfected HeLa cells were incubated with 5 mM N-acetyl-L-cysteine (NAC) for 5 h followed by treated with TNF-a/CHX for 12 h. Apoptotic cell death was assessed by flow cytometry as described in Material and methods. The percentage of apoptotic cells are shown as means7s.d. from three independent experiments.

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5796

Figure 8 Binding affinities of GSTP1-1 mutants to TRAF2. (a) Diagrams of the putative TRAF2-binding motifs (TWQE) in wild- type hGSTP1-1. (b) HEK293 cells were co-transfected with the indicated expression vectors, and after 36 h, the cells lysates were immunoprecipitated with anti-Xpress antibody, followed by immunoblot analysis with anti-Flag antibody. (c) Lysates of HEK293 cells (1 mg protein) expressing Flag-tagged TRAF2 were incubated with wild-type His-GSTP1-1 and its mutants immobilized on Ni2 þ – IDA–agarose beads in the lysis buffer at 41C for 8 h, respectively. The bound proteins were washed and eluted followed by immunoblot analysis using anti-Flag antibody. His-GSTP1-1 proteins were stained by Coomassie blue. (d) HeLa cells were co-transfected with 5 Â Jun2-Luc (0.2 mg), Flag-tagged TRAF2, Xpress-tagged GSTP1-1 wild-type or mutants as indicated. The luciferase activities were measured and values were normalized based on b-galactosidase (b-gal) activities. Values shown are averages (mean7s.d.) of one representative experiment in which each transfection was performed in triplicate. (e) HEK293 cells were co-transfected with indicated expression vectors, and after 36 h, the cells were lysed. The HA-tagged ASK1 was immunoprecipitated with anti-HA antibody (0.5 mg) from 200 mg of lysates, and the kinase activities (KA) were assessed using His-MKK7 (K149M) as substrate. (f) HEK293 cells were co- transfected with indicated expression vectors. After 36 h, cell apoptosis were assessed by flow cytometry. The graph shows the percentage of apoptotic cells. Data shown are averages of three independent experiments.

ROS-stimulated ASK1 activation in vivo (Yin et al., protected cells from ASK1–JNK cascade-dependent 2001), but does not affect the UV-induced ASK1 apoptosis elicited by TNF-a. In addition, unlike activity in vitro (Cho et al., 2001; Ryoo et al., 2004). GSTM1-1 that targeted on ASK1 and MEKK1, The present results demonstrated that GSTP1-1 directly GSTP1-1 acted on JNK and TRAF2 in stress-induced associated with TRAF2 but not TRAF6, and subse- MAPKs activation. The discrepancy of tissue distribu- quently inhibited TRAF2-elicited ASK1–JNK signaling tion and expression level between GSTM1-1 and cascade, suggesting that GSTP1-1 might regulate TNF- GSTP1-1 might account for this difference. a-activated TRAF2–ASK1 axis and subsequent cell Structural analysis in our experiments characterized apoptosis by interacting with TRAF2. the detailed mechanism of the physical and functional TRAF2 is considered to be a central regulator of interaction between GSTP1-1 and TRAF2. TRAF2 TNF-a signaling pathway as it acts as an important contains a conserved C-terminal homology region cellular adaptor protein to mediate ASK1 activation. termed the TRAF domain, and N-terminal RING- The role of TRAF2–ASK1 association in TNF-a- finger and several zinc-finger motifs. Both the TRAF triggered sustained activation of JNK and p38 has been domain and the RING-finger motif of TRAF2 are well documented (Hoeflich et al., 1999; Liu et al., 2000; required for TRAF2-mediated downstream signals. In Tobiume et al., 2001). TNF-a-induced production of TNF-a-activated TRAF2–ASK1–JNK cascade, the ROS triggers the dissociation of Trx from ASK1 and TRAF domain is essential for TRAF2–ASK1 associa- consequently liberalizes ASK1 for binding with TRAF2. tion, whereas the RING motif is required for the TRAF2 appears to activate ASK1 by enhancing and generation of ROS (Nishitoh et al., 1998; Hoeflich et al., stabilizing the oligomerization and autophosphorylation 1999; Liu et al., 2000). Furthermore, a consensus major of ASK1. In the present study, both overexpression and motif (P/S/A/T)x(Q/E)E was reported to exist in the RNA interfering experiments demonstrated that TRAF2-binding proteins including TNFR superfamily GSTP1-1 negatively regulated TNF-a-induced recruit- members (such as TNFR2, CD40,CD30, OX40, 4-1BB ment of ASK1 to TRAF2, while had no effect on the and LMP) and TRAF2-regulated molecules (such as TRAF2-MEKK1 association. GSTP1-1 inhibited sus- kinase and PKN1) (Xia et al., 2002; Gotoh tained activation of ASK1 and JNK, and subsequently et al., 2004). The present study showed that the TRAF

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5797 domain of TRAF2 and TRAF2-binding motif (TWQE signaling pathways (Pinkus et al., 1995, 1996; Fessler aa 38–41) in GSTP1-1 was necessary for the binding of et al., 2002; Xue et al., 2005). However, as described in TRAF2 to GSTP1-1. Glutathione S-transferase P1-1 the present study, GSTP1-1 had no effect on TNF-a- (TWAL) containing a mutated TRAF2-binding motif and TRAF2-induced NF-kB activation. During the (TWQE aa 38–41) not only showed obvious weak TNF-a treatment, the GSTP1-1 protein level was binding affinity to TRAF2 compared with GSTP1-1 constant up to 10 h and not affected by the presence (WT) but also revealed a limited ability to block of CHX. Furthermore, we did not find interaction of TRAF2-induced ASK1 activation and cell apoptosis. GSTP1-1 and TRAF6 even upon LPS treatment in It has been reported that endogenous TRAF2 co- macrophages (unpublished data). The diverse effects of precipitated with ASK1 in a TNF-a-dependent manner the GSTP1-1 might be owing to two distinct mechan- (Nishitoh et al., 1998; Hoeflich et al., 1999). In the isms:the ‘ROS scavenge’ and ‘ligand binding’, by which present study, we noticed that there is a specific GSTP1-1 functions as a regulator in anti-inflammatory association between GSTP1-1 and TRAF2 but no and antiapoptosis responses, respectively. association of TRAF2 and ASK1 in resting cells, after GSTP1-1 regulates TNF-a signaling pathway mainly TNF-a stimulation, the complex of GSTP1-1–TRAF2 via protein–protein direct interaction, but there are also obviously decreased, whereas the interaction of some other potential mechanisms involved in this TRAF2–ASK1 increased with the kinetics comparable process. GSTP1-1 undergo phosphorylation by the to the dissociation of GSTP1-1 from TRAF2. Taken Ser/Thr protein kinases, cAMP-dependent protein together, the observation described herein suggests a kinase and , resulting in a significant new regulatory function of GSTP1-1 in TNF-a triggered enhancement of its metabolic activity (Lo et al., 2004). signal cascades:by competing with ASK1 for binding to Whether GSTP1-1 could be phosphorylated by TNF-a the TRAF domain of TRAF2, GSTP1-1 thus inhibits stimulus and phospho-GSTP1-1 could affect proapop- TNF-a-induced TRAF2–ASK1–JNK cascade activa- totic cytokine-induced MAPKs signaling cascades re- tion and cell apoptosis. The endogenous GSTP1-1 main largely elusive. TNF-a-induced cells apoptosis associates with TRAF2 in unstimulated cells and mainly through two major signaling cascades:ASK1– dissociates from TRAF2 after TNF-a treatment, and JNK and TRADD–FADD–caspase-8 pathways (Locks- subsequently results in the association of ROS-liberated ley et al., 2001). Although NAC obviously reversed the ASK1 with TRAF2 and activation of downstream enhancement of TNF-a/CHX-induced cell death in signaling pathways. GSTP1-1-knockdown HeLa cells in our experiments, The inhibition effects of GSTP1-1 and GSTM1-1 on we could not exclude the possibility that GSTP1-1 ASK1–JNK signaling cascade were independent of their involves in the latter pathway. The relationship between GSH-conjugating activity (Adler et al., 1999; Cho et al., GSTP1-1 and caspases cascade has never been reported 2001; Ryoo et al., 2004). The tyrosine residue in the before. Whether GSTP1-1 is involved in death receptor- seventh amino-acid position of GSTP1-1 was confirmed mediated caspases activation is under investigation. to be critical for its enzyme activity (Kong et al., 1992). In conclusion, the results of the present study Two of our findings that GSTP1-1 (Y7F), an enzymatic elucidate a novel ligand-binding function of GSTP1-1 inactive mutant, acted as same as GSTP1-1 (WT) in in regulating kinase signaling and provide a new insight binding TRAF2 and regulating its signaling, and the for analysing the mechanism utilized by GSTP1-1 to cellular GSH concentration did not affect the amount of protect cells against different injury stimulus such as endogenous GSTP1-1–TRAF2 complex indicated that cytokines, UV or H2O2. Moreover, it is conceivable that GSTP1-1 enzymatic activity and cellular GSH level did GSTP1-1 functions as a general survival factor by not involve in the ‘TRAF2-binding’ regulatory function forming GSTP1-1–TRAF2 complex in tumor cells. of GSTP1-1. Previous studies suggested that TNF-a- Thus, inhibition of this complex may provide a novel triggered TRAF2–ASK1 interaction was dependent on strategy for antitumor therapy. ROS. Some ROS scavengers, such as NAC, inhibited the disassociation of ASK1-Trx and thus suppressed TNF-a-elicited cell apoptosis (Saitoh et al., 1998; Liu Materials and methods et al., 2000; Noguchi et al., 2005). However, our study demonstrated that the interaction of GSTP1-1 and DNA constructs TRAF2 was not influenced by NAC or BSO in resting pcDNA3-HA-GSTP1-1 has been described previously (Adler or TNF-a-treated HeLa cells, and suggested that the et al., 1999). pcDNA3.1-His-Xpress-GSTP1-1 was generated mechanism of TNF-a- induced disassociation of from pcDNA3-HA-GSTP1-1 by inserting hGSTP1-1 cDNA TRAF2–GSTP1-1 and ASK1-Trx may be different. into pcDNA3.1-His vector (Invitrogen, Carlsbad, CA, USA). We will test this hypothesis in the future studies. 5 Â Jun2-Luc, 2 Â NF-kB-Luc reporter vector, pcDNA3-HA- Our recent report showed that GSTP1 could down- ASK1, pCMV-Flag-TRAF2 wild type and other TRAF2 regulate the LPS-activated signaling pathways, includ- truncated constructs were generous gifts from Dr Ze’ev Ronai (The Burnham Institute, USA). pcDNA3.1-Xpress-MKK7 ing MAPKs, NF-kB and subsequent cytokine and pcDNA3.1-Xpress-MKK7 (K149M) expression vectors (Xue et al., 2005). A variety of cytotoxic agents and were kindly provided by Dr Zhenguo Wu (Hong Kong proinflammatory factors, including LPS, could activate University of Science and Technology). Bacterial expressed some transcription factors and increase cytosolic MKK7 (K149M) was produced and cloned into pET-28a GSTP1-1 level, which in turn regulated the stress (Novagen, Madison, WI, USA). pcDNA3.1-Xpress-GSTP1-1

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5798 (TWAL) and pcDNA3.1-Xpress-GSTP1-1 (Y7F) were con- immunoprecipitation, the lysates (500 mg) were incubated with structed with the QuickChange Site-Directed Mutagenesis kit 5 ml EzviewTM red anti-Flag M2 affinity gel for 2 h at 41C. The (Stratagene, La Jolla, CA, USA). The mutagenic primers beads were washed three times using the lysis buffer and then (Y7F-F, 50-CACCGTGGTCTTCTTCCCAGTTCG-30;Y7F-R, twice using tris-buffered saline buffer. The immunoprecipitates 50-CGAACTGGGAAGAAGACCACGGTG-30;andTWAL-F, were subjected to SDS–PAGE followed by transferring onto 50-CGTGGAGACGTGGGCCCTGGGCTCACTC-30; TWA polyvinylidene difluoride Western membranes. The immuno- L-R, 50-GTGTGAGCCCAGGGCCCACGTCACCACG-30) blot analyses were performed as described previously (Adler were designed to generate GSTP1-1 (Y7F) and GSTP1-1 et al., 1999). The antibody–antigen complexes were visualized (TWAL) mutants, respectively. Both expression vectors were by chemiluminescence method using Lumi-Light Western sequenced using T7 primer to confirm mutation. GSTP1-1 wild Blotting Substrate (Roche Applied Science) or using TMB type, Y7F and TWAL mutants were cloned into pET-28a for immunoblotting system (Promega, Madison, WI, USA). The bacterial expression. All plasmids were purified using the total density of the protein bands was calculated using Gel Endofree Plasmid Preparation kit (Qiagen). Doc2000 (Bio-Rad, Hercules, CA, USA). Aliquots of whole- cell lysates were subjected to immunoblotting analysis to Cell culture and transfection confirm appropriate expression of proteins. HEK 293 cells and cervical carcinoma HeLa cells obtained from Institute of and Cell Biology, the Chinese In vitro pull-down binding assays Academy of Sciences (Shanghai, People’s Republic of China), Recombinant polyhistidine-tagged GSTP1-1(WT), GSTP1- were cultured in Dulbecco’s modified Eagle’s medium (Invi- 1(TWAL) and GSTP1-1(Y7F) proteins were expressed in trogen) supplemented with 10% fetal bovine serum (HyClone, Escherichia. coli BL21 (DE3) using pET28a vector (Novagen). Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml strepto- Lysates of HEK293 cell transfected with Flag-tagged TRAF2 mycin at 371C with 5% CO2. Transient transfection was or Flag-tagged TRAF6 were incubated with His-GSTP1-1 performed with a modified calcium phosphate method or by immobilized on Ni2 þ –IDA–agarose beads (Novagen) in the the LipofectAMINE 2000 reagent (Invitrogen) according to lysis buffer at 41C for 8 h. The proteins of interest were the manufacturer’s instructions. In all cases, the total amount analysed by SDS–PAGE and immunoblotting using anti-Flag of DNA was normalized by the empty control plasmids. M2 antibody.

Antibodies and reagents Measurement of intracellular GSH level Mouse monoclonal antibody against HA-tag and polyclonal The GSH level were measured as described previously (Fan antibodies against JNK/SAPK, MKK4, phospho-JNK/SAPK et al., 2005) using Glutathione Assay kit (Calbiochem). (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-MKK4 (Ser257/Thr261), phospho-MKK7 (Ser271/ Thr275) and phospho-ASK1 (Thr845) were obtained from Cell Luciferase reporter assays Signaling Technology (Beverly, MA, USA). Antibodies HeLa cells cultured in 12-well plates were transiently against MKK6 (N-19), MEKK1 (1-9C-2A), TRAF2 (sc-876), transfected with c-Jun or NF-kB target sequence-linked- TRAF6 (H-274), IkBa (C-21) and normal mouse, rabbit or luciferase reporter plasmid (5 Â Jun2-Luc; 0.2 mgor2Â NF- goat immunoglobulin G were from Santa Cruz Biotechnology kB-Luc; 0.5 mg) together with indicated expression vectors. At (Santa Cruz, CA, USA). Polyclonal antibody against ASK1 36 h after transfection, cells were treated with TNF-a (50 ng/ was from Upstate (Lake Placid, NY, USA). Monoclonal ml) for 4 h and protein samples were prepared. The luciferase antibodies against GSTP1-1, JNK1, p38a, TRAF2 and activity was measured using Luciferase Assay System (Prome- recombinant human TNF-a were purchased from BD Phar- ga) and analysed by the Luminometer TD-20/20 (Turner Co. mingen or BD Transduction Laboratories (San Diego, CA, Ltd., Sunnyvale, CA, USA). pCMV-b-galactosidase (b-gal) USA). Monoclonal antibody against Xpress-tag was pur- expression vector (0.5 mg) was added in each transfection and chased from Invitrogen. Horseradish peroxidase-conjugated the b-gal assay was carried out as described previously (Adler secondary antibodies and CHX were obtained from Calbio- et al., 1999) to normalize the transfection efficiency. Each chem (La Jolla, CA, USA). Recombinant human IL-1b was transfection was performed at least twice with triplicates. purchased from Chemicon (Temecula, CA, USA). Anti-Flag TM M2 monoclonal antibodies, Ezview red anti-Flag M2 In vitro kinase assays affinity gel, NAC and BSO were obtained from Sigma (St The transfected HEK293 cells were lysed with lysis buffer Louis, MO, USA). containing 20 mM Tris (pH 7.5), 135 mM NaCl, 2 mM EDTA, 2mM DTT, 25 mM b-glycerophosphate, 2 mM sodium pyr- Co-immunoprecipitation and immunoblotting analysis ophosphate, 10 mM NaF, 10% glycerol, 1% Triton X-100, HEK293 or HeLa Cells were lysed in the lysis buffer 1mM sodium orthovanadate and 1 mM PMSF supplemented containing 20 mM Tris (pH7.5), 135 mM NaCl, 2 mM ethyle- with complete protease inhibitor cocktail. After centrifugation, nediaminetetraacetic acid (EDTA), 2 mM dithiothreitol (DTT), HA-ASK1 was immunoprecipitated with anti-HA monoclonal 25 mM b-glycerophosphate, 2 mM sodium pyrophosphate, antibody as described above. The agarose beads were rinsed 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, three times with the lysis buffer and once with the kinase assay 10 mM NaF and 1 mM phenylmethylsulfonyl fluoride (PMSF) buffer containing 25 mM Tris (pH 7.5), 5 mM b-glyceropho- supplemented with complete protease inhibitor cocktail sphate, 2 mM DTT, 10 mM MgCl2 and 1 mM sodium orthova- (Roche Applied Science, Indianapolis, IN, USA) at 41C. nadate. The activity of ASK1 was determined by incubation Lysates were centrifuged (15 000 g)at41C for 15 min. Proteins with bacterial expressed His-MKK7 (K149M) protein as (500 mg) were immunoprecipitated with indicated antibodies substrate at 301C for 25 min in 50 ml kinase assay buffer (0.5 mg), respectively. The precleared Protein A/G PLUS– supplemented with 100 mM ATP. The reaction was terminated agarose beads (Santa Cruz Biotechnology) were incubated by the addition of Laemmli sample buffer and the proteins with immunocomplexes for another 2 h and washed four were subjected to SDS–PAGE. The phosphorylated His- times with the lysis buffer. For Flag-tagged proteins MKK7 (K149M) was visualized by immunoblotting with

Oncogene GSTP1-1 modulates TNF-a signaling YWuet al 5799 phospho-specific polyclonal antibody to MKK7 (Ser271/ Apoptotic cell death assays Tyr275). HeLa or 293 cells were harvested by trypsinization, centrifuged and resuspended in phosphate-buffered saline. The cells were RNA interference fixed with 70% ethanol and stained with PI (10 mg/ml) and Small interfering RNA specific for human GSTP1-1 and RNase A (50 mg/ml) and incubated overnight. The cells were control (non-silencing) siRNA were purchased from Qiagen. analysed on a FACS Vantage SE flow cytometer (Becton (Product name:Hs_GSTP1_1_HP validated siRNA; catalog Dickinson, San Jose, CA, USA), and the data were analysed number:S100300349). The lyophilized siRNAs dissolved in using ModFitLT V2.0 DNA modeling software (Versity suspension buffer (Qiagen) as 20 mM solutions were heated to Software, Topsham, ME, USA). 901C for 1 min, followed by annealing at 371C for 60 min. HeLa cells were planted on six- or 96-well plates to 30–50% confluence and were transfected with the RNA oligonucleo- Acknowledgements tides using RNAiFectTM transfection reagent (Qiagen) accord- ing to the manufacturer’s instructions, resulting in a final RNA We particularly thank Dr Ze’ev Ronai and Dr Zhenguo Wu concentration of 100 nM. After 36 h, the cells were transfected for providing partial constructs used in this study, we also again with the same amount of siRNA together with thank Huaqun Chen for technical assistance. This work was expression or empty vectors using LipofectAMINE 2000. At supported by grants from the Major State Basic Research 30 h after the second transfection, the cells were treated with Development Program of China (No. 2002CB513000) and TNF-a (50 ng/ml) for the indicated time and prepared for National Nature Science Foundation of China (No. immunoprecipitation and immunoblot analysis. 30270527).


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