TRAF2 functions as an activator switch in the reactive oxygen species-induced stimulation of MST1

Free Radical Biology and Medicine 91 (2016) 105–113

Contents lists available at ScienceDirect

Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed

Original Contribution

TRAF2 functions as an activator switch in the reactive oxygen species-induced stimulation of MST1 Kyung-Hye Roh, Eui-Ju Choi n Laboratory of Cell Death and Human Diseases, Department of Life Sciences, Korea University, Seoul 02841, South Korea

art ic l e i nf o

a b s t r a c t

Article history: Received 13 November 2015 Received in revised form 8 December 2015 Accepted 10 December 2015 Available online 15 December 2015

Reactive oxygen species (ROS) have many physiological and pathological effects on diverse cellular events. In particular, excessive ROS causes oxidative stress that leads to cell death. The mammalian STE20-like kinase-1 (MST1), a multifunctional serine–threonine kinase, plays a pivotal role in oxidative stress-induced cellular signaling events. Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is also known to be essential for oxidative stress-induced cell death. Here, we showed that H2O2 induced the physical interaction between TRAF2 and MST1, and that this interaction promoted the homodimerization as well as the activation of MST1. Furthermore, TRAF2 was required for MST1 to mediate the H2O2-induced stimulation of c-Jun N-terminal kinase and p38 kinase as well as apoptosis. Taken together, our results suggest that TRAF2 functions as a key activator of MST1 in oxidative stressinduced intracellular signaling processes. & 2015 Elsevier Inc. All rights reserved.

Keywords: Hydrogen peroxide MST1 Oxidative stress Reactive oxygen species TRAF2

1. Introduction Uncontrolled production of reactive oxygen species (ROS) causes oxidative damage that eventually leads to cell death, and is associated with the pathogenesis of various diseases, including cancer, arteriosclerosis, ischemia-reperfusion injury, and many neurodegenerative diseases [1–3]. To clarify the mechanism of ROS-induced cytotoxicity is essential to understand the etiology of human diseases associated with oxidative stress in the molecular and cellular levels. However, intracellular signaling networks underlying oxidative stress-induced cell death are not yet fully understood, although many signal-regulating factors are shown to contribute to the oxidative stress-dependent biological events including cell death [4]. The mammalian STE20-like kinase-1 (MST1) is an ubiquitously expressed serine/threonine kinase and belongs to the group of germinal center kinases [5,6]. MST1 is composed of the NH2terminal kinase domain, an inhibitory domain, and the COOHterminal SARAH domain [7,8]. The SARAH domain is responsible for protein–protein interactions including homo- and heteromeric associations [9]. The homodimerization of MST1 contributes to the activation of the kinase [10]. MST1 can be activated when cells are exposed to a variety of cellular stress including oxidative stress [11,12]. Activated MST1 may stimulate the stress-activated mitogen-activated protein kinase (MAPK) pathways including the c-Jun n

Corresponding author. E-mail address: [email protected] (E.-J. Choi).

http://dx.doi.org/10.1016/j.freeradbiomed.2015.12.010 0891-5849/& 2015 Elsevier Inc. All rights reserved.

NH2-terminal kinase (JNK) and p38 MAPK pathways [11,13]. Many lines of evidence demonstrate that MST1 mediates ROS-induced apoptotic cell death in a variety of cell types [14–17]. Given its key role in apoptosis, MST1 has been suggested to be associated with the pathogenic mechanisms of many human disorders including amyotrophic lateral sclerosis [18], diabetes [19], and myocardial ischemia [20,21]. TNF receptor (TNFR)-associated factor 2 (TRAF2) is a member of the TRAF family proteins [22], which mediate the signal transduction initiated by the members of the TNF receptor superfamily [23–25]. TRAF2 contains the NH2-terminal Really Interesting New Gene (RING) finger domain, a central zinc finger loop, and the COOH-terminal TRAF domain [26]. The TRAF domain is critical for protein–protein interactions among TRAF2 and its upstream and downstream binding partners including TNFR associated death domain protein (TRADD), closely related mammalian members of the inhibitor of apoptosis protein (cIAP), and inositol-requiring enzyme 1 [27–29]. TRAF2 mediates the stimulation of the JNK pathway induced by TNFα [30] as well as by H2O2 [31]. Furthermore, ROS promotes the binding of TRAF2 to apoptosis signalregulating kinase 1 (ASK1), a MAPK kinase kinase (MAP3K) of the JNK and p38 pathways, thereby stimulating the ASK1-JNK/p38 signaling events [32]. In order to better understand the molecular mechanism for oxidative stress-induced signaling events, we here investigated the roles of TRAF2 and MST1 in H2O2-induced apoptotic cell death. We observed that H2O2 induces the physical association between TRAF2 and MST1, and that this association promotes the homodimerization and activation of MST1. Furthermore, TRAF2 was

106

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

required for MST1 to mediate the H2O2-induced stimulation of JNK and p38 MAPK as well as apoptosis. Thus, our findings suggest that TRAF2 functions as a key activator of MST1 in oxidative stressinduced intracellular signaling events.

2. Materials and methods 2.1. Cell culture and transfection Fibroblasts from wild-type and TRAF2 / mice were generous gifts from Soo Young Lee (Ewha Womans University, Korea). Fibroblasts from MST1 / mice were prepared as described previously [33]. Fibroblasts were cultured under a humidified atmosphere of 5% CO2 at 37 °C in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ ml). Where indicated, the cells were transfected with appropriate vectors with the use of lipofectamine 2000 (Invitrogen, Carlsbad, CA). Human embryonic kidney 293 (HEK293) cells were transfected with indicated expression plasmids with the use of polyethylenimine (Sigma, St Louis, MO). 2.2. Plasmids pME18S vector for Flag-MST1 was kindly provided by S Yonehara (Kyoto University, Japan). pET23b/ hexahistidine (His6)-tagged MST1(K59R), pET28A/His6-MST1(amino acid residues 1–420, 421–487), pHM6/hemagglutinin (HA) tagged-MST1(full-length, amino acid residues 1–420, and 421–487), and pcDNA3/Myc-MST1 were described previously [33,34,35]. To generate plasmids encoding variant regions of HA-tagged TRAF2 (full-length, amino acid residues 1–271, 87–271, 272–501), the cDNAs were amplified from full-length TRAF2 cDNA in pRK7 vector by polymerase chins reaction and subcloned into the HindⅢ and BamHⅠsites of the pcDNA3.0 vector. 2.3. Antibodies and reagents Rabbit polyclonal antibodies to MST1 and to phospho-p38 (Thr180/Tyr182) as well as mouse monoclonal antibody to phospho-JNK (Thr183/Tyr185) were obtained from Cell Signaling Technology (Boston, MA). Mouse monoclonal antibody to Flag epitope was from Sigma (St Louis, MO). Rabbit polyclonal antibodies to HA epitope, to GST, to p38α, and to JNK1/3, goat polyclonal antibody to MST1, and mouse monoclonal antibody to GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody to thioredoxin-1 (Trx-1) was from Ab frontier (Seoul, Korea). N-acetyl-L-cysteine (NAC) and 30% hydrogen peroxide solution were purchased from Sigma (St Louis, MO). 2.4. Co-immunoprecipitation and immunoblot analysis Cells were lysed in NETN lysis buffer (0.5% NP-40, 1 mM EDTA, 50 mM Tris–HCl, pH 8.0, 120 mM sodium chloride, 1 mM phenylmethyl sulfonyl fluoride, 2 μg/ml aprotinin, and 2 μg/ml leupeptin). Cell lysates were centrifuged at 12,000  g for 20 min at 4 °C, and the resulting supernatants were incubated at 4 °C for 12 h with the indicated antibodies and then for 1 h in the presence of protein G-conjugated Sepharose beads (GE Healthcare, Little Chalfont, UK). The beads were washed three times and the beadbound proteins were then subjected to SDS-PAGE. For immunoblot analysis, proteins on polyacrylamide gel were transferred to a polyvinylidene difluoride membrane. After being blocked with 5% bovine serum albumin, the membrane was incubated with appropriate primary antibodies at 4 °C for overnight and immunoreactive bands were visualized with horseradish peroxidase

Fig. 1. TRAF2 and MST1 mediate H2O2-induced apoptosis. Mouse fibroblast cells derived from wild-type, TRAF2 / and MST1 / mice were treated with 200 μM H2O2 for 16 h, fixed, and then examined for apoptosis by TUNEL assay. TUNEL-positive nuclei (arrowheads) are shown. Scale bar, 100 μm. (lower) Data from the TUNEL assay are means7S.D. of values from two independent experiments. *, Po0.05. NS, not significant.

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

107

Fig. 2. TRAF2 promotes MST1 activation induced by oxidative stress. (A) Wild-type and TRAF2 / mouse fibroblasts were incubated in the absence or presence of H2O2 (100 μM) for 30 min, then lysed. Cell lysates were subjected to immunoprecipitation with anti-MST1 antibody. The resulting precipitates were examined for MST1 activity by immune complex kinase assay with MBP as substrate. The intensity of bands of the phosphorylated MBP was quantified by densitometry, and the relative values of the intensity were shown in the right panel. Cell lysates were also immunoblotted with indicated antibodies. ◄, nonspecific. *, Po 0.05. NS, not significant. (B) HEK293 cells were transfected for 48 h with vectors encoding Flag-MST1 and HA-TRAF2. Cell lysates were subjected to immunoprecipitation with anti-Flag and the resulting precipitates were assayed for MST1 activity as in panel A.

conjugated secondary antibodies and enhanced chemiluminescence reagents (Thermo, Rockford, IL).

by SDS-PAGE and a Fuji BAS 7100 phosphoimager (Fujifilm, Tokyo, Japan).

2.5. Immune complex kinase assay

2.7. Apoptosis assay

Immune complex kinase assay was performed as described previously [36,37]. Briefly, cell lysates were subjected to centrifugation at 12,000  g, and the resulting supernatants were subjected to immunoprecipitation with appropriate antibodies. The resulting precipitates were incubated at 30 °C with agitation for 30 min in a 20 μl reaction solution that contained 20 mM Hepes, [γ-32p]ATP, and 1 μg of substrate protein. The reaction was terminated by adding 5 μl of SDS sample buffer. Phosphorylated proteins were separated by SDS-PAGE and analyzed with a Fuji BAS 7100 phosphoimager (Fujifilm, Tokyo, Japan). Myelin basic protein (MBP) (Millipore, Billerica, MA) was used as substrate of MST1.

Fibroblasts were examined for apoptosis by TUNEL assay with an in situ cell death detection kit (Roche, Penzberg, Germany). Cells were fixed, permeabilized, incubated with TUNEL-reaction mixture for 60 min at 37 °C, and then stained with DAPI. TUNELpositive nuclei were analyzed for apoptosis with an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a DP72CCD camera (Olympus, Tokyo, Japan). Images were postprocessed using Photoshop CS5 (Adobe, San Jose, CA). More than 100 nuclei in each group were counted in each experiment. Percentages of TUNEL-positive nuclei were calculated from two independent experiments. 2.8. In situ proximity ligation assay

2.6. In vitro binding TRAF2 gene in the pcDNA3.0 vector was translated in vitro in the presence of [35S]methionine with the use of a Quick Coupled TnT kit (Promega, Madison, WI). The 35S-labeled TRAF2 proteins were incubated at 4 °C first for 1 h with bacterially expressed His6MST1 variants (full-length, amino acid residues 1–420, and 421– 487) in a binding buffer [38], and then incubated for 1 h in the presence of Ni-NTA beads (Qiagen, Valencia, CA). Bead-bound proteins were washed three times with a washing buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, and 0.1% Tween 20), and eluted from the beads with a solution containing 50 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl, and 250 mM imidazole. Eluted 35S-labelled proteins were detected

The assay was performed with the use of a Duolink II fluorescence kit (Olink Bioscience, Uppsala, Sweden). Fibroblasts were grown on slide glasses in six-well culture plates, fixed, permeabilized, blocked, and then incubated with antibodies to Flag and to HA at 4 °C overnight. The cells were then subjected to an in situ proximity ligation assay with PLA probes, according to the manufacturer’s protocol. Immunofluorescence images of slides were taken with an Olympus BX53 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a DP72CCD camera (Olympus, Tokyo, Japan). Images were post-processed using Photoshop CS5 (Adobe, San Jose, CA). More than 50 cells were examined in each experiment and the protein–protein interaction was quantified from two independent experiments.

108

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

targeted to the sequences 5′-ATGACTGTCAGGATGTTGC-3′. Fibroblasts were transfected with the siRNA duplexes with the use of Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). 2.10. Statistical analysis Statistical significance (P-value) analysis was carried out with the Student's t-test with SPSS for windows version 12.0 (SPSS Inc., Chicago).

3. Results 3.1. TRAF2 and MST1 mediate oxidative stress-induced apoptosis MST1 and TRAF2 have been shown to play a role in oxidative stress-induced apoptotic cell death [14,31]. Indeed, we observed that H2O2 treatment markedly increased apoptosis in mouse embryonic fibroblasts and this increase was abolished in either TRAF2-deficient or MST1-deficient cells (Fig. 1A). The results thus suggested that both TRAF2 and MST1 mediate H2O2-induced apoptosis. 3.2. TRAF2 is required for MST1 activation Given that both TRAF2 and MST1 were involved in H2O2-induced apoptosis, we next examined whether TRAF2 might affect the H2O2-induced stimulation of MST1. As expected, H2O2 treatment resulted in the stimulation of MST1 in fibroblast cells. Intriguingly, the H2O2-induced stimulation of MST1 was markedly reduced in TRAF2-deficient cells (Fig. 2A). Furthermore, ectopically expressed TRAF2 increased the kinase activity of MST1 in HEK293 cells that had been transfected with vectors encoding Flag-tagged MST1 and HA-tagged TRAF2 (Fig. 2B). These results suggested that TRAF2 promotes the activation of MST1 in intact cells. 3.3. Hydrogen peroxide promotes interaction between MST1 and TRAF2

Fig. 3. H2O2 induces the dissociation of Trx-1 from MST1 as well as the association between MST1 and TRAF2. (A) Mouse fibroblast cells were incubated with 100 μM H2O2 for indicated time periods, then lysed. The lysates were examined for MST1 activity by immune complex kinase assay. The lysates were also subjected to immunoprecipitation with antibodies to TRAF2 or to MST1 and the resulting precipitates were immunoblotted with antibodies to MST1 or to Trx-1, respectively. (B) Mouse fibroblast cells were treated with 100 μM H2O2 for 30 min in the absence or presence of 1 mM NAC. Cell lysates were subjected to immunoprecipitation with either pre-immune mouse IgG or anti-TRAF2 antibody. The resulting precipitants were examined by immunoblot analysis with anti-MST1 antibody. Cell lysates were also assayed for MST1 activity by immune complex kinase assay.

Given that TRAF2 mediated the H2O2-induced stimulation of MST1 (Fig. 2), we next investigated if TRAF2 could physically associate with MST1 in intact cells. Mouse fibroblasts in culture were exposed to H2O2, and then examined for TRAF2-MST1 interaction as well as MST1 activity. Co-immunoprecipitation analysis data revealed that TRAF2 failed to interact with MST1 in the cells in basal conditions, but it physically associated with MST1 in the cells treated with H2O2 for 15 min and this association was further increased at 30 min (Fig. 3A). Concomitantly, MST1 activity was increased in the cells after H2O2 treatment for 15 or 30 min, compared to that of the untreated cells. Furthermore, NAC, a ROS scavenger, mitigated the H2O2-induced increase in the TRAF2MST1 interaction as well as MST1 activity (Fig. 3B). In comparison, thioredoxin-1 (Trx-1) bound to MST1 in the cells under basal conditions, and H2O2 treatment abolished the interaction between Trx-1 and MST1 in the cells (Fig. 3A). It is noteworthy that Trx-1 functions as an endogenous inhibitor of MST1 by means of the redox-sensitive binding to MST1 [33]. Collectively, our results suggested that ROS generation induces the recruitment of TRAF2 to MST1, and the TRAF2-MST1 complex formation promotes the activation of MST1.

2.9. RNA interference 3.4. The zinc finger domain of TRAF2 binds and activates MST1 Small interfering RNA (siRNA) oligonucleotides for mouse MST1 and GFP (control) were synthesized by Invitrogen (Invitrogen, Carlsbad, CA). The MST1 siRNA was targeted to the sequences 5′AATCGGACCTGCAGGAGATAA-3′ [35]. The GFP control siRNA was

Next, in order to further understand the interaction between TRAF2 and MST1, we searched for the regions of each protein responsible for the interaction. We transfected HEK293 cells with

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

109

Fig. 4. Identification of the regions of MST1 and TRAF2 responsible for the formation of TRAF2-MST1 complex. (A) HEK293 cells were transfected for 48 h with vectors encoding Myc-MST1 and HA-tagged TRAF2 variants (WT, 87–271, 272–501) as indicated. Cell lysates were subjected to immunoprecipitation with antibody to HA, and the resulting pellets were immunoblotted with antibody to Myc. Cell lysates were also subjected to immunoprecipitation with anti-Myc antibody and the resulting precipitates were examined for MST1 activity by immune complex kinase assay. (B) HEK293 cells were transfected for 48 h with vectors encoding HA-TRAF2 and Flag-tagged MST1 variants (WT, 1–326, 327–487) as indicated. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblot analysis with anti-HA antibody. (C) Purified His6-MST1 variants were incubated for 1 h with in vitro translated 35S-labeled TRAF2 and then applied to Ni-NTA beads. Bead-bound proteins were eluted and subjected to SDS-PAGE. 35S-labeled proteins on polyacrylamide gel were detected by autoradiography.

vectors for HA-tagged TRAF2 variants (full-length, amino acid residues 1–271, 87–271, 272–501) along with a vector for Myc epitope-tagged MST1, and then carried out co-immunoprecipitation. TRAF2(1–271) contains both a RING domain and a zinc finger domain, while TRAF2(87–271) contains a zinc finger domain only. TRAF2(272–501) contains a TRAF domain. Our results revealed that MST1 bound to full-length TRAF2, TRAF2(1–271), and TRAF2 (87–271), but not to TRAF2(272–501) (Fig. 4A). Furthermore, the kinase activity of MST1 was enhanced by TRAF2, TRAF2(1–271), and TRAF2(87–271) but not by TRAF2(272–501). Thus, these results suggest that the zinc finger domain of TRAF2 is the critical domain for both MST1 binding and activation.

3.5. TRAF2 binds to the SARAH domain of MST1 Next, to determine the regions of MST1 important for the interaction with TRAF2, we transfected HEK293 cells with vectors encoding HA-tagged MST1 variants (full-length, amino acid residues 1–420, 421–487) together with a vector for GST-TRAF2. MST1(1–420) contains both a kinase domain and an inhibitory domain, while MST1(421–487) contains a SARAH domain [9]. Coimmunoprecipitation data revealed that TRAF2 physically associated with full-length MST1 and MST1(421–487) but not with MST1(1–420) (Fig. 4B), suggesting that the SARAH domain in MST1 is critical for TRAF2 binding. The SARAH domain of MST1 has

110

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

Fig. 5. TRAF2 promotes MST1 homo-dimerization. (A) HEK293 cells were transfected for 48 h with plasmid vectors encoding Flag-MST1 and Myc-MST1 along with HATRAF2 Cell lysates were subjected to immunoprecipitation with anti-Flag antibody followed by immunoblot analysis with anti-Myc antibody. (B) Wild-type and TRAF2 / mouse fibroblasts were transfected for 48 h with vectors encoding Flag-MST1 and HA-MST1, and then were incubated in the absence or presence of 1 mM H2O2 for 30 min. For visualization of the MST1 homo-dimerization, the cells were fixed and subjected to the in situ PLA assay using antibodies to Flag and to HA. The fluorescence images of the stained cells were analyzed by fluorescent microscopy. Red dots represent MST1 homo-dimerization. The cells were also stained with anti-tubulin antibody (green) and DAPI (blue). Quantification of the PLA signals was shown in the lower panel. Scale bar, 20 μm. *, P o0.05. NS, not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

111

Fig. 6. TRAF2 is required for MST1-mediated stimulation of JNK and p38 MAPK as well as apoptotic cell death. (A, B) Wild-type and TRAF2 / mouse fibroblast cells were transfected with either GFP (control) or MST1 siRNA. In (A), after 24 h of transfection, the cells were left untreated or treated with 500 μM H2O2 for 30 min and lysed. The lysates were subjected to immunoblot analysis with antibodies to Thr183/Thr185-phosphorylated JNK (p-JNK), to Thr180/Tyr182-phosphorylated p38 (p-p38), to JNK1, to p38α, or to MST1. In (B), after 24 h of transfection, the cells were treated with 200 μM H2O2 for 16 h, fixed, and then examined for apoptosis with the use of TUNEL assay. Scale bar, 100 μm. (C) The TUNEL assay data in panel B were quantified and shown as means 7 S.D. of values from two independent experiments. *, P o0.05. NS, not significant.

been shown to be responsible for homomeric and heteromeric protein–protein interactions [7]. Interestingly, a reduced form of Trx-1 also binds to the SARAH domain of MST1, thereby inhibiting the homodimerziation and kinase activity of MST1, in a redoxdependent manner [33]. Next, we examined the binding between MST1 and TRAF2 in vitro. In this binding assay, S35-labeled TRAF2 bound to full-length MST1 and MST1(421–487) but not to MST1(1–420) (Fig. 4C). Thus, these results suggested that TRAF2 directly binds to the SARAH domain of MST1.

3.6. TRAF2 promotes MST1 homodimerization Given that TRAF2 binds the SARAH domain of MST1 and enhances the activity of MST1, we next examined whether TRAF2 could promote the homodimerization of MST1. MST1 homodimerization contributes to the mechanism underlying the activation of MST1 [39]. We transfected HEK293 cells with plasmid vectors for Flag-MST1 and Myc-MST1 without or with a vector for HA-TRAF2, and then performed co-immunoprecipitation. Our results revealed that HA-TRAF2 enhanced the binding between

112

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

Flag-MST1 and Myc-MST1 (Fig. 5A), suggesting that TRAF2 promotes MST1 homodimerization. Next, we performed proximity ligation assay (PLA) with using wild-type and TRAF2 / fibroblasts transfected with vectors for Flag-MST1 and Myc-MST1. PLA allows detection of protein–protein interactions in cells [40]. We observed that H2O2 induced the homodimerization of MST1 in wild-type fibroblast cells, but not in TRAF2 / cells (Fig. 5B). These results suggested that TRAF2 promoted H2O2-induced MST1 homodimerization.

targeting both MST1 and ASK1. In summary, our findings highlight a previously unrecognized role of TRAF2 in the mechanism of oxidative stress-induced MST1 activation. These results should provide us a new insight into understanding the mechanism of oxidative stress-induced signaling in normal physiological as well as pathological processes.

3.7. TRAF2 facilitates the MST1-mediated JNK and p38 activation

The work was supported by National Research Foundation of Korea Grants (2006-0093855, 2009-0080985, NRF-2015R1A4A1041919) funded by the Ministry of Science, ICT, and Future Planning of Korea, and by a Korea University grant (E.-J.C).

MST1 mediates the stimulation of the JNK and p38 MAPK pathways [11,13]. Given that TRAF2 is able to bind and activate MST1 as shown above, we decided to examine the contribution of TRAF2 to the MST1-mediated stimulation of JNK and p38. H2O2 induced an increase in the abundance of the active (phosphorylated) forms of JNK and 38 in wild-type fibroblasts transfected with control (GFP) siRNA, and this increase was markedly reduced in the cells expressing MST1 siRNA (Fig. 6). These results thus suggested that MST1 mediates the ROS-induced stimulation of JNK and 38 in the cells. In comparison, ablation of TRAF2 resulted in a marked suppression of the H2O2-induced increase in active (phosphorylated) JNK and 38 in TRAF2 / cells. Together, our results suggested that TRAF2 is required for MST1-mediated stimulation of JNK and p38. 3.8. TRAF2 is required for MST1-mediated apoptosis Given that TRAF2 physically associates with MST1 and thereby promotes the H2O2-induced stimulation of MST1 and its downstream kinases (Figs. 3 and 6A), we investigated if TRAF2 might be associated with the mechanism by which MST1 mediates H2O2induced apoptosis. We observed that H2O2 treatment markedly increased apoptotic cell death in mouse fibroblasts expressing control (GFP) siRNA, and this effect of H2O2 was abrogated in the cells expressing MST1 siRNA (Fig. 6B). Moreover, ablation of TRAF2 expression prevented H2O2-induced apoptosis in TRAF2-deficient fibroblasts. These results thus indicated that MST1 mediates the H2O2-induced apoptosis in wild-type fibroblasts, but not in TRAF2 / cells, suggesting that TRAF2 is required for MST1mediated apoptosis. We previously showed that Trx-1 is a negative regulator of MST1 [33]. Reduced form of Trx-1 binds to the SARAH domain of MST1 and thereby inhibits the activation of MST1. When ROS converts Trx-1 from the reduced form to the oxidized form, its oxidized form is dissociated from MST1. Then, free MST1 undergoes its homodimerization, which contributes MST1 activation [10]. However, a precise mechanism underlying the regulation of MST1 homodimerization is not understood clearly. In the present study, we demonstrate that TRAF2 binds to the SARAH domain of MST1 and promotes MST1 homodimerization. Given that the SARAH domain is responsible for MST1 homodimerization [9], the binding of TRAF2 to the SARAH domain of MST1 may be critical for the formation of the homomeric complexes of MST1. It would be intriguing to investigate if TRAF2 induces MST1 homodimerization through the oligomerization of TRAF2 itself. TRAF2 has been shown to bind and thereby stimulate the kinase activity of ASK1, a downstream target of MST1. Interestingly, distinct regions of TRAF2 appear to be involved in the activation of MST1 and ASK1. The zinc finger domain of TRAF2 is required for the activation of MST1, whereas The RING domain of TRAF2 is essential for the activation of ASK1 [32]. It is not clear whether TRAF2 is able to form a trimeric complex with MST1 and ASK1. Regardless of forming the trimeric complexes, TRAF2 appears to efficiently stimulate the JNK and p38 MAPK signaling pathways by

Acknowledgements

References [1] W. Droge, Free radicals in the physiological control of cell function, Physiol. Rev. 82 (1) (2002) 47–95. [2] S. Ohta, Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine, Pharmacol. Ther. 144 (1) (2014) 1–11. [3] S. Reuter, et al., Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49 (11) (2010) 1603–1616. [4] S.W. Ryter, et al., Mechanisms of cell death in oxidative stress, Antioxid. Redox Signal. 9 (1) (2007) 49–89. [5] C.L. Creasy, J. Chernoff, Cloning and characterization of a human protein kinase with homology to Ste20, J. Biol. Chem. 270 (37) (1995) 21695–21700. [6] I. Dan, N.M. Watanabe, A. Kusumi, The Ste20 group kinases as regulators of MAP kinase cascades, Trends Cell Biol. 11 (5) (2001) 220–230. [7] S.J. Rawat, J. Chernoff, Regulation of mammalian Ste20 (Mst) kinases, Trends Biochem. Sci. 40 (3) (2015) 149–156. [8] H. Glantschnig, G.A. Rodan, A.A. Reszka, Mapping of MST1 kinase sites of phosphorylation. Activation and autophosphorylation, J. Biol. Chem. 277 (45) (2002) 42987–42996. [9] C.L. Creasy, D.M. Ambrose, J. Chernoff, The Ste20-like protein kinase, Mst1, dimerizes and contains an inhibitory domain, J. Biol. Chem. 271 (35) (1996) 21049–21053. [10] R. Anand, et al., Biochemical analysis of MST1 kinase: elucidation of a C-terminal regulatory region, Biochemistry 47 (25) (2008) 6719–6726. [11] J.D. Graves, et al., Caspase-mediated activation and induction of apoptosis by the mammalian Ste20-like kinase Mst1, Embo J. 17 (8) (1998) 2224–2234. [12] L.K. Taylor, H.C. Wang, R.L. Erikson, Newly identified stress-responsive protein kinases, Krs-1 and Krs-2, Proc. Natl. Acad. Sci. USA 93 (19) (1996) 10099–10104. [13] S. Ura, et al., MST1-JNK promotes apoptosis via caspase-dependent and independent pathways, Genes Cells 6 (6) (2001) 519–530. [14] P.M. de Souza, M.A. Lindsay, Mammalian Sterile20-like kinase 1 and the regulation of apoptosis, Biochem. Soc. Trans. 32 (3) (2004) 485–488. [15] S.H. Ahn, et al., Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae, Cell 120 (1) (2005) 25–36. [16] M.K. Lehtinen, et al., A conserved MST-FOXO signaling pathway mediates oxidative-stress responses and extends life span, Cell 125 (5) (2006) 987–1001. [17] L. Xiao, et al., The c-Abl-MST1 signaling pathway mediates oxidative stressinduced neuronal cell death, J. Neurosci. 31 (26) (2011) 9611–9619. [18] J.K. Lee, et al., MST1 functions as a key modulator of neurodegeneration in a mouse model of ALS, Proc. Natl. Acad. Sci. USA 110 (29) (2013) 12066–12071. [19] A. Ardestani, et al., MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes, Nat. Med. 20 (4) (2014) 385–397. [20] D.P. Del Re, et al., Mst1 promotes cardiac myocyte apoptosis through phosphorylation and inhibition of Bcl-xL, Mol. Cell 54 (4) (2014) 639–650. [21] Y. Maejima, et al., Mst1 inhibits autophagy by promoting the interaction between Beclin1 and Bcl-2, Nat. Med. 19 (11) (2013) 1478–1488. [22] R.H. Arch, R.W. Gedrich, C.B. Thompson, Tumor necrosis factor receptor-associated factors (TRAFs)–a family of adapter proteins that regulates life and death, Genes Dev. 12 (18) (1998) 2821–2830. [23] V. Baud, M. Karin, Signal transduction by tumor necrosis factor and its relatives, Trends Cell Biol. 11 (9) (2001) 372–377. [24] L. Cabal-Hierro, P.S. Lazo, Signal transduction by tumor necrosis factor receptors, Cell Signal. 24 (6) (2012) 1297–1305. [25] G. Chen, D.V. Goeddel, TNF-R1 signaling: a beautiful pathway, Science 296 (5573) (2002) 1634–1635. [26] M. Rothe, et al., A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor, Cell 78 (4) (1994) 681–692. [27] H. Hsu, et al., TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways, Cell 84 (2) (1996) 299–308.

K.-H. Roh, E.-J. Choi / Free Radical Biology and Medicine 91 (2016) 105–113

[28] M. Rothe, et al., The TNFR2–TRAF signaling complex contains two novel proteins related to baculoviral inhibitor of apoptosis proteins, Cell 83 (7) (1995) 1243–1252. [29] F. Urano, et al., Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1, Science 287 (5453) (2000) 664–666. [30] Z.G. Liu, et al., Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death, Cell 87 (3) (1996) 565–576. [31] H.M. Shen, et al., Essential roles of receptor-interacting protein and TRAF2 in oxidative stress-induced cell death, Mol. Cell Biol. 24 (13) (2004) 5914–5922. [32] H. Liu, et al., Activation of apoptosis signal-regulating kinase 1 (ASK1) by tumor necrosis factor receptor-associated factor 2 requires prior dissociation of the ASK1 inhibitor thioredoxin, Mol. Cell Biol. 20 (6) (2000) 2198–2208. [33] J.S. Chae, et al., Thioredoxin-1 functions as a molecular switch regulating the oxidative stress-induced activation of MST1, Free Radic. Biol. Med. 53 (12) (2012) 2335–2343. [34] H.J. Oh, et al., Role of the tumor suppressor RASSF1A in Mst1-mediated

113

apoptosis, Cancer Res. 66 (5) (2006) 2562–2569. [35] H.J. Yun, et al., Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling, EMBO J. 30 (12) (2011) 2465–2476. [36] H.S. Park, et al., Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase, EMBO J. 20 (3) (2001) 446–456. [37] K. Ryoo, et al., Negative regulation of MEKK1-induced signaling by glutathione S-transferase Mu, J. Biol. Chem. 279 (42) (2004) 43589–43594. [38] S.G. Cho, et al., Identification of a novel antiapoptotic protein that antagonizes ASK1 and CAD activities, J. Cell Biol. 163 (1) (2003) 71–81. [39] D. Constantinescu Aruxandei, et al., Dimerization-induced folding of MST1 SARAH and the influence of the intrinsically unstructured inhibitory domain: low thermodynamic stability of monomer, Biochemistry 50 (51) (2011) 10990–11000. [40] O. Soderberg, et al., Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay, Methods 45 (3) (2008) 227–232.