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Nov 9, 2016 - Factor 1 (BIG1) Governs the Recruitment of Tumor. Necrosis Factor Receptor-Associated Factor 2 (TRAF2) to Tumor Necrosis Factor Receptor ...
International Journal of

Molecular Sciences Article

Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor 1 (BIG1) Governs the Recruitment of Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2) to Tumor Necrosis Factor Receptor 1 (TNFR1) Signaling Complexes Takuya Noguchi 1,2, *,† , Mei Tsuchida 2,† , Yosuke Kogue 2,† , Christian Spadini 1 , Yusuke Hirata 2 and Atsushi Matsuzawa 2, * 1 2

* †

Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland; [email protected] Laboratory of Health Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, 980-8578 Sendai, Japan; [email protected] (M.T.); [email protected] (Y.K.); [email protected] (Y.H.) Correspondence: [email protected] (T.N.); [email protected] (A.M.); Tel.: +81-22-795-6828 (T.N.); +81-22-795-6827 (A.M.); Fax: +81-22-795-6826 (T.N. & A.M.) These authors contributed equally to this work.

Academic Editor: Ritva Tikkanen Received: 26 September 2016; Accepted: 2 November 2016; Published: 9 November 2016

Abstract: Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a critical mediator of tumor necrosis factor-α (TNF-α) signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) to be recruited into TNF receptor 1 (TNFR1) signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK) activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I). Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2. Keywords: tumor necrosis factor-α (TNF-α); apoptosis; JNK; TRAF2; BIG1

1. Introduction Adenosine diphosphate (ADP)-ribosylation factor (ARF) is a small guanosine triphosphate (GTP)-binding protein that plays a key role in the regulation of membrane trafficking and the organization of the cytoskeleton [1,2]. The activation state of ARFs is reciprocally regulated by ARF-GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins) [3]. The ARF-GEFs have the Sec7 domain that catalyzes the exchange of guanine nucleotide on ARFs, and are divided into low- and high-molecular-weight ARF-GEFs [4]. Brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1) is classified as a high-molecular-weight ARF-GEF together Int. J. Mol. Sci. 2016, 17, 1869; doi:10.3390/ijms17111869

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with brefeldin A-inhibited guanine nucleotide-exchange factor 2 (BIG2) and Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), and these GEFs are also classified as brefeldin A (BFA)-sensitive ARF-GEFs [5–7]. BIG1 and BIG2 were originally isolated as a part of the macromolecular complex, and both preferentially activate ARF1 and ARF3 by catalyzing the replacement of ARF-bound guanosine diphosphate (GDP) with GTP to coordinate membrane trafficking between the Golgi apparatus and the plasma membrane [5,7–9]. The BIG-regulated membrane trafficking contributes to various cellular dynamics including cell spreading and adhesion, cell migration in wound healing, and neurite outgrowth [10–12]. The localization of BIG1 at the Golgi compartments has been observed in various culture cells, and the cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) promotes nuclear accumulation of BIG1 [13]. On the other hand, it has been reported that BIG2 is associated with not only Golgi compartments but also recycling endosomes, and regulates tubulation of endosomal membranes [14]. Interestingly, BIG1 fails to compensate for the function of BIG2, indicating that the tubulation of endosomal membranes is regulated by BIG2-specific mechanisms [15]. Moreover, BIG1 and BIG2 are paired with different binding partners, whereas they interact with each other [4]. Thus, a series of recent studies of the BIGs strongly suggest that BIG1 and BIG2 have distinct and redundant functions. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that induces a wide variety of cellular responses including cell growth, differentiation, immune responses, and cell death [16]. Two different receptors, named TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), can mediate signals from TNF-α. TNFR1 is ubiquitously expressed on almost all cells whereas only restricted cell types including neurons, endothelial cells, cardiac myocytes, thymocytes and mesenchymal cells express TNFR2. Engagement of TNFR1 by TNF-α rapidly leads to formation of a receptor complex referred to as the TNFR1 signaling complex (complex I), which elicits activation of the canonical nuclear factor-κB (NF-κB) pathways and the mitogen-activated protein (MAP) kinase pathways, such as the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways, that preferentially induce anti-apoptotic and pro-inflammatory responses. Although the gene activation as a primary signaling outcome of TNFR1 activation contributes to cellular survival, TNFR1 can also induce cell death including apoptosis and necroptosis under certain circumstances. TNFR1-induced apoptosis is mediated by the cytosolic complex (complex II) formed as a result of rearrangement of complex I [17,18]. Complex II is a Fas-associated protein with death domain (FADD)-based complex that functions as a signaling hub to activate caspase-8. Meanwhile, further rearrangement of complex II occurs when caspase-8 activation is blocked, resulting in the formation of a necrosome that promotes activation of the necrotic pathways [19]. Tumor necrosis factor receptor-associated factor 2 (TRAF2), belonging to the TRAF family of proteins, is a really interesting new gene (RING) finger protein that participates in TNF-α-induced cellular responses as a signaling component of both complex I and complex II [20,21]. Upon binding of TNF-α to TNFR1, TRAF2 is recruited to TNFR1 through the interaction with TNFR-associated death domain protein (TRADD), and then recruits cellular inhibitor of apoptosis 1 and 2 (cIAP1/2) proteins to complex I. cIAP1/2 catalyzes the ubiquitination of receptor-interacting protein 1 (RIP1), which in turn elicits further recruitment of the essential components including inhibitor of nuclear factor kappa-B kinase (IKK) complex, the linear ubiquitin assembly complex (LUBAC) and transforming growth factor β-activated kinase 1 (TAK) 1 into complex I [16,22]. The contribution of TRAF2 to the downstream signaling has been investigated by the gene knockout studies in mice [23,24]. In particular, these studies revealed that TRAF2 plays critical roles in activation of the canonical NF-κB and the JNK pathways. However, since TRAF2-mediated NF-κB activation is compensated by tumor necrosis factor receptor-associated factor 5 (TRAF5), another member of TRAF family proteins, TRAF2 knockout cells exhibit only a mild reduction of the NF-κB activation [23]. Nevertheless, TRAF2 knockout cells exhibit increased sensitivity to TNF-α-induced apoptosis, suggesting that TRAF2 is required for NF-κB-independent mechanisms that protect against TNF-α-induced apoptosis. Similar effects of TRAF2 on cell death have been demonstrated in Fas or tumor necrosis factor-related apoptosis-inducing

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ligand (TRAIL) receptor signaling [25,26]. Moreover, a recent report has demonstrated that TRAF2 suppresses the induction of apoptosis through the mechanism of K48-linked polyubiquitination of activated caspase-8, which can account for the NF-κB-independent mechanism by which TRAF2 exerts anti-apoptotic activity [27]. Compelling evidence indicates that cellular responses mediated by TNFR1 are highly associated with the membrane trafficking of TNFR1 [17,28,29]. TNFR1 is internalized into signaling vesicles termed TNFR1 receptosomes after complex I formation, which is an essential process for ensuring proper signal transduction [29,30]. However, the involvement of the ARF-GEFs in TNFR1-medated cellular processes is largely unknown, except that it has been demonstrated that BIG2 regulates the constitutive release of TNFR1 exosome-like vesicles [31]. In this study, we found that BIG1 is involved in TNF-α signaling. Knockdown of BIG1 showed similar phenotypes of TRAF2 deficient cells that had decreased activation of JNK and increased apoptosis in response to TNF-α. We also found that BIG1 is required for the recruitment of TRAF2 to both complex I and complex II. Taken together, these results indicate that BIG1 regulates TNFR1 signaling by targeting TRAF2. 2. Results 2.1. BIG1 Is Involved in TNF-α-Induced JNK Activation In order to examine whether BIG1 is involved in TNFR1 signaling, we performed BIG1 knockdown experiments using HT1080 fibrosarcoma or HeLa cells that predominantly express TNFR1 rather than TNFR2 [32]. Both independent small interfering RNAs (siRNA) against BIG1 significantly reduced both mRNA and protein expression of BIG1 (Figure 1A). When BIG1 expression was knocked down by these siRNAs in HT1080 cells, immunoblotting analysis using the phospho-specific antibodies that can monitor the activation states revealed that TNF-α-induced JNK activation was attenuated in BIG1 knockdown cells, while that of p38 MAP kinase was unchanged (Figure 1B). Similar findings were observed in BIG1 knockdown HeLa cells (Figure 1C). Moreover, we found that TNF-α-induced JNK activation was also attenuated in TRAF2 knockdown cells as previous studies have demonstrated (Figure 1D,E) [23,24]. These results suggest that BIG1 is involved in TNF-α-induced JNK activation in a similar manner to TRAF2. 2.2. BIG1 Is Dispensable for TNF-α-Induced Degradation of IκBα We next examined whether BIG1 knockdown affects the canonical NF-κB activation. In steady state condition, activation of the canonical NF-κB pathway is blocked by IκBα that inhibits NF-κB nuclear translocation, whereas, upon TNFR1 activation, IKK complex induces the phosphorylation-dependent degradation of IκBα, leading to the translocation and activation of NF-κB. As shown in Figure 2A, treatment with TNF-α promoted the degradation of IκBα, which occurred within 15 min and peaked at 30 min, and the expression levels of IκBα was returned to basal levels at 180 min, probably by the transcriptional activity of NF-κB. The kinetics of the degradation and the induction of IκBα were unaltered in both BIG1 knockdown HT1080 and HeLa cells (Figure 2A,B). Similar findings were observed in TRAF2 knockdown cells (Figure 2C,D). On the other hand, luciferase reporter assays of NF-κB displayed slightly reduced reporter activity in BIG1 knockdown cells when compared with control cells, although there was a statistically nonsignificant reduction (p = 0.16 vs. control) in the cells knocked down by BIG1 #2 siRNA (Figure 2E). These results suggest that BIG1 is dispensable for the degradation of IκBα, but it is possible that BIG1 is only partially involved in TNF-α-induced NF-κB activation through an alternative mechanism. Moreover, TRAF2 knockdown cells displayed a modest but significant reduction of the reporter activity without any effect on the degradation of IκBα, which is in agreement with data from previous studies using TRAF2-deficient cells (Figure 2C–E) [23,24]. Collectively, BIG1 knockdown cells displayed properties similar to those of TRAF2 in TNF-α-induced NF-κB activation, yet the extent of reduction in the reporter assay was limited in BIG1 knockdown cells.

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Figure BrefeldinA-inhibited A-inhibited guanine guanine nucleotide-exchange is is involved in in tumor Figure 1. 1.Brefeldin nucleotide-exchangefactor factor1 1(BIG1) (BIG1) involved tumor necrosis factor-α (TNF-α)-induced c-Jun N-terminal N-terminal kinases HT1080 or or HeLa Figure 1. Brefeldin A-inhibited guanine nucleotide-exchange factor 1activation. (BIG1) is (A) involved in tumor necrosis factor-α (TNF-α)-induced c-Jun kinases(JNK) (JNK)activation. (A) HT1080 HeLa cells were transfected with small interfering RNA (siRNA) for negative control or BIG1 (BIG1 #1 or or necrosis factor-α (TNF-α)-induced c-Jun N-terminal kinases (JNK) activation. (A) HT1080 or HeLa cells were transfected with small interfering RNA (siRNA) for negative control or BIG1 (BIG1 #1 cells were transfected with small interfering RNA (siRNA) for negative control or BIG1 (BIG1 #1 or BIG1 #2). After 48 h, the relative mRNA levels of Big1 and protein expression of BIG1 were determined BIG1 #2). After 48 h, the relative mRNA levels of Big1 and protein expression of BIG1 were determined #2). AfterRT-PCR 48 h, theand relative mRNA levels of Big1 and protein BIG1 were determined byBIG1 quantitative immunoblotting, respectively. Dataexpression shown areof the mean ± standard error by quantitative RT-PCR and immunoblotting, respectively. Data shown are the mean ± standard error RT-PCR immunoblotting, Data(C) shown aretransfected the mean ± standard errorfor of by thequantitative mean (SEM) (n = and 3); (B–E) HT1080 (B)respectively. or HeLa cells were with siRNA of the mean (SEM) (n = 3); (B–E) HT1080 (B) or HeLa cells (C) were transfected with siRNA for negative of the mean (SEM) (n = HT1080 3); (B–E) (D) HT1080 (B) or HeLa were transfected with siRNA for negative control or BIG1. or HeLa cells (E) cells were(C) transfected with siRNA for negative control or BIG1. HT1080 (D)HT1080 or HeLa (E) were transfected with siRNA for negative control or negative control or BIG1. (D)cells or HeLa cells (E) were transfected forwere negative control or tumor necrosis factor receptor-associated factor 2 (TRAF2). Afterwith 72 h,siRNA the cells treated tumor necrosis factor receptor-associated factor 2 factor (TRAF2). AfterAfter 72 h,72the cells were control or tumor necrosis factor receptor-associated (TRAF2). h, the cells weretreated treated with with 20 ng/mL TNF-α for the indicated periods. The cell 2extracts were subjected to immunoblotting 20 ng/mL the for indicated periods. The cell were subjected totoimmunoblotting with 20TNF-α ng/mL for TNF-α the indicated periods. The extracts cell extracts were subjected immunoblotting with with the indicated antibodies. The relative activity of JNK was quantified using ImageJ software. the indicated antibodies. The relative activity of JNK was was quantified using ImageJ with the indicated antibodies. The relative activity of JNK quantified using ImageJsoftware. software.

Figure 2. Cont.

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Figure2.2.BIG1 BIG1isisdispensable dispensable for for TNF-α-induced TNF-α-induced degradation (A–D) HT1080 (A)(A) or HeLa Figure degradationofofIκBα. IκBα. (A–D) HT1080 or HeLa cells(B) (B)were weretransfected transfectedwith with siRNA siRNA for (C)(C) or or HeLa cells (D)(D) were cells for negative negativecontrol controlororBIG1. BIG1.HT1080 HT1080 HeLa cells were transfectedwith withsiRNA siRNAfor fornegative negative control control or were treated with 20 20 ng/mL transfected or TRAF2. TRAF2.After After72 72h,h,the thecells cells were treated with ng/mL TNF-αforfor indicated periods. cell extracts subjected to immunoblotting with the TNF-α thethe indicated periods. TheThe cell extracts werewere subjected to immunoblotting with the indicated indicated antibodies; (E) HT1080 were transfected with indicated siRNA. After 24 h, the cells were antibodies; (E) HT1080 were transfected with indicated siRNA. After 24 h, the cells were transfected transfected with a plasmid mixacontaining a NF-κBreporter luciferase reporterand plasmid andluciferase a renilla luciferase with a plasmid mix containing NF-κB luciferase plasmid a renilla plasmid for plasmid for normalization together with indicated siRNA. After 48 h, cells were treated with 20 ng/mL normalization together with indicated siRNA. After 48 h, cells were treated with 20 ng/mL TNF-α TNF-α for 6 h. Firefly and renilla luciferase activities were quantified with dual luciferase reporter for 6 h. Firefly and renilla luciferase activities were quantified with dual luciferase reporter assay kit. assay kit. Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Data shown are the mean ± SEM (n = 3). Statistical significance was tested using an unpaired Student’s Student’s t-test (vs. control cells); * p < 0.05, *** p < 0.001. t-test (vs. control cells); * p < 0.05, *** p < 0.001.

2.3. Knockdown of BIG1 Increases Sensitivity to TNF-α-Induced Apoptosis 2.3. Knockdown of BIG1 Increases Sensitivity to TNF-α-Induced Apoptosis Since TNF-α responses in the early phase elicit the induction of anti-apoptotic proteins such as SinceFLICE-like TNF-α responses the early phase elicitand theTRAF2/5, induction anti-apoptotic proteins cellular inhibitory in protein (c-FLIP), cIAP1/2 theofactivation of cell death such as cellular FLICE-like inhibitory protein (c-FLIP), cIAP1/2 and TRAF2/5, the activation pathways is strongly suppressed [33,34]. However, the lack of the anti-apoptotic proteins usually ofallows cell death pathways is strongly suppressed [33,34]. However,For the lack ofTRAF2 the anti-apoptotic caspase-8 activation and subsequent induction of apoptosis. instance, knockout proteins usually allows caspase-8 activation and subsequent induction of apoptosis. For instance, mouse embryonic fibroblasts (MEFs) exhibit increased sensitivity to TNF-α-induced apoptosis, and TRAF2 knockout mousewith embryonic fibroblasts (MEFs) increased co-deletion of TRAF5 TRAF2 further increases its exhibit sensitivity [23,24].sensitivity As shown to in TNF-α-induced Figure 3A,B, TRAF2 knockdown reduced viability h afterfurther the treatment withitsTNF-α. Moreover, in TNF-αapoptosis, and co-deletion ofcell TRAF5 with24TRAF2 increases sensitivity [23,24]. As shown in Figure 3A,B, TRAF2 knockdown reduced cell viability 24 h after the treatment with TNF-α.

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Moreover, in TNF-α-treated TRAF2 knockdown cells, we increased p18 fragments treated TRAF2 knockdown cells, we observed increased p18observed fragments of caspase-8, which wasofa caspase-8, which was a result of autocatalytic cleavage (auto-cleavage) of caspase-8 induced after result of autocatalytic cleavage (auto-cleavage) of caspase-8 induced after the complex II formation the complex II These formation (Figure 3C,D). results of indicate knockdown of TRAF2 apoptosis promotes (Figure 3C,D). results indicate thatThese knockdown TRAF2that promotes TNF-α-induced TNF-α-induced apoptosis both HT1080 and HeLa cells. in both HT1080 and HeLa in cells. We next next examined examined whether whether knockdown of BIG1 affects the the sensitivity sensitivity to TNF-α-induced TNF-α-induced apoptosis. When When siRNA-transfected siRNA-transfected cells cells were were treated treated with with TNF-α, TNF-α, we found that knockdown of BIG1 significantly reduced cell viability viability to to aa similar similar extent extent as as TRAF2 TRAF2 knockdown knockdown cells (as shown in Figure 3A), when treated with a pan-caspase inhibitor Z-VAD-fmk 3A), and andthis thiswas wascompletely completelyrecovered recovered when treated with a pan-caspase inhibitor Z-VAD(fluoromethylketone) (Figure 4A). Moreover, the cleavage of caspase-8 followed followed by caspase-3 fmk (fluoromethylketone) (Figure 4A). Moreover, the cleavage of caspase-8 by activation caspase-3 was apparently acceleratedaccelerated in BIG1 knockdown cells (Figurecells 4B).(Figure On the 4B). otherOn hand, activation was apparently in BIG1 knockdown the knockdown other hand, of BIG2 didofnot have effectany oneffect TNF-α-induced apoptosis, although BIG2 BIG2 expression was knockdown BIG2 did any not have on TNF-α-induced apoptosis, although expression sufficiently suppressed (Figure 4C,D). These results showshow that BIG1 is required for protecting cells was sufficiently suppressed (Figure 4C,D). These results that BIG1 is required for protecting from TNF-α-induced apoptosis, and BIG2 is notisable compensate its functions. cells from TNF-α-induced apoptosis, and BIG2 not to able to compensate its functions. A

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Figure 3. Knockdown of TRAF2 increases sensitivity to TNF-α-induced apoptosis. (A,B) HT1080 (A) Figure 3. Knockdown of TRAF2 increases sensitivity to TNF-α-induced apoptosis. (A,B) HT1080 (A) or HeLa cells (B) were transfected with siRNA for negative control or TRAF2, and treated 72 h later or HeLa cells (B) were transfected with siRNA for negative control or TRAF2, and treated 72 h later with ± ± 50 the phenazine phenazine methosulfate methosulfate with 50 ng/mL ng/mLof ofTNF-α TNF-αfor for 24 24 h. h. Cell Cell viability viability was was monitored monitored with with the (PMS)/ 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)(PMS)/ 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, 2H-tetrazolium, inner salt (MTS) assay, assay, normalized normalized to to untreated untreated control control cells cells and and expressed expressed as as mean mean ± ± SEM inner salt (MTS) SEM (n (n == 3). 3). Statistical significance was tested using an unpaired Student’s t-test (vs. control cells); * p Statistical significance was tested using an unpaired Student’s t-test (vs. control cells); * p