c-jun n-terminal kinase regulates the interaction between 14-3-3 and bad in ethanol-induced cell...

c-Jun N-Terminal Kinase Regulates theInteraction Between 14-3-3 and Bad inEthanol-Induced Cell Death

Jae YoonHan, Eun Young Jeong, Yoon Sook Kim, Gu Seob Roh, Hyun Joon Kim,Sang Soo Kang, Gyeong Jae Cho, andWan Sung Choi*

Department of Anatomy and Neurobiology, Institute of Health Sciences, School of Medicine,Gyeongsang National University, Gyeongnam, South Korea

Activation of the c-jun N-terminal kinase (JNK) is knownto be an important step during ethanol-induced celldeath, but it has yet to be identified how JNK regulatesapoptosis. Therefore, we investigated the mechanismby which JNK induces cell death following ethanoltreatment. Ethanol (6 g/kg, 20% in saline) was adminis-tered subcutaneously to postnatal 7 day rat pups.Twelve hours after the first ethanol administration, ratpups were decapitated, and extracts of total proteinfrom cerebral cortices were prepared. Ethanol exposureinduced phosphorylation of JNK but did not affect theexpression levels of pro- and antiapoptotic proteins.Furthermore, interactions of phospho-JNK (p-JNK) with14-3-3 as well as with Bad were enhanced in the cere-bral cortices of ethanol-treated rats. Pretreatment withJNK inhibitor (SP600125) of SH-SY5Y cells inhibitedJNK phosphorylation and interaction between p-JNKand 14-3-3 resulting from ethanol. Furthermore, 14-3-3interaction with Bad was diminished in the cerebral cor-tices of ethanol-treated rats. These findings suggestthat JNK induces Bad release from 14-3-3 by inhibitingtheir interaction. After this event, Bad binds to Bcl-xL,releasing Bax from Bcl-xL and leading to cell death.We hypothesize that JNK may play an important roleduring ethanol-induced cell death via the inhibition ofantiapoptotic function of 14-3-3 as well as activation ofproapoptotic function of Bad. VVC 2008 Wiley-Liss, Inc.

Key words: ethanol; cell death; JNK; 14-3-3; Bad

The most deleterious effect of ethanol abuse isneural cell death. Especially, ethanol exposure during thebrain’s developing period has been known to induceapoptotic cell death (Ikonomidou et al., 2000). A con-sistent result was reported from a cell culture systemusing SH-SY5Y cells (Sakai et al., 2005), which showedthat thje cytotoxic effect of ethanol is more severe inundifferentiated cells compared with differentiated cells.Numerous mechanisms including mitochondrial dysfunc-tion have been suggested to contribute to ethanol-induced neural cell death. Previous studies suggest thatethanol-induced apoptotic cell death is an intrinsicpathway-mediated phenomenon involving Bax-induced

mitochondrial dysfunction (Young et al., 2003). The lossof mitochondrial membrane potential by Bax transloca-tion leads to cytochrome c release and caspase-3 activa-tion, leading to apoptotic cell death. Bax translocation isan important event during apoptosis and is regulated bythe pro- and antiapoptotic Bcl-2 family proteins (Greenand Reed, 1998; Wang, 2001).

14-3-3 proteins are a family of serine/threoninebinding proteins that are expressed ubiquitously andespecially enriched in the brain (Aitken et al., 1992).The 14-3-3 proteins have antiapoptotic functions andexert their antiapoptotic activity by directly sequesteringproapoptotic proteins such as Bad (Masters et al., 2002;Porter et al., 2006). Bad is regulated by phosphorylationat different serine residues. Bad is phosphorylated atSer112, -136, and -155 by survival factors (Datta et al.,1997; Harada et al., 1999; Zhu et al., 2002). Phospho-rylation of Bad by survival factors enhances its interac-tion with 14-3-3 and cytoplasmic sequestration in aninactive state. However, Bad phosphorylation at Ser128by c-jun N-terminal kinases (JNK) promotes the apo-ptotic effect of Bad (Donovan et al., 2002; Wang et al.,2007). After induction of apoptosis, Bad is activatedthrough the release from 14-3-3. Subsequently, Baddimerizes with Bcl-xL, triggering Bax release from Bcl-xL. This leads to Bax translocation to the mitochondria,cytochrome c release, and downstream apoptotic cascadeactivation (Datta et al., 2000). Thus, inactivation of Badis considered to be an important step in protecting cellsagainst death.

Contract grant sponsor: Korea Ministry of Education; Contract grant

number: KRF-2006-005-J04201; Contract grant sponsor: MRC Program

of MOST/KOSEF; Contract grant number: R13-2005-012-01001-1.

*Correspondence to: Wan Sung Choi, PhD, Department of Anatomy

and Neurobiology, Institute of Health Sciences, School of Medicine,

Gyeongsang National University, 92 Chilam-dong, Jinju, Gyeongnam

660-751, South Korea. E-mail: [email protected]

Received 29 November 2007; Revised 13 March 2008; Accepted 14

March 2008

Published online 2 June 2008 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21759

Journal of Neuroscience Research 86:3221–3229 (2008)

' 2008 Wiley-Liss, Inc.

JNK, a mitogen-activated protein kinase associatedwith apoptosis, is activated by various apoptotic stimuli(Kuan et al., 1999; Lee and Shukla, 2005). JNK mediatesthe apoptotic signaling pathway by phosphorylatingproapoptotic proteins such as Bax and Bad as well asantiapoptotic proteins such as 14-3-3 and Bcl-2(Donovan et al,2002; Schroeter et al., 2003; Tsurutaet al., 2004; Kim et al., 2006). 14-3-3 is known to be asubstrate of JNK, and its function is impaired by phos-phorylation on critical residues. A previous studyreported that JNK phosphorylated 14-3-3f at Ser184(Tsuruta et al., 2004). This promotes Bax dissociationfrom 14-3-3f, resulting in cell death. Consistently withthis result, a recent report showed that 14-3-3 phospho-rylation by JNK induced Bad release from 14-3-3 andantagonized the effects of AKT signaling (Sunayamaet al., 2005). Thus, JNK-mediated 14-3-3 phosphoryla-tion is thought to be a critical event that leads to celldeath.

Although JNK has been known to be implicated inmediating ethanol-induced cell death (Lee and Shukla,2005; Han et al., 2006), currently the mechanism isunclear. Thus, in the present study, we investigatedwhether ethanol-induced JNK activation induces Baddissociation from 14-3-3 and activates downstream celldeath pathway.

MATERIALS AND METHODS

Animals and Tissue Preparation

Seven-day-old Sprague-Dawley rats were used in allexperiments. Rats of this age fall in the middle of the braindevelopment period and showed peak sensitivity to ethanol-induced apoptotic neurodegeneration (Ikonomidou et al.,2000). Rats were housed in temperature-controlled conditionsunder a 14-hr light/10-hr dark photocycle (light on at 06:00),with food and water supplied ad libitum. All procedures wereperformed in accordance with the Guide for the care and use oflaboratory animals published by the Gyeongsang National Uni-versity (GLA-060530-R0008). Ethanol was prepared as a 20%(v/v) solution in normal saline and administered subcutane-ously to the rat pups on postnatal day 7. The whole dose (6.0g/kg) was delivered in two equal doses that were given 2 hrapart. Littermate controls received an equal volume of normalsaline. The blood concentration of ethanol was measuredusing a kit (Alcohol Reagent Set, Pointe Scientific, Inc.),according to the manufacturer’s procedure.

SH-SY5Y Neuroblastoma Cell Culture andEthanol Treatment

SH-SY5Y human neuroblastoma cells (ATCC, CRL-2266) were cultured in Dulbecco’s modified Eagle’s medium:Ham’s nutrient mixture F-12 (DMEM/F-12; Invitrogen, SanDiego, CA) supplemented with 10% fetal bovine serum, peni-cillin (100 U/ml), and streptomycin (100 lg/ml). The cul-tures were maintained at 378C in a humidified atmosphere of5% CO2 before experiments. For ethanol treatment of SH-SY5Y cells, the culture medium was removed and replacedwith fresh low-serum (0.5%) medium containing ethanol. All

experiments for ethanol treatment were carried out as previ-ously described to avoid ethanol volatility (Mitchell et al.,1998). Subconfluent monolayer SH-SY5Y cells were washedtwice with phosphate-buffered saline (PBS) and incubated for12 hr in low-serum DMEM/F-12 to diminish the effects ofgrowth factor contained in serum. Stock solutions (10 mM) ofthe specific JNK inhibitor SP600125 (Sigma, St. Louis, MO)was prepared in dimethyl sulfoxide (DMSO) and added to themedia (final 10 lM) 1 hr before ethanol treatment. FinalDMSO concentration did not exceed 0.1%, and SH-SY5Ycell viability was not affected at this concentration. Two hoursafter ethanol treatment, cells were washed with ice-cold PBSand cell lysates were prepared as described below.

Protein Preparation and Western Blot Analysis

Rat cerebral cortices and SH-SY5Y cells were lysed inlysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl,5 mM EDTA, 1% Nonidet P-40, and protease inhibitors(1 mM phenylmethylsulfonyl fluoride, 1 lg/ml aprotinin,1 lg/ml leupeptin). Lysates were sonicated several times for2–5 sec each and centrifuged at 12,000g for 20 min at 48C.Supernatants were collected, and the protein concentration ofeach lysate was determined using bicinchoninic acid (BCA)kit (Pierce, Rockford, IL) according to the manufacturer’sprotocol. Bovine serum albumin was used as a standard. Eachlysate was diluted appropriately with a sample buffer (0.15 MTris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% 2-mercap-toethanol). For Western blotting, samples were boiled for5 min. Equal amounts of protein were loaded and separatedby SDS-PAGE and transferred to nitrocellulose membranes.Membranes were washed in Tris-buffered saline containing0.1% Tween-20 and then incubated with the following anti-bodies: anti-JNK, antiphospho-JNK, anticleaved caspase-3(rabbit polyclonal, diluted 1:1,000; Cell Signaling Technology,Beverly, MA), anti-Bad Ser128 phosphospecific antibody(rabbit polyclonal, diluted 1:1,000; Chemicon, Temecula,CA), rabbit polyclonal anti-14-3-3f, rabbit polyclonal anti-Bad,mouse monoclonal anti-Bax, mouse monoclonal anti-Bcl-xL(diluted 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA),and rabbit polyclonal antipoly-(ADP-ribose) polymerase(PARP) antibody (diluted 1:5,000; BioMol Research Laborato-ries) as primary antibody. Membranes were incubated with sec-ondary antibody (1:10,000; Pierce, Rockford, IL). The ECLWestern blot analysis system (Amersham Pharmacia Biotech,Piscataway, NJ) was used for detection, according to the manu-facturer’s instructions. The intensity analysis was carried out inSigmaGel 1.0 and SigmaPlot 7.0 (SPSS Inc., Chicago, IL).

Immunoprecipitation

For immunoprecipitation analysis, 200 lg of total pro-tein was precleared with protein-A/G agarose beads (SantaCruz Biotechnology) to remove nonspecific binding proteins,then incubated with primary antibody overnight at 48C. Theimmune complex was precipitated with protein A/G agarosebeads for 2 hr at 48C. The protein-bead complex was thenwashed and collected by centrifugation. Samples were proc-essed as described for Western blot analysis.

3222 Han et al.

Journal of Neuroscience Research

Data Analysis

All data are presented as mean 6 SEM of three inde-pendent experiments. Statistical significance was determinedby using the Student’s t-test. Differences with a probability (P)less than 0.05 were considered statistically significant.

RESULTS

Effects of Ethanol on JNK Phosphorylation andPro- and Antiapoptotic Protein Expression

Cerebral cortices are the most sensitive area toethanol (Ikonomidou et al., 2000). In a previous study,we found that caspase-3 activation in rat cerebral corticespeaked at 12 hr, and the levels were maintained up to24 hr after the first ethanol administration (Han et al.,2005), suggesting that cell death is most robust at 12 hrafter the first ethanol administration. Therefore, rat cere-bral cortices were prepared at this time for Western blotand immunoprecipitation analysis. The blood concentra-tion of ethanol reached 674.8 6 23.7 mg/dl at 4 hr afterthe first ethanol treatment and remained above 200 mg/dl for more than 20 hr. To investigate whether ethanolaffects JNK phosphorylation and pro- and antiapoptoticprotein expression, we performed Western blot analysison total protein prepared from the cerebral cortices ofethanol- or saline-treated rats. We found that JNK phos-phorylation was significantly increased in the ethanolgroup compared with the saline controls (Fig. 1A). Theantiphospho-JNK primary antibody recognized twobands of 46 kDa and 54 kDa of phospho-JNK (p-JNK).The 46-kDa band was more detectable than the 54-kDaband (Fig. 1A), which is consistent with a previousreport (Villegas et al., 2006). Because total JNK was notaffected by ethanol treatment (Fig. 1B), this result sug-gests that ethanol treatment differentially induced p-JNKs. Moreover, ethanol treatment did not affect theexpression levels of 14-3-3f, Bad, Bax, or Bcl-xL (Fig.1B), suggesting that ethanol-induced cell death wasmediated by JNK activation and its downstream apopto-tic pathway rather than by the alteration of 14-3-3f,Bad, Bax, and Bcl-xL expression levels. Although Baxwas reported to increase in the cerebellum of ethanol-treated neonate rats (Nowoslawski et al., 2005; Siler-Marsiglio et al., 2005), this likely is due to differences inthe brain regions and the age of animals used, whichmay influence the sensitivity to ethanol.

Effect of Ethanol on p-JNK InteractionWith 14-3-3

14-3-3 proteins are known to antagonize the apo-ptotic signals by sequestering the proapoptotic proteins(Zhang et al., 1999). Recently, it was reported thatphosphorylation of 14-3-3 by activated JNK promotesthe dissociation of proapoptotic proteins from this pro-tein (Tsuruta et al., 2004; Sunayama et al., 2005). How-ever, there are no studies showing that JNK regulatesthe antiapoptotic function of 14-3-3 in ethanol-inducedcell death. Thus, to investigate whether activated JNKby ethanol regulates 14-3-3, we prepared the protein

Fig. 1. Effect of ethanol on JNK phosphorylation in rat cerebral cor-tices. Postnatal 7-day-old rat pups were treated subcutaneously withethanol (6 g/kg) or an equal volume of saline. Twelve hours afterthe first ethanol or saline treatment, rat pups were decapitated, andextracts of total protein from cerebral cortices were obtained for p-JNK, JNK, 14-3-3f, Bad, Bax, and Bcl-xL Western blot analysis. A:Representative Western blot showed the phosphorylation level ofJNK in the cerebral cortices of ethanol- or saline-treated rats. Thephosphorylation levels of JNK (54 and 46 kDa) were increased in thecerebral cortices of ethanol-treated rats compared with the salinecontrols. Densitometry was performed on the p-JNK (54 and 46kDa) bands and normalized to the a-tubulin bands. Densitometricanalysis is represented as arbitrary units (A.U.). B: Western blot anal-yses of anti- or proapoptotic proteins from the cerebral cortices ofethanol- or saline-treated rats. Each lane represents an individual ani-mal. a-Tubulin was used as an internal control. This experiment wasrepeated three times independently. Data (n 5 9) are presented asmean 6 SEM. *P < 0.05, **P < 0.01.

JNK Regulates 14-3-3 and Bad Interaction 3223


extract from the cerebral cortices of saline- or ethanol-treated rats and performed Western blot analysis onimmunoprecipitated p-JNK to detect the presence of14-3-3 (Fig. 2). The interaction of p-JNK with 14-3-3was increased in the cerebral cortices of the ethanolgroup compared with the saline controls. To confirmwhether the increment of this interaction is mediated byJNK activation after ethanol treatment, we treated SH-SY5Y cells with the JNK inhibitor SP600125. SH-SY5Y cells are undifferentiated cells that are widely usedfor studying neuronal cell death (Jung et al., 2007; Chenet al., 2008). SH-SY5Y cells had distinct level of viabil-ity (60% compared with control) at 100 mM ethanoltreatment for 6 hr. We chose 100 mM ethanol treatmentfor 2 hr to examine protein phosphorylation and theirinteraction, because these events happen before celldeath. As expected, the levels of p-JNK in SH-SY5Ycells exposed to SP600125, a JNK inhibitor, 1 hr beforeethanol treatment, were significantly lower comparedwith ethanol-only-treated cells (Fig. 3A). Furthermore,p-JNK interaction with 14-3-3 was inhibited bySP600125 (Fig. 3B).

Fig. 3. Effect of JNK inhibitor on p-JNK interaction with 14-3-3 inSH-SY5Y cells. SH-SY5Y cells were cultured for 12 hr in low-serum DMEM/F-12. The JNK inhibitor SP600125 was added to themedia (final 10 lM) 1 hr before ethanol treatment (100 mM). Twohours after ethanol treatment, the cells were washed with ice-coldPBS, and cell lysates were prepared. Representative Western blotsshowed the phosphorylation levels of JNK (A). The phosphorylationlevels of JNK (54 and 46 kDa) were increased after ethanol treat-ment, but this effect was inhibited in the presence of JNK inhibitor.Densitometry value of p-JNK was normalized to a-tubulin and rep-resented as arbitrary units (A.U.). B: Coimmunoprecipitation of p-JNK with 14-3-3f. Immunoprecipitation was performed with ananti-p-JNK antibody. Western blot analyses of immunoprecipitatedanti-p-JNK were performed with the 14-3-3f antibody. The interac-tion of p-JNK with 14-3-3 was inhibited in the presence of JNK in-hibitor. Densitometry analyses were normalized to IgG bands andrepresented as arbitrary units (A.U.). This experiment was repeatedthree times independently. Data are presented as mean 6 SEM.*P < 0.05, **P < 0.01.

Fig. 2. Effect of ethanol on p-JNK interaction with 14-3-3 in ratcerebral cortices. Postnatal 7-day-old rat pups were treated sub-cutaneously with ethanol (6 g/kg) or an equal volume of saline.Twelve hours after the first ethanol or saline treatment, rat pups weredecapitated and extracts of total protein from cerebral cortices wereprepared. A: Coimmunoprecipitation of p-JNK with 14-3-3f in ratcerebral cortices. Immunoprecipitation was performed with ananti-p-JNK antibody. Western blot analyses of immunoprecipitatedp-JNK were performed with anti-14-3-3f antibody. B: Coimmuno-precipitation of 14-3-3f with p-JNK in rat cerebral cortices. Im-munoprecipitation was performed with an anti-14-3-3 antibody.Western blot analyses of immunoprecipitated 14-3-3f were per-formed with anti-p-JNK antibody. The interaction of p-JNK with14-3-3f was increased in the cerebral cortices of ethanol-treated ratscompared with the saline controls. IgG bands are shown to confirmequality of antibody loading. Densitometric analyses were representedas arbitrary units (A.U.), normalized by IgG. This experiment wasrepeated three times independently. Data are presented as mean 6SEM. *P < 0.05, **P < 0.01.

3224 Han et al.


Effect of Ethanol on Bad Phosphorylation

It has been reported that JNK-induced Bad phos-phorylation at Ser128 residue promotes the apoptoticeffect of Bad (Donovan et al., 2002; Wang et al., 2007).Thus, to test the effect of ethanol on Bad phosphoryla-tion at Ser128, we performed Western blot analysis withanti-Bad Ser128 phosphospecific antibody and foundthat p-JNK interactions with Bad as well as p-Bad(Ser128) level were increased in the cerebral cortices ofethanol-treated rats (Fig. 4A,B). However, these changes

were significantly decreased in the presence of JNK in-hibitor in SH-SY5Y cells (Fig. 4C,D), suggesting thatBad phosphorylation at Ser128 is enhanced by p-JNK.

Effect of Ethanol on The Interaction BetweenPro- and Antiapoptotic Proteins

To test whether 14-3-3 phosphorylation affects itsinteraction with Bad, we performed Western blot analy-sis on immunoprecipitated 14-3-3f with anti-Badantibody. The association of Bad with 14-3-3f was

Fig. 4. Effect of ethanol on Bad phosphorylation in rat cerebral cor-tices and in SH-SY5Y cells. Postnatal 7-day-old rat pups were treatedsubcutaneously with ethanol (6 g/kg) or an equal volume of saline.Twelve hours after the first ethanol or saline treatment, rat pups weredecapitated, and extracts of total protein from cerebral cortices wereprepared (A,B). A: Western blot analyses of Bad phosphorylation atSer128 in rat cerebral cortices. Each lane represents an individual ani-mal. Densitometric analyses are represented as arbitrary units (A.U.),normalized by a-tubulin. B: Coimmunoprecipitation of p-JNK withBad in rat cerebral cortices. Western blot analyses of immunoprecipi-tated p-JNK were performed with anti-Bad antibody. IgG bandswere shown to confirm equality of antibody loading. SH-SY5Y cells

were cultured for 12 hr in low serum DMEM/F-12 (C,D). TheJNK inhibitor SP600125 was added to the media (final 10 lM) 1 hrbefore ethanol treatment (100 mM). Two hours after ethanol treat-ment, the cells were washed with ice-cold PBS, and cell lysates wereprepared. Representative Western blots showed Bad phosphorylationat Ser128 in SH-SY5Y cells (C). D: Coimmunoprecipitation of Badwith p-JNK in SH-SY5Y cells. Western blot analyses of immunopre-cipitated anti-p-JNK were performed with the anti-Bad antibody.Densitometry analyses was normalized to IgG bands and representedas arbitrary units (A.U.). This experiment was repeated three timesindependently. Data are presented as mean 6 SEM. *P < 0.05, **P< 0.01.



significantly decreased in the cerebral cortices of etha-nol-treated rats compared with the saline controls (Fig.5A). Bad binds more strongly to Bcl-xL than to Bcl-2 inmammalian cells and reverses the death repressor activityof Bcl-xL (Yang et al., 1995). Therefore, to examine

whether Bad dissociated from 14-3-3 binds to Bcl-xL,Western blot analysis on immunoprecipitated Bcl-xL wasperformed to detect the presence of Bad. Bad binding toBcl-xL was significantly increased in the cerebral corticesof ethanol-treated rats compared with the saline controls(Fig. 5B), which is consistent with previous studies(Siler-Marsiglio et al., 2006; Wang et al., 2007). Bax, aproapoptotic protein of the Bcl-2 family, plays an im-portant role in the regulation of mitochondria-dependentapoptosis (Korsmeyer et al., 2000) and is negativelyregulated by Bcl-xL through its sequestration in thecytoplasm (Yang et al., 1995). Therefore, Bax releasefrom Bcl-xL may enhance mitochondria-dependentapoptosis. As expected, Bax interaction with Bcl-xL wassignificantly decreased in the cerebral cortices ofethanol-treated rats compared with the saline controls(Fig. 5C), suggesting that ethanol activates Bad throughits dissociation from 14-3-3, and then this leads to acti-vation of downstream cell death pathways.

Effects of Ethanol on Caspase-3 Activation andPARP Cleavage

Bax released from Bcl-xL is translocated to mito-chondria, leading to cytochrome c release and caspase-3activation (Datta et al., 2000; Young et al., 2005). Cas-pase-3 is a critical executioner of apoptosis and activatedby proteolytic processing into p17 fragments (D’Melloet al., 2000). Therefore, to determine whether caspase-3is activated after ethanol treatment, Western blot analysison cleaved caspase-3 was performed. The full-length cas-pase-3 (32 kDa) was significantly decreased in the etha-nol-treated rats, whereas the cleaved caspase-3 (17 kDa)was increased compared with the saline controls (Fig.6A), which is consistent with a previous report (Younget al., 2005). Furthermore, caspase-3 is responsible forthe proteolytic cleavage of many key proteins, such asthe nuclear enzyme PARP (Janicke et al., 1998). There-fore, we examined whether PARP was cleaved afterethanol treatment. As expected, PARP cleavage wasincreased 3.4-fold in the ethanol-treated group comparedwith the saline controls (Fig. 6B).

DISCUSSION

Ethanol administration to immature rat pups indu-ces a robust apoptotic cell death through mitochondrialdysfunction (Olney et al., 2002; Young et al., 2005),and this cell death was blocked in homozygous Bax-de-ficient mice, suggesting that ethanol induces cell deathby Bax-induced mitochondrial dysfunction (Younget al., 2003; Nowoslawski et al., 2005). Interactionbetween pro- and antiapoptotic proteins regulates mito-chondria-dependent apoptosis, and the balance betweenpro- and antiapoptotic proteins is an important factorin the ethanol-induced cell death. Also, JNK plays animportant role in ethanol-induced cell death (Lee andShukla, 2005).

In the present study, we found that the 14-3-3interaction with activated JNK was increased in the

Fig. 5. Effect of ethanol on the interaction between pro- and antia-poptotic proteins in rat cerebral cortices. Postnatal 7-day-old rat pupswere treated subcutaneously with ethanol (6 g/kg) or an equal vol-ume of saline. Twelve hours after the first ethanol or saline treat-ment, rat pups were decapitated, and extracts of total protein fromcerebral cortices were prepared. A: Effect of ethanol on 14-3-3finteraction with Bad in rat cerebral cortices. Immunoprecipitationwas performed with an anti-14-3-3f antibody. Western blot analysesof immunoprecipitated 14-3-3f were performed with the anti-Badantibody. B: Effect of ethanol on Bad interaction with Bcl-xL in ratcerebral cortices. Immunoprecipitation was performed with an anti-Bcl-xL antibody. Western blot analyses of immunoprecipitated Bcl-xL were performed with the anti-Bad antibody. C: Effect of ethanolon Bcl-xL interaction with Bax in rat cerebral cortices. Immunopre-cipitation was performed with an anti-Bcl-xL antibody. Western blotanalyses of immunoprecipitated Bcl-xL were performed with theanti-Bax antibody. IgG bands were shown to confirm equality ofantibody loading. Densitometry analyses were normalized to IgGbands and represented as arbitrary units (A.U.). This experiment wasrepeated three times independently. Data (n 5 9) are presented asmean 6 SEM. *P < 0.05, **P < 0.01.

3226 Han et al.


cerebral cortices of ethanol-treated rats. Because JNKactivation is an essential step for ethanol-induced celldeath, we thought that activated JNK might inhibit theantiapoptotic function of 14-3-3. 14-3-3 proteins exerttheir antiapoptotic function by sequestering proapoptoticproteins such as Bad and Bax in the cytoplasm in aninactive state (Nomura et al., 2003; Porter et al., 2006).Bad is dissociated from 14-3-3 in response to apoptoticstimuli, and Bad dephosphorylation precedes this dissoci-ation (Datta et al., 2000). Bad is phosphorylated at

Ser112 and Ser136 by survival factors such as PKA andAKT, respectively (Datta et al., 1997; Harada et al.,1999). Thus, suppression of survival factors woulddecrease Bad phosphorylation followed by Bad dissocia-tion from 14-3-3. In addition, recent studies showedthat antiapoptotic function of 14-3-3 proteins was abol-ished through their phosphorylation by activated JNK(Tsuruta et al., 2004; Sunayama et al., 2005), and 14-3-3phosphorylation and cell death were inhibited by JNKinhibitor (Tsuruta et al., 2004). Thus, 14-3-3 was pro-posed to be a major target of JNK in cell death induc-tion and our results are consistent with these reports(Tsuruta et al., 2004; Sunayama et al., 2005).

Ethanol-induced Bad phosphorylation at Ser128was blocked by JNK inhibitor in the present study, sug-gesting that activated JNK phosphorylates Bad at Ser128and promotes its activation. Recent studies havereported that JNK phosphorylates Bad at Ser128 andpromotes the apoptotic effect of Bad by antagonizingsurvival factor-induced Bad inactivation (Donovan et al.,2002; Wang et al., 2007). Concordantly, we showedthat ethanol suppresses the activation of survival factorsand promotes JNK activation (Han et al., 2006). Thus,our findings suggest that ethanol induces Bad phospho-rylation at distinct sites by regulating survival factors andJNK.

Fig. 7. Schematic diagram of JNK action on neural cell in ethanol-induced cell death. Ethanol administration to immature rats inducesJNK activation. Then, activated JNK induces phosphorylation of 14-3-3 and Bad at Ser128 in a concerted fashion, resulting in Bad disso-ciation from 14-3-3. After Bad release from 14-3-3, the downstreamcell death pathway is activated, leading to cell death.

Fig. 6. Effects of ethanol on caspase-3 activation and PARP cleavagein rat cerebral cortices. Postnatal 7-day-old rat pups were treated sub-cutaneously with ethanol (6 g/kg) or an equal volume of saline.Twelve hours after the first ethanol or saline treatment, rat pups weredecapitated, and total protein from cerebral cortices was extractedand subjected to Western blot analyses with activated caspase-3 (A)and PARP (B). Each lane represents an individual animal; 30 lg oftotal protein was loaded, and a-tubulin was used as an internal con-trol. This experiment was repeated three times independently. Data(n 5 9) are presented as mean 6 SEM. *P < 0.05, **P < 0.01.



It has been reported that activation of JNK is animportant step during ethanol-induced cell death (McAl-hany et al., 2000; Lee and Shukla, 2005; Han et al.,2006). Nevertheless, the role of JNK during ethanol-induced cell death was not clear. In the present study,we found that ethanol induced JNK activation and pro-moted p-JNK interaction with 14-3-3 and Bad, resultingin Bad release from 14-3-3 and activation of subsequentcell death pathway. Additionally, we have shown thatJNK promotes cell death through its binding to Bcl-2(Han et al., 2006). Therefore, we propose that JNK par-ticipates in ethanol-induced cell death by the inhibitionof antiapoptotic proteins as well as activation of proa-poptotic Bad (Fig. 7).

ACKNOWLEDGMENTS

We thanks to Dr. Catherine Rivier and Dr. LeeSoon (Salk Institute) for the kind review and suggestions.

REFERENCES

Aitken A, Collinge DB, van Heusden BP, Isobe T, Roseboom PH,

Rosenfeld G, Soll J. 1992. 14-3-3 proteins: a highly conserved, wide-

spread family of eukaryotic proteins. Trends Biochem Sci 17:498–501.

Chen G, Ma C, Bower KA, Shi X, Ke Z, Luo J. 2008. Ethanol pro-

motes endoplasmic reticulum stress-induced neuronal death: Involve-

ment of oxidative stress. J Neurosci Res 86:937–946.

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg

ME. 1997. Akt phosphorylation of BAD couples survival signals to the

cell-intrinsic death machinery. Cell 91:231–241.

Datta SR, Katsov A, Hu L, Petros A, Fesik SW.Yaffe MB, Greenberg

ME. 2000. 14-3-3 proteins and survival kinases cooperate to inactivate

BAD by BH3 domain phosphorylation. Mol Cell 6:41–51.

D’Mello SR, Kuan CY, Flavell RA, Rakic P. 2000. Caspase-3 is

required for apoptosis-associated DNA fragmentation but not for cell

death in neurons deprived of potassium. J Neurosci Res 59:24–31.

Donovan N, Becker EB, Konishi Y, Bonni A. 2002. JNK phosphoryla-

tion and activation of BAD couples the stress-activated signaling path-

way to the cell death machinery. J Biol Chem 277:40944–40949.

Green DR, Reed JC. 1998. Mitochondria and apoptosis. Science 281:

1309–1312.

Han JY, Joo Y, Kim YS, Lee YK, Kim HJ, Cho GJ, Choi WS, Kang

SS. 2005. Ethanol induces cell death by activating caspase-3 in the rat

cerebral cortex. Mol Cells 20:189–195.

Han JY, Jeong JY, Lee YK, Roh GS, Kim HJ, Kang SS, Cho GJ, Choi

WS. 2006. Suppression of survival kinases and activation of JNK medi-

ate ethanol-induced cell death in the developing rat brain. Neurosci

Lett 398:113–117.

Harada H, Becknell B, Wilm M, Mann M, Huang LJ, Taylor SS, Scott

JD, Korsmeyer SJ. 1999. Phosphorylation and inactivation of BAD by

mitochondria-anchored protein kinase A. Mol Cell 3:413–422.

Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz

K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney

JW. 2000. Ethanol-induced apoptotic neurodegeneration and fetal alco-

hol syndrome. Science 287:1056–1060.

Janicke RU, Ng P, Sprengart ML, Porter AG. 1998. Caspase-3 is

required for alpha-fodrin cleavage but dispensable for cleavage of other

death substrates in apoptosis. J Biol Chem 273:15540–15545.

Jung CH, Hong MH, Kim JH, Lee JY, Ko SG, Cho K, Seog HM.

2007. Protective effect of a phenolic-rich fraction from Schisandra chi-

nensis against H2O2-induced apoptosis in SH-SY5Y cells. J Pharm Phar-

macol 59:455–462.

Kim BJ, Ryu SW, Song BJ. 2006. JNK- and p38 kinase-mediated phos-

phorylation of Bax leads to its activation and mitochondrial transloca-

tion and to apoptosis of human hepatoma HepG2 cells. J Biol Chem

281:21256–21265.

Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH.

2000. Pro-apoptotic cascade activates BID, which oligomerizes BAK or

BAX into pores that result in the release of cytochrome c. Cell Death

Differ 7:1166–1173.

Kuan CY, Yang DD, Samanta Roy DR, Davis RJ, Rakic P, Flavell RA.

1999. The Jnk1 and Jnk2 protein kinases are required for regional spe-

cific apoptosis during early brain development. Neuron 22:667–676.

Lee YJ, Shukla SD. 2005. Pro- and anti-apoptotic roles of c-Jun N-ter-

minal kinase (JNK) in ethanol and acetaldehyde exposed rat hepato-

cytes. Eur J Pharmacol 31:31–45.

Masters SC, Subramanian RR, Truong A, Yang H, Fujii K, Zhang H,

Fu H. 2002. Survival-promoting functions of 14-3-3 proteins. Biochem

Soc Trans 30:360–365.

McAlhany RE Jr, West JR, Miranda RC. 2000. Glial-derived neurotro-

phic factor (GDNF) prevents ethanol-induced apoptosis and JUN ki-

nase phosphorylation. Brain Res Dev Brain Res 119:209–216.

Mitchell JJ, Paiva M, Heaton MB. 1998. Optimal 96-well plate set-up to

avoid ethanol volatility when assessing ethanol cytotoxicity. Alcohol

15:137–139.

Nomura M, Shimizu S, Sugiyama T, Narita M, Ito T, Matsuda H, Tsuji-

moto Y. 2003. 14-3-3 Interacts directly with and negatively regulates

pro-apoptotic Bax. J Biol Chem 278:2058–2065.

Nowoslawski L, Klocke BJ, Roth KA. 2005. Molecular regulation of

acute ethanol-induced neuron apoptosis. J Neuropathol Exp Neurol

64:490–497.

Olney JW, Wozniak DF, Jevtovic-Todorovic V, Farber NB, Bittigau P,

Ikonomidou C. 2002. Drug-induced apoptotic neurodegeneration in

the developing brain. Brain Pathol 12:488–498.

Porter GW, Khuri FR, Fu H. 2006. Dynamic 14-3-3/client protein

interactions integrate survival and apoptotic pathways. Semin Cancer

Biol 16:193–202.

Sakai R, Ukai W, Sohma H, Hashimoto E, Yamamoto M, Ikeda H,

Saito T. 2005. Attenuation of brain derived neurotrophic factor

(BDNF) by ethanol and cytoprotective effect of exogenous BDNF

against ethanol damage in neuronal cells. J Neural Transm 112:1005–

1013.

Schroeter H, Boyd CS, Ahmed R, Spencer JP, Duncan RF, Rice-Evans

C, Cadenas E. 2003. c-Jun N-terminal kinase (JNK)-mediated modula-

tion of brain mitochondria function: new target proteins for JNK sig-

nalling in mitochondrion-dependent apoptosis. Biochem J 372:359–

369.

Siler-Marsiglio KI, Paiva M, Madorsky I, Pan Q, Shaw G, Heaton MB.

2005. Functional mechanisms of apoptosis-related proteins in neonatal

rat cerebellum are differentially influenced by ethanol at postnatal days

4 and 7. J Neurosci Res 81:632–643.

Siler-Marsiglio KI, Madorsky I, Pan Q, Paiva M, Neeley AW, Shaw G,

Heaton MB. 2006. Effects of acute ethanol exposure on regulatory

mechanisms of Bcl-2-associated apoptosis promoter, bad, in neonatal rat

cerebellum: differential effects during vulnerable and resistant develop-

mental periods. Alcohol Clin Exp Res 30:1031–1038.

Sunayama J, Tsuruta F, Masuyama N, Gotoh Y. 2005. JNK antagonizes

Akt-mediated survival signals by phosphorylating 14-3-3. J Cell Biol

170:295–304.

Tsuruta F, Sunayama J, Mori Y, Hattori S, Shimizu S, Tsujimoto Y,

Yoshioka K, Masuyama N, Gotoh Y. 2004. JNK promotes Bax translo-

cation to mitochondria through phosphorylation of 14-3-3 proteins.

EMBO J 23:1889–1899.

Villegas SN, Njaine B, Linden R, Carri NG. 2006. Glial-derived neuro-

trophic factor (GDNF) prevents ethanol (EtOH) induced B92 glial cell

3228 Han et al.


death by both PI3K/AKT and MEK/ERK signaling pathways. Brain

Res Bull 71:116–126.

Wang X. 2001. The expanding role of mitochondria in apoptosis. Genes

Dev 15:2922–2933.

Wang XT, Pei DS, Xu J, Guan QH, Sun YF, Liu XM, Zhang GY.

2007. Opposing effects of Bad phosphorylation at two distinct sites by

Akt1 and JNK1/2 on ischemic brain injury. Cell Signal 19:1844–1856.

Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. 1995.

Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and

promotes cell death. Cell 80:285–291.

Young C, Klocke BJ, Tenkova T, Choi J, Labruyere J, Qin YQ,

Holtzman DM, Roth KA, Olney JW. 2003. Ethanol-induced neuronal

apoptosis in vivo requires BAX in the developing mouse brain. Cell

Death Differ 10:1148–1155.

Young C, Roth KA, Klocke BJ, West T, Holtzman DM, Labruyere J,

Qin YQ, Dikranian K, Olney JW. 2005. Role of caspase-3 in ethanol-

induced developmental neurodegeneration. Neurobiol Dis 20:608–614.

Zhang L, Chen J, Fu H. 1999. Suppression of apoptosis signal-regulating

kinase 1-induced cell death by 14-3-3 proteins. Proc Natl Acad Sci

USA 96:8511–8515.

Zhu Y, Yang GY, Ahlemeyer B, Pang L, Che XM, Culmsee C, Klumpp

S, Krieglstein J. 2002. Transforming growth factor-beta 1 increases bad

phosphorylation and protects neurons against damage. JNeurosci 22:

3898–3909.



c-jun n-terminal kinase regulates the interaction between 14-3-3 and bad in ethanol-induced cell...

Documents