jnk-dependent phosphorylation of c-jun on serine 63 ... · signal transduction pathways, which lead...

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JNK-dependent Phosphorylation of c-Jun on Serine 63 Mediates

Nitric Oxide-induced Apoptosis of Neuroblastoma cells

Lei Li, Zhiwei Feng1 and Alan G. Porter*

Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic

of Singapore

* Corresponding author. Mailing address: 6874-3761 or 6874-3777. Fax: 6779-1117E-mail: [email protected]

1Present address : National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433.

Running title: c-Jun phosphorylation promotes NO-induced apoptosis

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 14, 2003 as Manuscript M310415200 by guest on A

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Summary

c-Jun N-terminal kinases (JNKs) potentiate transcriptional activity of c-Jun by phosphorylating

serines 63 and 73. Moreover, JNK and c-Jun can modulate apoptosis. However, an involvement of

nitric oxide (NO)-induced phosphorylation of c-Jun on Ser-63 and Ser-73 in apoptosis has not been

explored. We report that in SH-Sy5y neuroblastoma cells, NO induced apoptosis following JNK

activation and phosphorylation of c-Jun almost exclusively on Ser-63. Importantly, NO-induced

apoptosis and caspase-3 activity were inhibited in cells stably transformed with dominant-negative

c-Jun in which Ser-63 is mutated to alanine (S63A), but not in cells transformed with dominant-

negative c-Jun (S73A). Ser-63 of c-Jun (but not Ser-73) was required for NO-induced, c-Jun-

dependent transcriptional activity. NO-induced apoptosis, Ser-63 phosphorylation of c-Jun and

caspase-3 activity were all inhibited in SH-Sy5y cells transformed with dominant-negative jnk. A

caspase-3 inhibitor prevented apoptosis but not c-Jun phosphorylation. In a different neuroblastoma

cell line, NO-induced Ser-63 phosphorylation of c-Jun and apoptosis were blocked by a specific

JNK inhibitor. We conclude that NO-inducible apoptosis is mediated by JNK-dependent Ser-63

phosphorylation of c-Jun upstream of caspase-3 activation in neuroblastoma cells.

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Introduction

In the central nervous system, NO generation results in part from successive events of

enhanced glutamate release, NMDA receptor stimulation, Ca2+ influx and NO synthase activation

(1-3). There is much evidence that excessive NO generation during strokes, ischemia or

neurodegenerative diseases contributes to neuronal cell death (4, 5). NO can exert its cytotoxic

effects in diverse cell types via generation of highly reactive free radicals like peroxynitrite, which

damages DNA, proteins and lipids by oxidation (3, 6, 7). Such damage in turn triggers downstream

signal transduction pathways, which lead to apoptosis or necrosis (6). However, the death pathways

that are activated in neurons in response to massive NO production are not well understood. As NO

can stimulate the activity of the transcription factor AP-1 in neurons (8, 9), one such death pathway

might involve the AP-1-dependent regulation of cell death or survival genes.

c-Jun, a prominent member of the AP-1 transcriptional factor family, has been implicated in

the regulation of a wide range of biological processes including apoptosis, which it can promote or

counteract, depending on the tissue, the developmental stage and the nature of the death stimulus

(10, 11). Its transcriptional activities are regulated by changes in the level of c-jun expression as

well as posttranslational modifications of the c-Jun protein. In particular, phosphorylation of Ser-63

and Ser-73 in the N-terminal transactivation domain of c-Jun, which is mediated primarily by the c-

Jun N-terminal kinases (JNKs) (12), substantially enhances the activity of c-Jun as a transcriptional

factor (13, 14). c-Jun N-terminal phosphorylation on Ser-63 and Ser-73 can be either pro- or anti-

apoptotic (15, 16). c-Jun phosphorylation is thought to be required for the anti-apoptotic function of

c-Jun during hepatogenesis (17). The precise role of c-Jun phosphorylation in genotoxin-induced

apoptosis (UV, DNA-damaging agents) is controversial, but c-Jun phosphorylation is pro-apoptotic

in neurons subjected to kainate, a low potassium concentration, or NGF deprivation (15, 18-20).


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Bim, Hrk and Fas ligand are among the proteins whose upregulation upon neurotrophin withdrawal

is transcriptionally controlled, at least in part, by c-Jun phosphorylation (19, 21, 22).

The mitogen-activated protein (MAP) kinases include the JNKs and the p38 MAP kinases,

which are activated by diverse cellular stress including inflammatory cytokines, heat shock and UV

irradiation (10, 23, 24). JNK and p38 activities have been implicated in cell death associated with

glutamate excitotoxicity (25, 26). Two previous reports suggested that NO activates p38 MAP

kinase, triggering significant apoptosis in neuronal cells (27, 28). JNK-mediated c-Jun

phosphorylation is important for apoptosis of starved neuronal cells (19), and the JNK3 isoform is

required for kainate-induced cytotoxicity in the CNS (29). Mouse fibroblasts derived from jnk1 -/-

jnk2 -/- double knock out embryos that lack all JNK activity are less sensitive to apoptosis induced

by UV. The brains of these embryos exhibit altered morphologies due to de-regulated apoptosis,

which surprisingly is increased in some brain regions, but decreased in others (30). Thus, much

evidence suggests that c-Jun phosphorylation is often but not always pro-apoptotic, particularly in

neuronal cells.

We recently reported that in SH-Sy5y cells, the constitutive activity of c-Jun/AP-1 in the

absence of detectable AP-1 DNA-binding is required for the expression of the neural cell adhesion

molecule NCAM140 (31). This basal c-Jun/AP-1-dependent synthesis of NCAM140 counteracts

NO-induced apoptosis. Here, we investigated whether NO induces c-Jun phosphorylation and

regulates apoptosis through the JNK-c-Jun pathway, as do other cellular stressors. Notably, we

found that JNK-dependent phosphorylation of c-Jun on Ser-63 promotes NO-induced apoptosis of

neuroblastoma cells. A speculative model is proposed which can account for the pro- and anti-

apoptotic action of c-Jun/AP-1 within a single neuroblastoma cell.


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EXPERIMENTAL PROCEDURES

Materials and Plasmid Constructions -- The human neuroblastoma cell lines SH-Sy5y and

SHEP were obtained from Eva Feldman (University of Michigan, USA) and from Evelyne Goillot

(Laboratoire d'Immunologie, Centre Leon Berard, Lyon, France), respectively. The polyclonal

antibodies against phospho c-Jun, phospho JNK, c-Jun and JNK were from Cell Signaling

Technology. The antibody against actin was from Santa Cruz Biotechnology, Inc. DEVD-afc and z-

DEVD-fmk were obtained from BACHEM. Lipofectin and opti-MEM were purchased from Life

Technologies, Inc. The cell proliferation reagent, WST-1, was purchased from Roche. The D-TAT

and D-JNKI1 peptides were from Alexis Biochemicals (Switzerland). The dual-luciferase assay kit

and β-gal assay kit were from Promega. All the other reagents used in this research were from

Sigma.

The plasmid encoding dominant-negative c-Jun (denoted JunAA) was obtained from Dan

Mercola (Sidney Kimmel Cancer Center, San Diego, CA, USA). The plasmids bearing the

dominant-negative S63A or S73A mutations in c-Jun were constructed by PCR-based mutagenesis

based on the plasmid JunAA. The primers used were: S63A (F) 5’-GCT CAA GCT GGC GTC

TCC CGA GCT GG-3’; S63A (B) 5’-CCA GCT CGG GAG ACG CCA GCT TGA GC-3’; S73A

(F) 5’-CCT CCT CAC CTC TCC CGA CG-3’; S73A (B) 5’-CGT CGG GAG AGG TGA GGA

GG-3’, respectively. The Gal4-c-Jun transactivator and Gal4-luciferase reporter plasmids were

purchased from Stratagene. The modified Gal4-c-Jun plasmids (S63A or S73A) were constructed

by cloning the transactivation domain of c-Jun (amino acids 1-221) bearing either the S63A or

S73A mutations into the pFA-CMV vector from Stratagene. The pGL3-AP1 reporter plasmid and

RPL-TK plasmid were kindly provided by S. Dhakshinamoorthy (Institute of Molecular and Cell

Biology, Singapore). The plasmid encoding dominant-negative (DN)-JNK1 was provided by Roger


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J. Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School,

Worcester, MA), and the plasmid encoding DN-JNK2 was provided by Dr. Shengcai Lin (Institute

of Molecular and Cell Biology, Singapore).

Cell Culture and transfection -- Both SH-Sy5y and SH-EP cells were maintained in

Dulbecco’s modified medium containing 10% fetal bovine serum, 100 units/ml penicillin and 100

��

proteins, either exogenous or derived from the bovine serum. Normally the cells were split after 48

h in culture. The JunAA, S63A, S73A, DN-jnk1 and DN-jnk2 plasmids were transfected into SH-

Sy5y cells using Lipofectin following the manufacturer’s instructions. The stably transfected cells

�� !"�� #"�$��%��

��&418 (DN-jnk1$��'��()-jnk2).

Reporter Assays -- For the Gal4-c-Jun reporter assay, 50 ng of Gal4-c-Jun activator plasmids

�� !"�� #"�� $�� *� &��+-��*�� *� ,-

galactosidase plasmid were co-transfected into SH-Sy5y cells in 6-well plates (1-2 × 105 cells/plate).

40 hours later, cells were treated with 2 mM SNP for the indicated times. Medium was removed

and cells were washed three times with ice-��-. ��/�� 0�� +��l reporter

� ��**��0��,-�� 1��$��2��3��*��

��*��*��0�� (-

20e luminometer. An aliquot of the same samp��,-galactosidase activity for

normalizing luciferase activity obtained above. For the AP-�� *� �&4"-AP1

reporter plasmid and 10 ng of RPL-TK plasmid were cotransfected in S63A or S73A or vector

control cells in 6-well plates. 40 hours later, cells were treated with 2 mM SNP for the indicated


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times and harvested as described above. Lysates were diluted ten times, and firefly luciferase

��0�� *�� *� *�*� � ��ciferase substrate.

The Renilla luciferase activity, as internal control, was measured by adding Stop&Glo solution in

the same tube.

Cell Death Assays -- To measure cell death by WST-1 or LDH release assay, cells (1-2 ×

104 /well) were plated in 96-well plates and treated with SNP for up to 15 h. WST-1 was added to

the culture medium at a 1:10 dilution and incubated at 37°C for 1 h or till the color of the medium

turned red (incubation time can vary according to the cell number in the culture). The absorbance

was measured at a wavelength of 420 nm. To carry out the LDH release assay, the supernatants of

the cells were collected, and the cell layer was lysed with an equal volume of lysis buffer (DME

plus 0.1% Triton X-100). LDH activity in the supernatant and the lysate was quantitated. The

cytotoxicity was calculated as percentage of LDH release by the ratio of supernatant/ (lysate +

supernatant).

Caspase Activity Assay -- The activity of caspase-3-like proteases was measured using

microtiter plates as described (28, 32) with modifications. After SNP treatment, the cells were lysed

in lysis buffer (20 mM Hepes, pH 7.5, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA,

��5�6&�� $�� –

80°C. The samples were diluted 1:10 with reaction buffer (60 µM fluorogenic substrate DEVD-afc

in 50 mM HEPES, pH 7.4, 1% sucrose, 0.1% CHAPS, 10 mM DTT) in a final volume of 100 µl

and incubated at 37°C for 30 min. Released afc was kinetically measured with a fluorescent

spectrophotometer set at excitation wavelength of 400 nm and emission wavelength of 505 nm. For


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normalization, protein concentrations of the corresponding samples were estimated simultaneously

by using the BCA reagents from Pierce Chemical�/�� 7��$�

× min.

Western Blot Analysis – 105�� 3�� ( ��**��!3�%��5��-

HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue and 50 mM freshly added DTT).

Sonication was needed to shear the genomic DNA and reduce the viscosity of the lysate. The

sonicated lysate was then heated at 99°C for 5 min and subjected to centrifugation at 14,000 rpm for

%��+8/��%��*��9� ( -12% polyacrylamide gels and

transferred onto PVDF membranes (Millipore). Detection of bands was performed using the

Phototope®-HRP Western Blot Detection System (Cell Signaling).

Peptide Inhibition Assay -- The inhibition assay was carried out in SHEP neuroblastoma

cells. D-TAT and D-JNKI1 peptides were added at final concentrations of 20, 50 and 100 uM into

the medium. After 24 h, the medium was refreshed with peptides, and SNP was added at final

concentration of 2 mM. At various times thereafter, cell death and c-Jun phosphorylation were

measured as described above.


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Results

NO induces JNK activation, c-Jun phosphorylation on Ser-63 and apoptosis in SH-Sy5y

cells -- Various NO donors have been widely used to study oxidative stress and cellular responses

by mimicking endogenous NO generation (33). SH-Sy5y neuroblastoma cells are highly sensitive to

cell death induced by various NO donors including SNP at concentrations in the range 0.5-2.5 mM,

and the mode of cell death under these conditions is apoptosis (28, 31, 34). As previously described

(31), a time-dependent increase in cell death was observed beginning around 8 h after addition of

SNP to the SH-Sy5y cells (Fig. 1A), and this increase correlated with the appearance of significant

JNK kinase activity at 6 to 7.5 h after addition of SNP (Fig. 1B, upper panel).

Since JNKs are the main upstream kinases for c-Jun N-terminal phosphorylation (12), we

next tested whether the Ser-63 and Ser-73 residues of c-Jun were phosphorylated following SNP

treatment of SH-Sy5y cells by using phospho Ser-63 and phospho Ser-73 specific antibodies. A

strong and sustained c-Jun phosphorylation on Ser-63 was observed at 6 to 8 h after SNP addition,

whereas phosphorylation on Ser-73 was virtually undetectable (Fig. 1C). In contrast, UV irradiation

of SH-Sy5y cells resulted in similar levels of Ser-63 and Ser-73 phosphorylation (Fig. 1C). Thus,

JNK activation and selective phosphorylation of c-Jun on Ser-63 both occurred around the onset of

NO donor-induced apoptosis. Ser-63 phosphorylation and the death of SH-Sy5y cells both occurred

at 1.5 mM and 2 mM SNP. Concentrations of SNP lower than 1.5 mM neither induced cell death

nor elicited Ser-63 phosphorylation of c-Jun (data not shown), indicating that c-Jun is

phosphorylated only at toxic concentrations of SNP. Excessive concentrations of SNP higher than

2.5 mM resulted in detectable Ser-73 phosphorylation that closely correlated with the onset of

appreciable necrosis, indicating that predominant Ser-63 phosphorylation is an apoptosis-related

phenomenon (data not shown).


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NO-induced apoptosis is blocked in S63A and JunAA stable cells but not in S73A stable cells

-- To investigate whether c-Jun phosphorylation contributes to NO-induced apoptosis, we stably

transfected SH-Sy5y cells with plasmids encoding various dominant negative forms of c-Jun. In one

form, Ser-63 was mutated to alanine (denoted S63A), while in another Ser-73 was mutated to

alanine (denoted S73A). In a third form, both Ser-63 and Ser-73 were mutated to alanines (JunAA),

which compromises the ability of c-Jun to transactivate target genes (15).

To exclude the possibility that highly overexpressed S63A, S73A or JunAA might quench

JNK activity by sequestering JNK in an abortive complex, we chose for further analysis

independent clones in which S63A (Fig. 2A, top panel) or JunAA (Fig. 2A, lower panel) or S73A

(data not shown) are expressed at levels only slightly in excess of endogenous c-Jun. Normal

phosphorylation of endogenous c-Jun on Ser-63 in response to UV was still observed in two

independent clones of S63A stable cells (Fig. 2B); and as expected, the endogenous Ser-73

phosphorylation of c-Jun in response to UV was more intense in S63A stable cells compared with

the vector control cells (Fig. 2C). Analogous results were obtained in UV-treated S73A cells (data

not shown). In addition, NO-induced phosphorylation of endogenous c-Jun on Ser-63 still occurred

in two independent JunAA clones (Fig. 2D). These data indicate that the expression of c-Jun

mutated to S63A and/or S73A did not compromise endogenous JNK activity.

We then compared the sensitivities of the above three different stable cells and vector

control cells to NO donors and UV radiation. At any concentrations of SNP that were sufficient to

induce apoptosis, several independent clones of S63A and JunAA stable cells showed markedly

increased resistance to cell death compared with vector control cells (Fig. 3A). Importantly, various

S73A stable cell lines failed to show resistance to NO compared to vector control cells (Fig. 3A). In

contrast, neither S63A nor S73A stable cells were resistant to UV-induced cell death, whereas


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JunAA cells only showed a marginal increase in resistance to UV (Fig. 3B). These data provide

evidence that Ser-63 phosphorylation of c-Jun is important in NO-induced, but not UV-induced cell

death.

Ser-63 of c-Jun is required for c-Jun- and AP-1-mediated transactivation in response to NO

-- Dual phosphorylation of Ser-63 and Ser-73 has been previously found to lead to c-Jun-dependent

transactivation (14), and accordingly mutation of both serines reduces the ability of c-Jun to

transactivate target genes (15). Because we found NO caused c-Jun phosphorylation predominantly

on Ser-63, and since S63A and JunAA stable cells showed markedly increased resistance to cell

death, we next asked whether Ser-63 phosphorylation alone can potentiate c-Jun and AP-1

transactivation. Using a Gal4-c-Jun reporter system, we found that Gal4-c-Jun (wild type) as well as

Gal4-c-Jun (S73A) were transactivated up to 4-fold in SH-Sy5y cells upon NO stimulation (Fig.

4A). However, Gal4-c-Jun (S63A) and Gal4-c-Jun (JunAA) were completely inactive in

transactivation (Fig. 4A). In parallel experiments, transient transfections with AP-1 reporter

plasmids revealed that NO-induced AP-1 activation of up to ~2.7 fold occurred in SH-Sy5y and

S73A cells, but was absent in S63A cells (Fig. 4B). Thus, our combined data from the c-Jun and

AP-1 reporter assays indicate that the presence of Ser-63 (but not Ser-73) is required for NO-

induced c-Jun/AP-1 transactivation. These results also indicate that S63A and the JunAA constructs

function as dominant-negatives by inhibiting gene transcription mediated by endogenous c-Jun.

Caspase-3 contributes to NO-induced cell death and is inhibited in S63A stable cells --

Caspase-3 was found to be important for NO-induced apoptosis of SH-Sy5y cells, since prevention

of caspase-3 activity by z-DEVD (a selective caspase-3 inhibitor) promoted cell survival (Fig. 5A).


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At z-DEVD concentrations that reduce cell death by 50% (Fig. 5A) and completely inhibit caspase-

3 (Fig. 5B), the levels of NO-induced Ser-63 phosphorylation of c-Jun were similar to those in the

absence of z-DEVD (Fig. 5C), indicating that caspase 3 may act downstream of c-Jun

phosphorylation. Two bands of phospho c-Jun were occasionally observed, (Fig. 5C and Fig. 6) as

has been noted previously (19, 35). We next assayed caspase-3 activity in S63A and S73A stable

cells after NO donor treatment and found that caspase-3 activity was efficiently inhibited in S63A

cells compared to vector control cells, while S73A cells showed similar (or slightly enhanced)

caspase-3 activity under the same conditions (Fig. 5D). Since we showed that caspase-3 contributes

to NO-induced apoptosis of SH-Sy5y cells, these results provide additional evidence that c-Jun

phosphorylation on Ser-63 (but not Ser-73) mediates NO-induced apoptosis, and indicate that

caspase-3 contributes to apoptosis downstream of c-Jun phosphorylation.

Phosphorylation of c-Jun on Ser-63 and apoptosis are blocked in dominant-negative jnk

stable cells -- To provide more direct proof that JNK(s) play a role in c-Jun phosphorylation in

response to NO, we stably transfected SH-Sy5y cells with DN-jnk1 or DN-jnk2 plasmids. At least

two independent clones of both DN-jnk1 and DN-jnk2 stable cells exhibited a greatly diminished or

absent phosphorylation of endogenous c-Jun on Ser-63 (Fig. 6A and B), indicating that JNKs are

responsible for NO donor-stimulated c-Jun phosphorylation in SH-Sy5y cells. Various

independently isolated DN-jnk1 and DN-jnk2 clones also showed increased resistance to apoptosis

at three concentrations of NO donor (Fig. 7A) that was quantitatively similar to that observed in

S63A cells (Fig. 3A). A marked decrease of caspase-3 activity, indicative of increased survival, was

also observed in these DN-jnk1 and DN-jnk2 cells (Fig. 7B). This combined evidence suggests that


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inhibition of JNK leads to enhanced cell survival through the suppression of JNK-dependent Ser-63

phosphorylation of c-Jun and the inhibition of caspase-3.

Evidence that JNK-mediated c-Jun phosphorylation on Ser-63 is a general phenomenon in

NO-induced apoptosis of neuroblastoma cells -- It was important to find out whether JNK-mediated

c-Jun phosphorylation on Ser-63 also plays a general role in NO-induced apoptosis in

neuroblastoma cell lines, and to employ an alternative strategy to block JNK. Using SHEP

neuroblastoma cells (36, 37), we found strong Ser-63 phosphorylation of c-Jun beginning at 8 h

after SNP treatment, whereas Ser-73 phosphorylation was again virtually undetectable (Fig. 8A). As

with SH-Sy5y cells, UV irradiation caused both Ser-63 and Ser-73 phosphorylation of c-Jun in SH-

EP cells (Fig. 8A). A cell-permeable peptide, D-JNKI1 that specifically inhibits JNK activity (38)

effectively blocked both Ser-63 phosphorylation (Fig. 8B) and death of SH-EP cells (Fig. 8C). In

contrast, a control cell-permeable peptide (D-TAT) neither prevented Ser-63 phosphorylation nor

the death of SHEP cells (Fig. 8B and 8C, respectively). These results offer additional evidence that

the JNK family of protein kinases phosphorylates c-Jun in NO-induced apoptosis and argue that

JNK-mediated c-Jun phosphorylation on Ser-63 plays an important general role in triggering the

death of neuroblastoma cells.


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Discussion

Previous reports have indicated that excessive generation of NO might be coupled to the

activation of signal transduction cascades involving stressed-activated protein kinases and

transcription factors. The activation of p38 MAP kinase occurs in NO-donor induced apoptosis of

various neuronal cells (27, 28), which in one case involved p38 acting upstream of Bax to trigger

the intrinsic (mitochondria) death pathway (28). There are many reports that NO can regulate AP-1

in the brain (8, 39), and various other studies have demonstrated that c-Jun/AP-1 can modulate

apoptosis induced by diverse agents (40-44). However, the existence of NO-induced JNK-c-Jun

signaling and subsequent gene regulation in apoptosis has not been explored until now. In neurons,

the JNK-c-Jun pathway is pro-apoptotic during neurotrophin factor withdrawal, kainate treatment,

and potassium deprivation (15, 18, 19). This involves Ser-63 and Ser-73 phosphorylation, and it is

accepted that the transcriptional activation of c-Jun in cell growth and development depends strictly

on the dual phosphorylation of these amino acids (14, 15).

It is, therefore, notable we now show that Ser-63 (but not Ser-73) phosphorylation of c-Jun

mediates NO-induced apoptosis of neuroblastoma cells. Our evidence came from several

complementary lines of investigation. Firstly, NO induced a strong activation of JNK, and only

toxic concentrations of SNP induced phosphorylation of c-Jun on Ser-63 prior to and at the onset of

apoptosis. Secondly, S63A and JunAA stable cells (but not S73A cells) exhibited significantly

increased resistance to apoptosis triggered by NO as measured by cell death and caspase-3 assays.

Moreover, DN-jnk1 and DN-jnk2 stable cells showed increased resistance to NO correlating with

markedly reduced Ser-63 phosphorylation and caspase-3 activation. Our data suggest that JNKs are

primarily responsible for phosphorylating c-Jun on Ser-63, which is further supported by our

unpublished observation that a specific p38 MAP kinase inhibitor failed to block Ser-63


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phosphorylation of c-Jun. Thirdly, in a different approach, a highly specific JNK-inhibitory peptide

blocked both exclusive Ser-63 phosphorylation and NO-induced apoptosis of SHEP neuroblastoma

cells.

Might one or both of the JNKs (1 and 2) directly phosphorylate substrates other than c-Jun

and thereby contribute to apoptosis? Although still controversial, there is evidence that JNK-

mediated phosphorylation of p53, p66shcA or Bcl-2 family members is pro-apoptotic in various

different contexts (45-49). However, we showed here that JNK activity is present at normal levels in

S63A, JunAA and S73A cells, arguing that alternative potential JNK death substrates other than c-

Jun would still be phosphorylated under conditions in which S63A protects from NO killing. Thus,

we believe that Ser-63 of c-Jun is the important target in the NO-inducible killing pathway. It is not

known why NO induces only Ser-63 phosphorylation, but it is worth speculating. There are 10

known isoforms of JNKs, and NO might activate one isoform that only targets Ser-63. Alternatively,

NO might activate a Ser-73 phosphatase; or putative NO-mediated chemical modification (e.g.

nitrosylation) of c-Jun could preferentially block Ser-73 phosphorylation.

What is the pathway by which Ser-63 phosphorylation of c-Jun connects to caspase-3

activation and mediates apoptosis? There are two major possibilities. First, Ser-63 phosphorylation

might activate c-Jun, which then transactivates death genes (or suppresses protective genes). This is

strongly supported by the results of the Gal4-c-Jun and AP-1 reporter assays which show that the

S63A mutation alone abolishes transactivation. These assays also indicate that c-Jun is a crucial

component of an AP-1 complex activated by NO, in agreement with antibody supershift

experiments demonstrating c-Jun protein is abundant in AP-1 complexes after NO stimulation (31).

Speculatively, Ser-63 phosphorylation might regulate a different set of target genes compared with

dual phosphorylated or non-phosphorylated c-Jun, resulting in a shift to pro-cell death gene


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expression. It is worth considering the known c-Jun-regulated genes that are thought to play roles in

neuronal apoptosis (11, 50-54). Among the stronger candidates are hrk, bim and fasl, which are pro-

apoptotic (19, 21, 22, 55-57), but other possible genes are bcl3 and GAP43 (58, 59). The second,

perhaps less likely, possibility is Ser-63 phosphorylation may lead to apoptosis through

transcriptional repression due to actions of c-Jun that suppress or antagonize other transcription

factors (60).

In our previous study, we showed that SH-Sy5y cells expressing a different DN-c-Jun

(TAM-67 in which the transactivation domain of c-Jun is deleted) are more sensitive to NO-induced

apoptosis (31). This is in complete contrast to the S63A, JunAA and DN-jnk constructs used in this

study, all of which render neuroblastoma cells more resistant to apoptosis. It is important to explain

the opposing effects of the dominant-negative c-Jun (TAM-67) and S63A/ JunAA/ DN-jnk

neuroblastoma cells in an attempt to understand the role(s) of c-Jun/AP-1 in NO-induced apoptosis.

A speculative model is presented in Fig. 9 that can account for this apparent paradox. The TAM-67

dominant-negative protein is known to efficiently inhibit general AP-1-mediated transcription (61,

62). TAM-67 cells become more sensitive to NO at least in part through the inhibition of an

important NCAM140-mediated cell survival pathway activated by a constitutive level of c-Jun/AP-

1 function (31) (Fig. 9A). The synthesis of NCAM140 does not presumably require Ser-63

phosphorylation of c-Jun, because it occurs in the absence of NO stimulation, which we showed in

the present study is essential to activate the JNK-phospho-c-Jun pathway. Support for a

phosphorylation-independent function of c-Jun comes from several other directions. For example,

mutant mice in which the c-Jun locus is replaced by JunAA are healthy and fertile, which is in

contrast to the embryonic lethality of Jun-/- mice (15, 63, 64). In addition, JunAA itself can regulate

transcription by acting as a suppresser and antagonizer of other transcription factors (60).


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In contrast to TAM-67 cells (31), we found that the NCAM140 protein is still synthesized at

normal levels in NO-resistant S63A, JunAA and in DN-jnk1 and DN-jnk2 cells (unpublished data).

This indicates the constitutive c-Jun/AP-1-dependent NCAM140 survival pathway is intact in these

cells (Fig. 9B). In other words, the S63A, JunAA and DN-jnk constructs block the pro-apoptotic

JNK-c-Jun pathway without affecting the synthesis of neuroprotective NCAM-140, so the cells are

resistant to apoptosis compared to SH-Sy5y cells and TAM-67 cells (Fig. 9B).

Consistent with our previous and present studies, it was reported that c-Jun can protect

undifferentiated rat PC12 neuronal cells from apoptosis independently of c-Jun phosphorylation, but

in the fully differentiated cells JNK signaling can induce apoptosis and c-Jun mediates this response

(42). However, NO was not one of the apoptosis paradigms used. Together with our earlier report

(31), we now have evidence that c-Jun/AP-1 can fulfill opposite functions in a single

undifferentiated neuroblastoma cell, which we speculate to occur in the following context. A

constitutive or basal activity of c-Jun/AP-1 factor(s) (independent of c-Jun phosphorylation on Ser-

63) is able to counteract relatively low levels of NO - in part through the constant expression of

neuroprotective NCAM140 (31). In contrast, a toxic concentration of NO will lead to c-Jun

phosphorylation on Ser-63 by JNK that triggers apoptosis via yet discovered c-Jun targets.


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Acknowledgements -- We thank Eva Feldman, University of Michigan, for SH-SY5Y cells and

Evelyne Goillot, Laboratoire d'Immunologie, Centre Leon Berard, Lyon, France for SHEP cells. We

are also grateful to Dan Mercola, Sidney Kimmel Cancer Center, San Diego, CA, USA, for the

plasmid encoding JunAA; Roger J. Davis, Howard Hughes Medical Institute, University of

Massachusetts Medical School, Worcester, MA, USA, for the plasmid encoding dominant-negative

JNK1; and Shengcai Lin of our institute for the plasmid encoding dominant-negative JNK2.

Christiane Volbracht and Hannes Hentze are acknowledged for helpful comments, and Hannes

Hentze for assistance with the artwork. This work was generously supported by the Institute of

Molecular and Cell Biology, Singapore through funds made available by A*STAR. A.G.P. is an

adjunct staff member of the Department of Surgery, National University of Singapore.


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References

1. Ankarcrona, M., Dypbukt, J. M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S. A.,

and Nicotera, P. (1995) Neuron 15, 961-973

2. Leist, M. and Nicotera, P. (1998) Exp.Cell Res. 239, 183-201

3. Lipton, S. A. (1999) Cell Death.Differ. 6, 943-951

4. Coyle, J. T. and Puttfarcken, P. (1993) Science 262, 689-695

5. Torreilles, F., Salman-Tabcheh, S., Guerin, M., and Torreilles, J. (1999) Brain Res.Brain

Res.Rev. 30, 153-163

6. Kroncke, K. D., Fehsel, K., and Kolb-Bachofen, V. (1997) Nitric.Oxide. 1, 107-120

7. Schulz, J. B., Matthews, R. T., and Beal, M. F. (1995) Curr.Opin.Neurol. 8, 480-486

8. Bogdan, C. (2001) Trends Cell Biol. 11, 66-75

9. Haby, C., Lisovoski, F., Aunis, D., and Zwiller, J. (1994) J Neurochem. 62, 496-501

10. Leppa, S. and Bohmann, D. (1999) Oncogene 18, 6158-6162

11. Shaulian, E. and Karin, M. (2002) Nat.Cell Biol. 4, E131-E136

12. Morton, S., Davis, R. J., McLaren, A., and Cohen, P. (2003) EMBO J 22, 3876-3886

13. Pulverer, B. J., Kyriakis, J. M., Avruch, J., Nikolakaki, E., and Woodgett, J. R. (1991)

Nature 353, 670-674


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

20

14. Smeal, T., Binetruy, B., Mercola, D. A., Birrer, M., and Karin, M. (1991) Nature 354, 494-

496

15. Behrens, A., Sibilia, M., and Wagner, E. F. (1999) Nat.Genet. 21, 326-329

16. Wisdom, R., Johnson, R. S., and Moore, C. (1999) EMBO J 18, 188-197

17. Ganiatsas, S., Kwee, L., Fujiwara, Y., Perkins, A., Ikeda, T., Labow, M. A., and Zon, L. I.

(1998) Proc.Natl.Acad.Sci.U.S.A 95, 6881-6886

18. Harada, J. and Sugimoto, M. (1999) Jpn.J Pharmacol. 79, 369-378

19. Le Niculescu, H., Bonfoco, E., Kasuya, Y., Claret, F. X., Green, D. R., and Karin, M. (1999)

Mol.Cell Biol. 19, 751-763

20. Yamagishi, S., Yamada, M., Ishikawa, Y., Matsumoto, T., Ikeuchi, T., and Hatanaka, H.

(2001) J Biol.Chem. 276, 5129-5133

21. Harris, C. A. and Johnson, E. M., Jr. (2001) J Biol.Chem. 276, 37754-37760

22. Whitfield, J., Neame, S. J., Paquet, L., Bernard, O., and Ham, J. (2001) Neuron 29, 629-643

23. Canman, C. E. and Kastan, M. B. (1996) Nature 384, 213-214

24. Ichijo, H. (1999) Oncogene 18, 6087-6093

25. Chung, K. C., Shin, S. W., Yoo, M., Lee, M. Y., Lee, H. W., Choe, B. K., and Ahn, Y. S.

(2000) J Neurochem. 75, 9-17


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

21

26. Kawasaki, H., Morooka, T., Shimohama, S., Kimura, J., Hirano, T., Gotoh, Y., and Nishida,

E. (1997) J Biol.Chem. 272, 18518-18521

27. Cheng, A., Chan, S. L., Milhavet, O., Wang, S., and Mattson, M. P. (2001) J Biol.Chem. 276,

43320-43327

28. Ghatan, S., Larner, S., Kinoshita, Y., Hetman, M., Patel, L., Xia, Z., Youle, R. J., and

Morrison, R. S. (2000) J Cell Biol. 150, 335-347

29. Yang, D. D., Kuan, C. Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakic,

P., and Flavell, R. A. (1997) Nature 389, 865-870

30. Kuan, C. Y., Yang, D. D., Samanta Roy, D. R., Davis, R. J., Rakic, P., and Flavell, R. A.

(1999) Neuron 22, 667-676

31. Feng, Z., Li, L., Ng, P. Y., and Porter, A. G. (2002) Mol.Cell Biol. 22, 5357-5366

32. Hentze, H., Schmitz, I., Latta, M., Krueger, A., Krammer, P. H., and Wendel, A. (2002)

J.Biol.Chem. 277, 5588-5595

33. Brune, B., von Knethen, A., and Sandau, K. B. (1999) Cell Death.Differ. 6, 969-975

34. Oh-Hashi, K., Maruyama, W., Yi, H., Takahashi, T., Naoi, M., and Isobe, K. (1999)

Biochem.Biophys.Res.Commun. 263, 504-509

35. Leppa, S., Saffrich, R., Ansorge, W., and Bohmann, D. (1998) EMBO J 17, 4404-4413

36. Baker, D. L., Reddy, U. R., Pleasure, D., Thorpe, C. L., Evans, A. E., Cohen, P. S., and Ross,

A. H. (1989) Cancer Res. 49, 4142-4146


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

22

37. Fulda, S., Susin, S. A., Kroemer, G., and Debatin, K. M. (1998) Cancer Res. 58, 4453-4460

38. Bonny, C., Oberson, A., Negri, S., Sauser, C., and Schorderet, D. F. (2001) Diabetes 50, 77-

82

39. Kataoka, K. and Yanase, H. (1998) Neurosci.Res. 32, 103-117

40. Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R., and Johnson, E. M., Jr. (1994)

J Cell Biol. 127, 1717-1727

41. Kihiko, M. E., Tucker, H. M., Rydel, R. E., and Estus, S. (1999) J Neurochem. 73, 2609-

2612

42. Leppa, S., Eriksson, M., Saffrich, R., Ansorge, W., and Bohmann, D. (2001) Mol.Cell Biol.

21, 4369-4378

43. Potapova, O., Basu, S., Mercola, D., and Holbrook, N. J. (2001) J Biol.Chem. 276, 28546-

28553

44. von Knethen, A., Callsen, D., and Brune, B. (1999) Mol.Biol.Cell 10, 361-372

45. Buschmann, T., Potapova, O., Bar-Shira, A., Ivanov, V. N., Fuchs, S. Y., Henderson, S.,

Fried, V. A., Minamoto, T., Alarcon-Vargas, D., Pincus, M. R., Gaarde, W. A., Holbrook, N.

J., Shiloh, Y., and Ronai, Z. (2001) Mol.Cell Biol. 21, 2743-2754

46. Fan, M., Goodwin, M., Vu, T., Brantley-Finley, C., Gaarde, W. A., and Chambers, T. C.

(2000) J Biol.Chem. 275, 29980-29985


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

23

47. Kharbanda, S., Saxena, S., Yoshida, K., Pandey, P., Kaneki, M., Wang, Q., Cheng, K., Chen,

Y. N., Campbell, A., Sudha, T., Yuan, Z. M., Narula, J., Weichselbaum, R., Nalin, C., and

Kufe, D. (2000) J Biol.Chem. 275, 322-327

48. Le, S., Connors, T. J., and Maroney, A. C. (2001) J Biol.Chem. 276, 48332-48336

49. Yamamoto, K., Ichijo, H., and Korsmeyer, S. J. (1999) Mol.Cell Biol. 19, 8469-8478

50. Mattson, M. P., Culmsee, C., and Yu, Z. F. (2000) Cell Tissue Res. 301, 173-187

51. Pennypacker, K. (1997) Histol.Histopathol. 12, 1125-1133

52. Pennypacker, K. R. (1995) J Fla.Med.Assoc. 82, 551-554

53. Shaulian, E. and Karin, M. (2001) Oncogene 20, 2390-2400

54. Tong, L., Toliver-Kinsky, T., Taglialatela, G., Werrbach-Perez, K., Wood, T., and Perez-

Polo, J. R. (1998) J Neurochem. 71, 447-459

55. Kasibhatla, S., Brunner, T., Genestier, L., Echeverri, F., Mahboubi, A., and Green, D. R.

(1998) Mol.Cell 1, 543-551

56. Kolbus, A., Herr, I., Schreiber, M., Debatin, K. M., Wagner, E. F., and Angel, P. (2000)

Mol.Cell Biol. 20, 575-582

57. Morishima, Y., Gotoh, Y., Zieg, J., Barrett, T., Takano, H., Flavell, R., Davis, R. J.,

Shirasaki, Y., and Greenberg, M. E. (2001) J Neurosci. 21, 7551-7560

58. Haas, C. A., Hollerbach, E., Deller, T., Naumann, T., and Frotscher, M. (2000)

Eur.J.Neurosci. 12, 4233-4242


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

24

59. Rebollo, A., Dumoutier, L., Renauld, J. C., Zaballos, A., Ayllon, V., and Martinez, A. (2000)

Mol.Cell Biol. 20, 3407-3416

60. Herdegen, T. and Waetzig, V. (2001) Oncogene 20, 2424-2437

61. Brown, P. H., Chen, T. K., and Birrer, M. J. (1994) Oncogene 9, 791-799

62. Thompson, E. J., Gupta, A., Stratton, M. S., and Bowden, G. T. (2002) Mol.Carcinog. 35,

157-162

63. Hilberg, F., Aguzzi, A., Howells, N., and Wagner, E. F. (1993) Nature 365, 179-181

64. Johnson, R. S., van Lingen, B., Papaioannou, V. E., and Spiegelman, B. M. (1993) Genes

Dev. 7, 1309-1317


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

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Figure Legends

FIG. 1. JNK activation correlates with c-Jun phosphorylation on Ser-63 in response to NO

during apoptosis in SH-Sy5y cells. (A) Cells were treated with 2 mM SNP for the indicated times

and the percentage of dead cells was determined. Values are the mean and the SD is determined

from three experiments performed in triplicate. (B) Cells were treated with 2 mM SNP for the

indicated times, and total proteins in the cell lysates were separated on 0.1% SDS-12%

polyacrylamide gels. JNK phosphorylation was measured by using an antibody against phospho

JNK. The JNK protein was revealed by Western blotting as a loading control. (C) Cells were

treated with 2 mM SNP for the indicated times, lysed and proteins were separated as described in B.

c-Jun NH2-terminal phosphorylation on Ser-63 and Ser-73 was assessed by using antibodies

specific for phospho Ser-63 or phospho Ser-73. In C, left two lanes, SH-Sy5y cells treated with UV

(100 J/m2) acted as a positive control for dual Ser-63 and Ser-73 phosphorylation. c-Jun protein was

revealed by Western Blotting as a loading control.

FIG. 2. Stable expression of S63A or JunAA does not inhibit endogenous c-Jun

phosphorylation in SH-Sy5y cells. (A) Expression levels of the exogenous c-Jun proteins in the

stable cell lines compared to vector control (V.C.). Upper panel, two clones of S63A. Lower panel,

two clones of JunAA. (B) Two independent clones of S63A stable cell and the vector control (V.C.)

cell were treated with 100 J/m2 UV, and the cells were harvested after 1 h. Total proteins in the cell

lysates were fractionated on 0.1% SDS-12% polyacrylamide gels and subjected to immunoblot

analysis using an antibody against phospho Ser-63. (C) Two independent clones of S63A stable cell

and the vector control cell line (V.C.) were treated with 100 J/m2 UV, and total proteins were

prepared as in B for immunoblot analysis using an antibody against phospho Ser-73. (D) Two


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independent clones of JunAA stable cell and the vector control cell line (V.C.) were treated with 2

mM SNP for 10 hr. Total proteins were prepared as in B for immunoblot analysis using an antibody

against phospho Ser-63. Actin was visualized as a loading control in all three panels.

FIG. 3. Stable expression of S63A and JunAA increases resistance of SH-Sy5y cells to NO. (A)

S63A, S73A and JunAA stable cells and vector control (V.C) cells were treated with SNP for 15 h

at the different indicated concentrations. The percentage of dead cells was measured. Values are the

mean and the SD was determined from three independent clones of S63A, S73A or JunAA each in

triplicate. (B) S63A, S73A and JunAA stable cells and vector control cells (V.C.) were treated with

UV at the different indicated doses. After 24 h, the percentage of dead cells was measured. Values

are the mean and the SD was determined from three independent clones of S63A, S73A or JunAA

each in triplicate.

FIG. 4. Ser-63 of c-Jun is required for c-Jun- and AP-1-mediated transactivation in response

to NO. (A) Different Gal4-c-Jun constructs bearing the unmodified c-Jun sequence, or S63A, S73A

or JunAA mutations in the transactivation region of c-Jun, were cotransfected with a luciferase

�� ,-gal into SH-Sy5y cells. 40 hr later, the cells were treated with 2

�5� )-�*��0��4��*��0�� ,-gal activity

was also measured as an internal control. WT, wild-type. (B) The AP-1 reporter plasmid was

cotransfected with the RPL-TK plasmid into S63A, S73A and vector control (V-AA) stable cells.

40 hr later, the cells were treated with 2 mM SNP for the indicated times and harvested. Firefly

luciferase activity was measured and Renilla luciferase activity was measured as an internal control.


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FIG. 5. Caspase-3 contributes to NO-induced apoptosis of SH-Sy5y cells downstream of c-Jun

phosphorylation. (A) SH-Sy5y cells were treated with 2 mM SNP in the absence or presence of the

indicated concentrations of caspase-3 inhibitor z-DEVD for 15 h, and the percentage of dead cells

was measured. (B) SH- % ��3��5� )-��*�3��5�

z-DEVD for the indicated times, and caspase-3 activity was measured. In A and B, values are the

mean and the SD is determined from three experiments performed in triplicate. (C) SH-Sy5y cells

were treated with 2 mM SNP in the absence or presence of z-DEVD for the indicated times, and

total proteins from the cell lysates were fractionated on 0.1% SDS-12% polyacrylamide gels, then

subjected to Western blot analysis using an antibody against phospho Ser-63. (D) S63A and S73A

stable cells and vector control (V.C) cells were treated with 2 mM SNP for the indicated times and

caspase-3 activity was measured by using a fluorogenic caspase-3 substrate DEVD-afc as described

in “Experimental Procedures”. Values are the mean and the SD is determined from three

independent clones of S63A or S73A each in triplicate.

FIG. 6. Dominant-negative-jnk1 and –jnk2 inhibit NO-induced Ser-63 phosphorylation in SH-

Sy5y cells. Two independent clones of (A) DN-jnk1 and (B) DN-jnk2 stable cells and vector control

cells (V-JNK1 or V-JNK2) were exposed to 2 mM SNP for up to 10 h. Total proteins in the cell

lysates were fractionated on 0.1% SDS-12% polyacrylamide gels, then subjected to immunoblot

analysis using an antibody against phospho Ser-63. c-Jun was visualized as a loading control.

FIG. 7. Dominant-negative-jnk1 and –jnk2 inhibit NO-induced apoptosis and caspase-3

activation in SH-Sy5y cells. (A) DN-jnk2 or DN-jnk1 stable cells and the vector control cells (V-

JNK) were exposed to different concentrations of NO donor SNP for 15 h. The percentage of dead


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cells was measured. (B) DN-jnk2 or DN-jnk1 stable cells and the vector control cells were exposed

to 2 mM SNP for the indicated times, and caspase-3 activity was measured as described in

“Experimental Procedures”. In both A and B, Values are the mean and SD is determined from three

independent clones of DN-jnk2 or DN–jnk1 cells, each in triplicate.

FIG. 8. A specific JNK inhibitor D-JNKI1 blocks c-Jun phosphorylation on Ser-63 and

inhibits apoptosis in SHEP neuroblastoma cells. (A) SHEP cells were treated with 2 mM SNP for

the indicated times. Total proteins in the cell lysates were fractionated on 0.1% SDS-12% SDS-

PAGE and subjected to Western blot analysis using specific antibodies against phospho Ser-63 or

phospho Ser-73. SHEP cells treated with UV (100 J/m2) acted as a positive control for dual Ser-63

and Ser-73 phosphorylation. c-Jun phosphorylation is indicative of JNK activation. (B) SHEP cells

were treated with 2 mM SNP plus different indicated concentrations of a cell-permeable JNK

inhibitory peptide (D-JNKI1) or control peptide (D-TAT) for 10 h. c-Jun phosphorylation on Ser-63

was evaluated as described in A. (C) SHEP cells were treated with 2 mM SNP plus different

indicated concentrations of D-JNKI1 or D-TAT peptides for 15 h, and the percentage of cell death

was measured. Values are the mean and the SD is determined from experiments performed in

triplicate.

FIG. 9. A speculative model of how different dominant-negative forms of c-Jun (TAM-67 and

S63A/ JunAA) have opposite effects on the sensitivity of SH-Sy5y cells to NO. (A) TAM-67

overexpression in SH-Sy5y cells leads to a general suppression of c-Jun/AP-1-dependent

transcription, including protective genes such as NCAM140 (31), regardless of phosphorylation of

c-Jun on Ser-63 and Ser-73. Therefore, TAM-67 cells are sensitized to apoptosis. (B) S63A and


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JunAA only block pro-apoptotic phospho-c-Jun dependent events such as caspase-3 activation,

whereas the phospho c-Jun-independent neuroprotective NCAM140 is still synthesized in the S63A

stable cells. Therefore, S63A and JunAA cells are more resistant to apoptosis than SH-Sy5y cells.

DN-jnk (-1 or -2) inhibits the activity of JNK, leading to the failure to phosphorylate c-Jun, which

therefore has the equivalent effect as S63A and JunAA.


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Fig 1

A

B

C

0 4 6 8 9 10 11

SNP [h]

PhosphoSer-63

PhosphoSer-73

c-Jun

UV

PhosphoJNK

JNK

0 UV 4 6 7 7.5 8 9 10

SNP [h]

0 4 8 120

25

50

75

SNP [h]

Cyt

otox

icit

y[%

]±± ±±

SD


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Fig 2

V.C S63A-6

S63A-8

B

actin

PhosphoSer-63

+ + + [UV]

V.C S63A-6

S63A-8A

c-Jun

c-Jun

AA49

V.C AA62

C

actin

PhosphoSer-73

+ + + [UV]

V.CS63A-6

S63A-8 D

- + - + - + [SNP]

V.C AA62

AA49

actin

PhosphoSer-63

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Fig 3

A

B

0 1.5 2 2.50

25

50

75V.CS63AS73AJunAA

SNP [mM]

Cyt

otox

icity

[%] ±± ±±

SD

0 100 200 3000

25

50

75V.CS63AS73AJunAA

UV [J/m2]

Cyt

otox

icity

[%] ±± ±±

SD


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0 6 8 100

1

2

3V-AAS63A-8S73A-3

AP-1 Reporter Assay

SNP [h]

Fold

Incr

ease

inLu

cife

rase

Act

ivit

y

Fig 4

A

B

0 6 8 100.0

2.5

5.0

7.5WTS63AS73AJunAA

Gal4-c-Jun Reporter Assay

SNP [h]

Fold

Incr

ease

inlu

cife

rase

acti

vity


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Fig 5

A B

C

0 8 10 8 10 SNP [h]

PhosphoSer-63

c-Jun

+ z-DEVD

D

0

25

50

75

SNP

z-DEVD

-

- -

+ +

+

+

+

+

+10µµµµM 20µµµµM 50µµµµM

Cyt

otox

icit

y[%

]±± ±±

SD

0 8 10 120

500

1000

1500V.CS63AS73A

SNP [h]

Cas

pase

3ac

tivi

ty[µµ µµ

U/m

g]±± ±±

SD

0

250

500

750

z-DEVD +-

SNP [h]

+-

0 8 10 8 10

Cas

pase

3ac

tivi

ty(µµ µµ

mol

/mgx

min

)

+


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Fig 6

PhosphoSer-63

c-Jun

A

SNP [h]V-J

NK1

JNK1-3

JNK1-4

0 10 0 10 0 10

PhosphoSer-63

c-Jun

B

JNK2-

1

JNK2-

5

JNK2-

8

V-JNK2

0 10 0 10 0 10 0 10 SNP [h]


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Fig 7

A

B

0 4 8 120

500

1000

1500V-JNKDN-JNK2DN-JNK1

SNP [h]

Cas

pase

3ac

tivi

ty[ µµ µµ

U/m

g]±± ±±

SD

0 1.5 2 2.50

25

50

75

100V-JNKDN-JNK2DN-JNK1

SNP [mM]

Cyt

otox

icit

y[%

]±± ±±

SD


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Fig 8

PhosphoSer-63

PhosphoSer-73

c-Jun

0 4 6 8 10UV

SNP [h]

PhosphoSer-63

c-Jun

20 50 100 20 50 100

D-TAT D-JNKI1

A

B

C

SNP+ + + + +Peptide [µµµµM]0

+

0 20 50 1000

10

20

30

40 D-TATD-JNKI1

peptide [µµµµM]

Cyt

otox

icit

y[%

]±± ±±

SD

-


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TAM-67

NO

JNK

Apoptosis

c-JUN

FIG. 9

DN-jnk

Survival

Basalc-JUN/ AP-1

NCAM140

P

P S63

S63A,JunAA

Caspase-3

TAM-67 cells moresensitive to NO

DN-jnk or S63A cellsmore resistant to NO

?

A B


http://www.jbc.org/

Lei Li, Zhiwei Feng and Alan G. Porterapoptosis of neuroblastoma cells

JNK-dependent phosphorylation of c-Jun on serine 63 mediates nitric oxide-induced

published online November 14, 2003J. Biol. Chem.

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jnk-dependent phosphorylation of c-jun on serine 63 ... · signal transduction pathways, which lead...

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