Biochem. J. (2012) 447, 261–269 (Printed in Great Britain)
261
doi:10.1042/BJ20120598
DJ-1 promotes the proteasomal degradation of Fis1: implications of DJ-1
in neuronal protection
Qiang ZHANG*†, Junbing WU*†, Rong WU*†, Jun MA*†, Guiping DU*†, Renjie JIAO*, Yong TIAN*, Zheng ZHENG‡
and Zengqiang YUAN*1
*State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, †College of Life Sciences, Graduate School of
CAS, Beijing 100049, China, and ‡Beijing Institute of Geriatrics, Xuanwu Hospital of Capital Medical University, Beijing 100853, China
Mutations in DJ-1/PARK7 (Parkinson protein 7) have been
identified as a cause of autosomal-recessive PD (Parkinson’s
disease) and the antioxidant property of DJ-1 has been shown
to be involved in the regulation of mitochondrial function and
neuronal cell survival. In the present study, we first found that
the DJ-1 transgene mitigated MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine)-induced DA (dopamine) neuron cell death
and cell loss. We then observed that the protein levels of DJ-1
were significantly decreased, whereas levels of Fis1 [fission 1
(mitochondrial outer membrane) homologue] were noticeably
increased in the striatum of MPTP-treated mice. In addition to
our identification of RNF5 (RING-finger protein-5) as an E3-
ligase for Fis1 ubiquitination, we demonstrated the involvement
of the DJ-1/Akt/RNF5 signalling pathway in the regulation of
Fis1 proteasomal degradation. In other experiments, we found
that Akt1 enhances the mitochondrial translocation and E3ligase activity of RNF5, leading to Fis1 degradation. Together,
the identification of Fis1 degradation by DJ-1 signalling in the
regulation of oxidative stress-induced neuronal cell death supplies
a novel mechanism of DJ-1 in neuronal protection with the
implication of DJ-1 in a potential therapeutic avenue for PD.
INTRODUCTION
DJ-1, Pink1 [PTEN (phosphatase and tensin homologue deleted
on chromosome 10)-induced putative kinase 1] and Parkin, the
three core PD-related proteins, can form a PPD (Pink1–Parkin–
DJ-1) complex to promote degradation of mis-folded proteins
[22]. The Pink1/Parkin pathway can also regulate mitochondrial
morphology in Drosophila [23].
In mammals, the dynamin-like protein Drp1 (dynamin 1-like)
and the mitochondrial outer membrane protein Fis1 [fission
1 (mitochondrial outer membrane) homologue] participate in
mitochondrial fission or fragmentation. Fis1 interacts with
and recruits Drp1 oligomers to the mitochondria and induces
mitochondrial fragmentation. It has been shown that an increased
level of cellular Fis1 strongly promotes mitochondrial fission and
fragmentation, and Fis1 knockdown induces an elongated/fused
mitochondrial morphology and inhibits apoptosis [24–26]. Fis1
protein levels are significantly increased in AD (Alzheimer’s
disease) and are related to a reduction in mitochondrial number
and abnormal subcellular distribution [27], suggesting that the
Fis1 protein could be involved in oxidative stress-induced
neuronal cell death. Post-translational modifications of Drp1,
including phosphorylation, SUMOylation, S-nitrosylation and
ubiquitination, have been reported [28–31], indicating that
mitochondrial fission is influenced by the cellular signalling
events by a number of signalling pathways. However, in contrast
with Drp1, the molecular mechanism underlying Fis1 protein
regulation remains elusive.
Recently, several groups reported that DJ-1 is involved in the
regulation of mitochondrial morphology and functions in different
PD (Parkinson’s disease) is the most common locomotion
disorder and the second most common neurodegenerative disease.
Clinically, PD patients suffer from resting tremor, rigidity,
bradykinesia and altered gait [1,2]. A severe dopaminergic neuron
loss and a decrease in striatal dopamine levels underlie the motor
deficits observed in PD patients [3,4]. Mutations in the DJ-1
gene [PARK7 (Parkinson protein 7)] cause autosomal-recessive
PD [5,6]. DJ-1 is a chaperone-like protein involved in antioxidant
and transcriptional regulation [7–9].
Previously, it has been reported that overexpression of DJ-1
leads to Akt activation and cell survival in mammalian cells [10].
In neurons, knockdown of DJ-1 by siRNA (short interfering RNA)
resulted in increased apoptosis in response to oxidative stress, ER
(endoplasmic reticulum) stress or proteasome inhibition [9,11].
DJ-1 deficiency leads to hypersensitivity to oxidative stress and
environmental toxins related to PD in both mice and Drosophila
[12–16]. Viral-directed overexpression of DJ-1 in the nigrostriatal
area confers resistance to MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine)-induced dopamine neuron death and rescues
locomotion defects [12,17].
Upon oxidative stress, DJ-1 translocates to the mitochondria,
serving a neuroprotective role [18–20]. Oxidation of Cys106 has
been shown to promote mitochondrial translocation of DJ-1 and
is critical for DJ-1 function in neuronal protection. In addition, the
disease-related Cys106 alanine residue (C106A) mutant is unable
to serve a neuroprotective role [18,21]. It has also been shown that
Key words: DJ-1, fission 1 (mitochondrial outer membrane)
homologue (Fis1), Parkinsonism, signal transduction.
Abbreviations used: DA, dopamine; DN, dominant-negative; Drp1, dynamin-related protein 1; EGF, epidermal growth factor; ER, endoplasmic reticulum;
Fis1, fission 1 (mitochondrial outer membrane) homologue; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HA,
haemagglutinin; HDM2, double minute 2 protein; HEK, human embryonic kidney; MDM2, Mdm2, p53 E3 ubiquitin protein ligase homologue; MEF,
mouse embryonic fibroblast; MITOL, membrane-associated ring finger (C3HC4) 5; MPP + , 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine; NAC, N -acetylcysteine; PD, Parkinson’s disease; PI3K, phosphoinositide 3-kinase; Pink1, PTEN (phosphatase and tensin homologue
deleted on chromosome 10)-induced putative kinase 1; RNAi, RNA interference; RNF5, RING-finger protein-5; shRNA, short hairpin RNA; SNc, substantia
nigra pars compacta; TH, tyrosine hydrolase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling; UPS, ubiquitin–proteasome
system.
1
To whom correspondence should be addressed (email [email protected]).
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Q. Zhang and others
model systems [32–34]. Kamp et al. [34] reported that DJ-1
inhibits alpha-synuclein-induced mitochondrial fragmentation in
Caenorhabditis elegans. Thomas et al. [35] showed that DJ-1
knockdown induces mitochondrial fragmentation. This, along
with other evidence, led to the conclusion that DJ-1 works
parallel to the PINK1/parkin pathway to maintain mitochondrial
function upon oxidative stress, suggesting that DJ-1 may regulate
mitochondrial function independent of PINK/Parkin. In addition,
Aleyasin et al. [36] showed that Akt activation is required
for the protective function of DJ-1 in oxidative stress-induced
cell death. Taken together, these findings suggest that DJ-1/Akt
signalling is involved in neuroprotection through the regulation
of mitochondrial function.
In the present study, we have identified a new link between
PD-related genes and the mitochondrial outer membrane protein.
We observed decreased Fis1 protein levels in MPTP-induced PD
mice and that transgenic DJ-1 attenuates DA (dopaminergic)
neuronal apoptosis induced by MPTP treatment. In a second
line of experiments, we found that DJ-1 activates protein kinase
Akt and promotes RNF5 (RING-finger protein-5)-dependent Fis1
degradation. These results indicate that DJ-1 regulates Fis1 proteasomal degradation in response to oxidative stress with the
implication of a protective role of DJ-1 in neurodegenerative
diseases.
EXPERIMENTAL
Plasmids, RNAi (RNA interference) and antibodies
A polyclonal anti-GFP (green fluorescent protein) antibody
was purchased from Invitrogen. The anti-Myc and anti-HA
(haemagglutinin) antibodies were purchased from Santa Cruz
Biotechnology). The anti-Fis1 and anti-FLAG antibodies were
purchased from Sigma and the anti-Drp1 antibody from BD
Biosciences. The monoclonal anti-DJ-1 antibody was from
Stressgen or self-raised by using the recombinant human DJ1 protein as the antigen (with assistance from the Dr JiangPin Jin laboratory at Wayne State University). The anti-RNF5
antibody, wild-type and C42S RNF5 expression plasmids, and
shRNA (short hairpin RNA) against RNF5 were kindly supplied
by Dr Hong-Bing Shu (Wuhan University, Wuhan, China) [37].
Fis1 was cloned from pcDNA3-Myc-Fis1 and inserted into the
pEGFP-C2 vector. The shRNA targeting sequence for human DJ1 was 5 -GTAAAG TTACAACACACCC-3 [10].
Cell culture and transfection
SH-SY5Y and HEK (human embryonic kidney)-293T cell lines
were cultured in DMEM (Dulbecco’s modified Eagle’s medium)
containing 10 % FBS (fetal bovine serum) in a 5 % CO2
incubator at 37 ◦ C. Transfection was performed according to
the manufacturer’s protocol (LipofectamineTM 2000, Invitrogen).
Briefly, the DNA/LipofectamineTM mixture was added directly
to cells and incubated for 24–48 h for gene expression. For the
RNAi assays, cells were transfected and grown for 72 h before
harvesting.
Immunoprecipitation and Western blotting
Immunoprecipitation and Western blotting were performed as
described previously [38]. Briefly, for immunoprecipitation,
cells were incubated for 15 min in lysis buffer [50 mM Hepes,
1 % Nonidet P40, 0.1 % deoxycholate, 0.05 % SDS, 150 mM
NaCl, 0.1 M NaF, 1 mM EGTA, 10 % glycerol, 1 mM DTT
(dithiothreitol), 1 mM Na3 VO4 , 2 mg/ml aprotinin and 1 mM
c The Authors Journal compilation c 2012 Biochemical Society
PMSF] on ice and then centrifuged for 15 min at 12 000 g at
4 ◦ C. The supernatant was incubated with antibody and Protein
G beads for 2–4 h. The immunoprecipitates were denatured in
loading buffer followed by SDS/PAGE (10 % gel) electrophoresis
and Western blotting.
DJ-1 transgenic mice, the MPTP-induced PD model and cell death
assay
DJ-1 transgenic mice were generated in the animal facility of the
Institute of Biophysics, Chinese Academy of Sciences by using a
CMV (cytomegalovirus) promoter-driven human DJ-1 construct.
The animals were maintained in the Animal Care Facility
of the Institute of Biophysics and all experiments involving
animals were approved by the Institutional Animal Care and Use
Committee of the Institute of Biophysics, Chinese Academy of
Sciences.
For the generation of the PD mouse model, 2-month-old
wild-type and DJ-1 transgenic male mice were injected with
five doses of MPTP (30 mg/kg, intraperitoneal every 24 h)
or normal salting. At 5 days after the injection, mice were
anaesthetized and perfused with normal salting followed by 4 %
paraformaldehyde/PBS (pH 7.4). The brains were removed and
post-fixed 3 days in 4 % paraformaldehyde. After this, paraffinembedded 5 μm serial coronal sections were cut throughout the
brain including the substantia nigra.
TUNEL (terminal deoxynucleotidyltransferase-mediated
dUTP nick-end labelling) staining was performed using
the ApopTag® Flurescein In Situ Apoptosis Detection kit
(Chemicon). Briefly, the sections were deparaffinized, then
pre-treated with proteinase K (20 μg/ml) for 15 min at room
temperature (25 ◦ C). After washing with PBS twice for 5 min
each time, the sections were incubated with equilibration buffer
(90416, Millipore) for at least 10 s at room temperature. Then, the
sections were incubated with the working strength TdT (Terminal
deoxynucleotidyl Transferase) enzyme in a humidified chamber
at 37 ◦ C for 1 h. After the incubation, the sections were washed
with PBS three times (1 min each). The sections were then
incubated with working strength anti-digoxigenin conjugate in a
humidified chamber for 30 min at room temperature (avoiding
exposure to light). After washing with PBS four times for 2 min
each time, the sample DNA was labelled using Hoechst 33258.
To count the TH + (tyrosine hydrolase) cells in the SNc
(substantia nigra pars compacta), mice were anaesthetized deeply
with pentobarbital (135 mg/kg of body weight) and transcardially
perfused with PBS followed by 4 % paraformaldehyde. The
brains were post-fixed and around 20 sections were collected
serially crossing the SNc. Sections were incubated overnight
at 4 ◦ C in anti-TH antibody (dilution 1:100; Cell Signaling
Technology) followed by a room temperature incubation with
goat anti-(mouse IgG), diluted 1:200 for 2 h. Sections were then
incubated with DAB (diaminobenzidine). TH-immunopositive
neurons were counted in the serial sections (every second section
for a total of 10 sections) of the SNc.
Statistical analysis
Statistical analysis of the data was performed with a twotailed Student’s t test or one-way ANOVA followed by Fisher’s
PLSD (partial least-squares difference) post-hoc test using Origin
software (Version 8). The data are presented as means +
− S.E.M.
except for analyses of luciferase assays where means +
− S.D. are
shown and the number of experiments are indicated in each
Figure. *P < 0.05, **P < 0.01 or ***P < 0.001 denotes statistical
significance.
DJ-1-mediated signalling in the regulation of Fis1
Figure 1
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DJ-1 transgene protects cells from MPTP-induced DA neuronal cell death and cell loss
(A and B) Brain sections from wild-type (WT) and DJ-1 transgenic mice (TG) treated with normal salting (N.S.) or MPTP were analysed by TUNEL assay. (B) DJ-1 transgene prevents MPTP-induced
DA neuronal apoptosis (**P < 0.01 using one-way ANOVA, n = 3). (C and D) DA neurons were stained in the substantia nigra with the anti-TH antibody. TH-immunopositive cells was measured
as described in the Experimental section. The genotyping data of DJ-1 transgenic mice are shown in the right-hand panel of (C). The primers used for genotyping were: human DJ-1 forward,
5 -taccaggaggtaatctgggcgc-3 and human DJ-1 reverse, 5 -gtctttaagaacaagtggagcc-3 . (D) MPTP significantly induced DA neuron loss (**P < 0.01 using one-way ANOVA, n = 3), and the DJ-1
transgene mitigated the MPTP-induced neuronal cell loss (**P < 0.01 using one-way ANOVA, n = 3). (E) Lysates of nigrostriatal tissue from DJ-1 transgenic or wild-type mice were immunoblotted
with anti-Fis1, anti-Drp1, anti-DJ-1 (self-raised monoclonal antibody) or anti-actin antibodies. Fis1 protein levels decreased in DJ-1 mice compared with the wild-type mice. (F) Lysates of
nigrostriatal tissue from DJ-1 transgenic or wild-type mice with or without MPTP treatment were immunoblotted with anti-Fis1, anti-DJ-1 (self-raised monoclonal antibody), anti-FLAG or anti-GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) antibodies as indicated. MPTP treatment reduces DJ-1 protein levels and DJ-1 transgene can attenuate the rise of Fis1 protein levels induced by MPTP
treatment.
RESULTS
The DJ-1 transgene protects DA neurons from MPTP-induced
apoptosis and DA neuron loss
It has been extensively reported that DJ-1 has a neuroprotective
function in neurons [12,17]. Consistently, we observed that the
DJ-1 transgene significantly inhibited MPTP-induced DA neuron
apoptosis in mice by using a TUNEL assay (Figures 1A and
1B). Consistent with the results of the TUNEL assays, we found
that transgenic DJ-1 mitigated MPTP-induced DA neuronal loss
by staining DA neurons in the substantia nigra with the antiTH antibody (Figures 1C and 1D). Together, we reported that
the DJ-1 transgene protects DA neuronal apoptosis and cell
loss in MPTP-induced PD mice. Interestingly, we found that
the Fis1 protein level in substantia nigra decreased in DJ-1
transgenic mice (Figure 1E). In addition, we observed that
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Figure 2
Q. Zhang and others
DJ-1 expression promotes Fis1 degradation
(A) Lysates of MEFs from wild-type (WT) or DJ-1 transgenic (TG) mice were subjected to immunoblotting with antibodies against Fis1, Drp1, phospho-Ser473 Akt1 (p-Akt1), Akt1, DJ-1 or GAPDH.
MEFs from DJ-1 transgenic mice exhibited higher levels of Akt1 Ser473 phosphorylation and decreased Fis1 protein levels compared with the wild-type mice. (B) Lysates of HEK-293T cells transfected
with DJ-1 shRNA or its control vector were immunoblotted with antibodies against Fis1, Drp1, DJ-1 (Stressgen) or actin. Knockdown of DJ-1 increases Fis1, but not Drp1, protein levels. Molecular
mass is shown on the left-hand side in kDa. (C) Reverse transcripts of the total RNA extracted from SHSY-5Y cells, which were stably transfected with sh-DJ-1 or the control vector, were followed by
a real-time PCR assay. The real-time primers for Fis1 were: 5F, 5 -CACGCAGTTTGAGTACGCCT-3 and 3R, 5 -CCGCTGTTCCTCCTTGCTC-3 . RLU, relative light unit.
the DJ-1 transgene attenuates MPTP-induced up-regulation
of the levels of Fis1 protein in the striatum of the PD model
(Figure 1F) and MPTP treatment reduces levels of the DJ-1 protein
in mice (Figure 1F). Consistently, we found that MPP + (1methyl-4-phenylpyridinium) could increase Fis1 protein level in
HT22 cells and DJ-1 overexpression attenuates MPP + -induced
up-regulation of Fis1 protein levels (Supplementary Figure S1
at http://www.BiochemJ.org/bj/447/bj4470261add.htm). Taken
together, our results suggest that exogenous expression of DJ-1 in
mice regulates Fis1 protein levels and DJ-1 transgene attenuates
MPTP-induced DA neuronal apoptosis and cell loss.
DJ-1 regulates Fis1 protein levels
Consistent with the results that MPTP could reduce the DJ-1
protein level in the murine striatums (Figure 1F), we also observed
lower expression levels of Fis1 protein in DJ-1 transgenic
MEF (mouse embryonic fibroblast) cells compared with the
control MEFs (Figure 2A), indicating that DJ-1 down-regulates
Fis1 expression in both neuronal cells and other cell types.
The expression of DJ-1 RNAi effectively reduced the protein
level of DJ-1 in HEK-293T cells and DJ-1 knockdown increased
Fis1 protein levels (Figure 2B, and Supplementary Figures S2A
and S2B at http://www.BiochemJ.org/bj/447/bj4470261add.htm).
Interestingly, we observed that DJ-1 up-regulates the mRNA level
of Fis1 (Figure 2C). It has been shown that Fis1 protein levels
are changed in neurodegenerative diseases [27], and that DJ-1
promotes the degradation of mis-folded proteins [22]. Together,
these findings suggest that DJ-1 regulates Fis1 protein levels. It
has been shown that Fis1 is a major regulator of mitochondrial
dynamics [24]. We also observed that overexpression of Fis1induced mitochondrial fragmentation in HT22 cells and wildtype DJ-1, but not the C106A mutant, blocks Fis1-induced
mitochondrial fragmentation (Supplementary Figure S3 at
http://www.BiochemJ.org/bj/447/bj4470261add.htm).
DJ-1 regulates Fis1 protein levels through the PI3K
(phosphoinositide 3-kinase)/Akt signalling pathway
DJ-1 has been shown to activate PI3K/Akt signalling and
promote cell survival [10]. We next confirmed that DJ-1 functions
as the activator of PI3K/Akt signalling. DJ-1 overexpression
increased Akt1 phosphorylation at Ser473 under both starvation
c The Authors Journal compilation c 2012 Biochemical Society
and EGF (epidermal growth factor) treatment conditions,
whereas LY294002, a specific PI3K inhibitor, inhibited DJ-1induced PI3K/Akt signalling activation as indicated by the
phosphorylation of Akt1 at Ser473 (Figure 3A). Consistently,
knockdown of DJ-1 alleviates EGF-induced Akt1 activation
(Figure 3B). We also found that C106A mutant DJ-1 failed to
activate Akt (Figure 3C), indicating that the anti-oxidation activity
of DJ-1 is required for PI3K/Akt signalling activation. Taken
together, this suggests that DJ-1 activates PI3K/Akt signalling.
Additionally, we found that expression of wild-type
DJ-1 decreased Fis1 protein levels and the expression of the DJ-1
C106A mutant failed to reduce Fis1 levels (Figure 3D). This
raises the question, does PI3K/Akt signalling involve the DJ1/Fis1 pathway? In response, we observed that expression of
DN (dominant-negative) Akt1 significantly blocks DJ-1-induced
Fis1 protein level reduction (Figure 3E). We also observed DJ1 deficiency induces increased Fis1 protein levels, which can
be rescued by NAC (N-acetylcysteine) in the DJ-1 knockdown
SH-SY5Y stable cell line (Figure 3F). Together, these results
suggest that DJ-1/Akt signalling regulates Fis1 protein and the
anti-oxidation activity of DJ-1 is required for Akt activation and
Fis1 degradation.
RNF5 is the E3 ligase for Fis1 degradation
RNF5 [also known as RMA1 (RING finger protein with
membrane anchor 1)] has been reported to regulate a variety
of biological processes, including protein quality control in
the ER, cell movement, tumorigenesis and myopathological
degeneration [39,40]. Previously, RNF5 has been shown to be
involved in the negative regulation of virus-triggered signalling
by targeting STING {stimulator of interferon genes; also known
as MITA [mediator of IRF3 (interferon regulatory factor 3)
activation] (mitochondrial membrane adaptor protein)} and
MAVS [mitochondrial antiviral signalling protein; also known as
VISA (virus-induced signalling adaptor)] for ubiquitination and
degradation at the mitochondria [37,41]. As a putative E3 ligase,
there are very few reported substrates, especially mitochondriarelated proteins. In our experiments, we found that DJ-1 induced
Fis1 protein degradation. We then asked whether mitochondrialocalized RNF5 could function as an E3 ligase for Fis1
degradation. As expected, we observed that expression of wildtype RNF5 markedly induced Fis1 degradation, whereas the C42S
RNF5 mutant lacking E3 ligase activity [37] greatly reduced Fis1
DJ-1-mediated signalling in the regulation of Fis1
Figure 3
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DJ-1 activates the PI3K/Akt signalling pathway
(A) Lysates of COS7 cells transfected with a plasmid expressing Akt1 together with the DJ-1 plasmid or its control vector, were starved and treated with EGF and/or LY294002 (LY), subjected to
immunoprecipitation (IP) with an anti-HA antibody and then immunoblotted with an antibody against phospho-Ser473 Akt1 (p-Ser473 Akt) or Akt1. The ratio of phospho-Ser473 Akt1/total Akt1 is
quantified and showed in the lower panel (**P < 0.01 using one-way ANOVA, n = 3). DJ-1 activated basal Akt1 and enhanced EGF-induced Akt1 activation. (B) HA-immunoprecipitates of lysates of
HEK-293T cells transfected with HA–Akt1 and DJ-1 RNAi or the control vector were immunoblotted with an anti-(phospho-Ser473 Akt1) antibody. Input were immunoblotted with anti-(phospho-Ser473
Akt1), anti-HA–Akt1, anti-DJ-1 or anti-GAPDH antibodies. The last two samples were treated with EGF for 1 h before harvest. The amount of phospho-Ser473 Akt1 decreased upon DJ-1 knockdown.
The ratio of phospho-Ser473 Akt1/total Akt1 was quantified and is shown in the lower panel (**P < 0.01 using one-way ANOVA, n = 3). (C) Lysates of HeLa cells transfected with wild-type (WT) or
C106A DJ-1 plasmids or its control vector, followed by treatment with EGF, were immunoblotted with anti-Akt1 or anti-(phospho-Ser473 Akt1) antibodies. Wild-type, but not the C106A mutant, DJ-1
activates Akt1 and enhances EGF-induced Akt1 activation. (D) Lysates of HEK-293T cells transfected with Fis1 together with wild-type DJ-1, DJ-1 C106A or the control vector were immunoblotted
with antibodies against GFP or actin. Wild-type, but not the C106A mutant, DJ-1 reduced Fis1 protein levels. (E) Lysates of cells transfected with Fis1 together with FLAG–DJ-1 and DN Akt1 or
the control vector were immunoblotted with anti-GFP–Fis1, anti-FLAG–DJ-1 anti-HA–Akt1 or anti-actin antibodies. DN Akt1 blocks DJ-1-induced reduction of the Fis1 protein level. The ratio of
GFP–Fis1/actin was quantified and is shown in the lower panel (**P < 0.01 using one-way ANOVA, n = 3). (F) Lysates of SH-SY5Y cells stably transfected with DJ-1 shRNA or control vector were
immunoblotted with antibodies against Fis1, DJ-1, Akt1, phospho-Ser473 Akt1 or GAPDH. Cells were incubated with NAC (1 mM) for 48 h before harvest. Knockdown of DJ-1 increases Fis1 protein
levels and NAC treatment reduced Fis1 protein levels induced by DJ-1 deficiency. The ratio of Fis1/GAPDH was quantified and is shown in the lower panel (***P < 0.001 using one-way ANOVA,
n = 3).
degradation (Figure 4A). In vivo ubiquitination assays showed
that the ubiquitination of Fis1 was increased when co-transfected
with wild-type, but not C42S, RNF5 (Figure 4B), suggesting
that RNF5 is an E3 ligase for Fis1 ubiquitination. Consistent
with the finding that RNF5 expression induced Fis1 degradation,
RNF5 knockdown using RNF5 RNAi increased Fis1 protein levels
(Figure 4C). In other experiments, we found that DJ-1 can enhance
the interaction between RNF5 and Fis1 (Supplementary Figure
S4A at http://www.BiochemJ.org/bj/447/bj4470261add.htm) and
increased the auto-ubiquitination of RNF5 (Figure S4B),
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Q. Zhang and others
Figure 4 RNF5 is the E3 ligase of Fis1 and mediates DJ-1 induced Fis1
degradation
(A) Lysates of HEK-293T cells transfected with Fis1 together with wild-type (WT) RNF5,
RNF5-C42S or the control vector were immunoblotted with antibodies against Myc (Fis1),
GFP–RNF5 or actin. RNF5 expression induced Fis1 degradation. (B) GFP immunoprecipitates
(IP) of lysates of HEK-293T cells transfected with Fis1 and HA–ubiquitin plasmids together with
RNF5, RNF5-C42S or the control vector, and immunoblotted with an anti-HA antibody. Cells
were treated with 10 μM MG132 for 3 h before harvest. Expression of RNF5 increased Fis1
ubiquitination. (C) Lysates of HeLa cells transfected with RNF5 shRNA or control plasmids were
immunoblotted with anti-Fis1, anti-DJ-1, anti-RNF5 or anti-GAPDH antibodies. Knockdown of
RNF5 led to an increase in Fis1 protein levels. Molecular mass is shown on the left-hand side
in kDa.
suggesting that DJ-1 may enhance RNF5 E3 ligase activity. Taken
together, we conclude that RNF5 functions as an E3 ligase in DJ1-mediated Fis1 degradation.
Akt/RNF5 signalling regulates the ubiquitination of Fis1
To determine the importance of Akt activation in RNF5mediated Fis1 ubiquitination, we expressed Akt1 together with
RNF5 and Fis1. We found that expression of Akt1 increased
RNF5-directed Fis1 degradation and MG132 treatment rescued
Akt1-mediated reduction of Fis1 protein levels (Figure 5A).
However, we expressed C42S mutant RNF5 with Akt1 and
found that RNF5 C42S abolished Akt1-induced Fis1 degradation
(Figure 5B), indicating that RNF5 acts downstream of Akt in
the regulation of Fis1 degradation. Consistently, the expression
of wild-type Akt1, but not DN Akt1, increased the interaction
c The Authors Journal compilation c 2012 Biochemical Society
Figure 5 DJ-1/Akt1 signalling functions as upstream of RNF5 in the Fis1
degradation pathway
(A) Lysates of HEK-293T cells transfected with RNF5 and Fis1 plasmids, together with Akt1
or control vector, were immunoblotted with anti-GFP–Fis1, anti-FLAG–RNF5, anti-HA–Akt1 or
anti-actin antibodies. Expression of Akt1 promoted RNF5 and Fis1 degradation. (B) Lysates of
HEK-293T cells transfected with plasmids of GFP–Fis1 together with Akt1 and RNF5-C42S or
the control vector were immunoblotted with an anti-GFP–Fis1 antibody. RNF5 C42S blocked
Akt1-induced Fis1 degradation. (C) GFP-immunoprecipitates (IP) of lysates of HEK-293T cells
transfected with Myc–Fis1 and RNF5 plasmids, together with wild-type (WT) or DN Akt1 or the
control vector after immunoblotting with an anti-Myc–Fis1 antibody. Cells were treated with
10 μM MG132 for 3 h before harvesting. Expression of Akt increased the interaction between
RNF5 and Fis1. Molecular mass is shown on the left-hand side in kDa.
between Fis1 and RNF5 (Figure 5C). In other experiments, we
observed that DN Akt1 blocked the auto-ubiqutination of RNF5
(Supplementary Figure S5 at http://www.BiochemJ.org/bj/447/
bj4470261add.htm).
Since PI3K/Akt signalling has been reported to activate several
E3 ligases, including MDM2 (Mdm2, p53 E3 ubiquitin protein
ligase homologue)/HDM2 (double minute 2 protein) [41] and
Nedd4 (neural-precursor-cell-expressed developmentally downregulated 4) [42] through phosphorylation, we sought to examine
the possibility that RNF5 is a downstream target of Akt signalling.
We did not observe RNF5 phosphorylation by Akt1 kinase with
in vitro kinase assays (results not shown). However, we found that
expression of wild-type, but not DN Akt1, dramatically enhanced
RNF5 mitochondrial translocation (Figure 6), suggesting Akt may
DJ-1-mediated signalling in the regulation of Fis1
Figure 6
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Akt1 increases RNF5 mitochondrial translocation
(A) GFP–RNF5 and mito-Red were transfected together with Akt1 or DN Akt1 or the control vector in HeLa cells followed by fluorescent microscopy analysis. Akt1 increased the mitochondrial
localization of RNF5. (B) Cytoplasmic (Cyto) and mitochondrial (Mito) fractions from the HeLa cells transfected with GFP–RNF5 together with Akt1 or DN Akt1 or the control vector (Ctrl) were
immunoblotted with anti-GFP, anti-HA or the indicated antibodies. Wild-type (WT) Akt1 increased the mitochondrial abundance of RNF5. The ratio of GFP–RNF5/GAPDH or Tom20 (translocase of
outer mitochondrial membrane 20 homologue) was quantified and is shown in the lower panel (***P < 0.001 using one-way ANOVA, n = 3).
regulate Fis1 degradation through increasing the mitochondrial
localization of RNF5 and the interaction between RNF5
and Fis1 proteins. In another line of experiments, we
observed that wild-type, but not C106A, DJ-1 increases the
mitochondrial localization of RNF5 (Supplementary Figure S6A
at http://www.BiochemJ.org/bj/447/bj4470261add.htm). In addition, C106A DJ-1 compromises Akt1 expression-mediated RNF5
mitochondrial translocation (Supplementary Figure S6B). Taken
together, these results indicate that Akt1 activation is critical for
RNF5-mediated Fis1 ubiquitination and that RNF5 acts downstream of DJ-1/Akt signalling in the regulation of Fis1 protein.
DISCUSSION
In the present study, we have discovered DJ-1 promotes RNF5mediated Fis1 proteasomal degradation by activating PI3K/Akt
signalling and inhibits Fis1-induced mitochondrial fragmentation
and neuronal cell death (Supplementary Figure S7). Moreover,
we have established the physiological significance of these
findings using transgenic mice. The mice expressing DJ-1 are
protected from oxidative stress-induced DA neuronal apoptosis
(Figures 1A–1D).
Multiple lines of evidence suggest that mitochondrial
dysfunction plays a critical role in neurodegenerative diseases. It
has been reported that mitochondrial morphology and function
are dynamically regulated by a family of proteins, including
Drp1 and Fis1, which promote mitochondrial fission/
fragmentation, and OPA1 [optic atrophy 1 (autosomal dominant)]
and MFN1/2 (mitofusin1/2), which promote mitochondrial
fusion [43]. PD-related proteins, such as DJ-1, Parkin, Pink1
and LRRK2 (leucine-rich repeat kinase 2), interact with
mitochondria. Interestingly, mutations of these proteins associated
c The Authors Journal compilation c 2012 Biochemical Society
268
Q. Zhang and others
with pathological conditions have been linked to mitochondrial
dysfunction and Parkinsonism [44,45]. In the present study, we
show that DJ-1 promotes mitochondrial outer membrane protein
Fis1 degradation in the MPTP-induced PD model. These results
provide new evidence that DJ-1 protects neurons from oxidation
through direct regulation of mitochondrial protein Fis1. An
important question for future studies is whether other PD-related
proteins that are involved in the regulation of mitochondrial
morphology act through Fis1.
The UPS (ubiquitin–proteasome system) has been shown to be
responsible for the removal of mis-folded proteins from various
subcellular compartments [46]. Previously, UPS-dependent
degradation of mitochondrial regulators has been documented
[47]. For example, an E3 ligase called MITOL (mitochondrial
ubiquitin ligase) or MARCH5 [membrane-associated ring finger
(C3HC4) 5], is a membrane-bound protein that has been shown
to regulate mitochondrial fission through ubiquitination of the
mitochondrial fission-related proteins, Fis1 and Drp1 [47]. The
RING finger ubiquitin ligase RNF5 is conserved from worms to
humans and has been shown to be involved in muscle physiology,
cell adhesion and motility, innate immunity, and protein quality
control [48,49]. RNF5 has been identified as an ER-bound E3
ligase and is also localized in the mitochondria [37,49]. Similar
to MITOL [47], RNF5 knockdown in the present study induced
mitochondrial fission, indicating that mitochondrial morphology
may be dynamically regulated by more than one E3 ligase. In the
present study we identified RNF5 as a mitochondrial E3 ligase for
Fis1, which functions as a downstream target of Akt signalling.
Our findings also revealed that PI3K/Akt enhances RNF5
ligase activity and increased RNF5-mediated Fis1 degradation.
Unlike MDM2 or HDM2, that are regulated by Akt1 through
phosphorylation, we failed to detect RNF5 phosphorylation
with in vitro Akt kinase assays. However, we observed that
Akt1 promoted RNF5 mitochondrial translocation (Figure 6),
suggesting that Akt1 regulates RNF5 function by influencing the
localization of RNF5 and the interaction with its substrate.
In conclusion, elucidation of DJ-1 signalling as a key mediator
of the regulation of Fis1 protein levels and DA neuron survival
raises the important question of whether activation of DJ-1
signalling may provide a potential therapeutic avenue for
neurodegenerative diseases.
AUTHOR CONTRIBUTION
Qiang Zhang, Junbing Wu and Jun Ma performed the biochemistry experiments; Rong
Wu, Yong Tian and Guiping Du performed the animal experiments; Zengqiang Yuan wrote
the paper with assistance from Qiang Zhang, Renjie Jiao and Zheng Zheng; and Zengqiang
Yuan supervised the project.
ACKNOWLEDGEMENTS
We thank Dr J. Ren for helpful discussions and Dr Joy Fleming and members of the Yuan
laboratory for critical reading of the paper prior to submission and helpful discussions.
We thank Dr Jiang-Pin Jin from for the help in the production of anti-DJ-1 monoclonal
antibody. We also thank the Pathology Facility at the Institute of Biophysics; Dr M.R.
Cookson for the expression plasmids of WT and C106A DJ-1; Dr Hong-Bing Shu for
providing RNF5 expression plasmids, shRNA and antibodies; Dr J. Wu for providing
HT-22 cells; and Dr Q. Chen for providing Fis1 plasmid and antibody.
FUNDING
This work was supported by the National Science Foundation of China [grant numbers
81125010 and 81030025 (to Z.Y.)] and the Ministry of Science and Technology of China
[grant numbers 973-2009CB918704 and 973-2012CB910701 (to Z.Y.)].
c The Authors Journal compilation c 2012 Biochemical Society
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Received 11 April 2012/6 August 2012; accepted 8 August 2012
Published as BJ Immediate Publication 8 August 2012, doi:10.1042/BJ20120598
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 447, 261–269 (Printed in Great Britain)
doi:10.1042/BJ20120598
SUPPLEMENTARY ONLINE DATA
DJ-1 promotes the proteasomal degradation of Fis1: implications of DJ-1
in neuronal protection
Qiang ZHANG*†, Junbing WU*†, Rong WU*†, Jun MA*†, Guiping DU*†, Renjie JIAO*, Yong TIAN*, Zheng ZHENG‡
and Zengqiang YUAN*1
*State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China, †College of Life Sciences, Graduate School of
CAS, Beijing 100049, China, and ‡Beijing Institute of Geriatrics, Xuanwu Hospital of Capital Medical University, Beijing 100853, China
EXPERIMENTAL
Mitochondrial morphology analysis
We imaged mitochondria with the ×100 oil-immersion lens
of a Zeiss (SN 3832000344) microscope. First, we plated
HT22 cells on to 0.13–0.17 mm coverslips pre-treated with
0.1 % gelatin overnight. HT22 cells were then transfected
with mito-DsRed for mitochondrial labelling. At 24–36 h after
transfection, cells were fixed in 4 % PFA for 10–20 min at room
temperature. After incubation in Hoechst 33258 (1:3000 dilution)
for 5 min to label the nucleus, coverslips were mounted with
anti-fluorescence quench reagent (P0126, Beyotime). The signal
of the DsRed (excitation wavelength = 530 nm and emission
wavelength = 580 nm) or GFP (excitation wavelength = 475 nm
and emission wavelength = 509 nm) and Hoechst (emission
maximum at 461 nm) were detected by fluorescence microscopy.
Analysis of mitochondrial length was performed as described
previously [1]. Briefly, to obtain quality pictures of mitochondrial,
a 0.2 μm Z-stack is optimal for fixed samples. We took eight
Z-planes from one cell then merged the Z-planes into a single
image. A total of ten random cells per one sample were
analysed by confocal microscopy and the mitochondrial length
was measured using ImageJ software (http://rsbweb.nih.gov/ij/).
The mitochondria that measured <0.8 mm were considered as
fragmentated.
Figure S2
Figure S1
DJ-1 can attenuate MPP+-induced up-regulation of Fis1
Lysates of HT22 cells transfected with GFP–DJ-1 or its control vector were treated with 1.5 mM
MPP + for 4 h and immunoblotted with antibodies against Fis1, DJ-1, Akt1, phospho-Ser473
Akt1 (p-S473 Akt1) or GAPDH. The ratio of Fis1/GAPDH was quantified and is shown in the
lower panel (**P < 0.01 using one-way ANOVA, n = 3).
Fis1 is regulated at the protein level by DJ-1
(A) SH-SY5Y cells were stably transfected with DJ-1 RNAi or the control vector. At 72 h after transfection, cells were treated with 50 μg/ml cycloheximide (CHX) for the times indicated. The lysates
were then subjected to immunoblotting with anti-Fis1, anti-DJ-1 or anti-GAPDH antibodies. (B) Quantitative analysis of the level of Fis1 compared with the level of GAPDH. (C) HeLa cells were
transfected with GFP–Fis1 and wild-type (WT) DJ-1, DJ-1 C106A or the control vector. At 24 h after transfection, cells were treated with 10 μM MG132 for 4 h and the lysates were subjected to
immunoblotting with anti-GFP or anti-GAPDH antibodies.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
Q. Zhang and others
Figure S3
DJ-1 inhibits mitochondrial fragmentation induced by Fis1
(A) The mitochondrial morphology of HT22 cells was analysed by fluorescent microscopy. A plasmid expressing mito-Red was transfected together with the plasmids encoding DJ-1, DJ-1 C106A,
Fis1 or the control vector (Ctrl). Expression of Fis1 led to a significant increase in the percentage of cells displaying fragmented mitochondria. Co-expression of DJ-1 reduced Fis1-induced
mitochondrial fragmentation. (B) Quantification of mitochondrial fragmentation in HT22 cells treated and analysed as in (A). No less than 100 cells per condition were analysed. All of the histograms
show means +
− S.D. Wild type DJ-1, but not the C106A mutant, inhibited Fis1-induced mitochondrial fission. (**P < 0.01 using one-way ANOVA, n = 10).
Figure S4
DJ-1 increases the interaction between RNF5 and Fis1
(A) GFP immunoprecipitates (IP) of the lysates of HEK-293T cells were transfected with the
plasmids of RNF5 and Fis1 together with or without DJ-1, and immunoblotted with an antibody
against Myc–Fis1. Cells were treated with 10 μm MG132 for 3 h before harvest. DJ-1 increases
the binding between Fis1 and RNF5. The ratio of Myc-Fis1/total cell lysis Myc-Fis1 was
quantified and showed in the lower panel (**P < 0.01 using one-way ANOVA, n = 3). *, IgG.
(B) Lysates of HEK-293T cells transfected with the plasmids of RNF5 and Fis1 together with
DJ-1 or the control vector were immunoblotted with antibodies against RNF5, Fis1, DJ-1 or
actin. Cells were treated with 10 μM MG132 for 3 h before harvest. DJ-1 can promote the
auto-ubiquitination (auto-ub) of RNF5.
c The Authors Journal compilation c 2012 Biochemical Society
DJ-1-mediated signalling in the regulation of Fis1
Figure S5
DN–Akt inhibits RNF5 auto-ubiquitination
GFP immunoprecipitates (IP) of the lysates of HEK-293T cells transfected with Myc–ub
(ubiquitin) together with GFP–RNF5 and DN Akt1 or the vector control were inmmunoblotted
with Myc to detect RNF5 auto-ubiquitination. Cells were treated with 10 μM MG132 for 3 h
before harvesting. *, IgG. Molecular mass is given on the left-hand side in kDa.
Figure S6
DJ-1/Akt pathway regulates RNF5 mitochondrial translocation
(A) HT22 cells were co-transfected with GFP–RNF5, wild-type (WT) DJ-1 or C106A DJ-1 for
24 h followed by fluorescent microscopy analysis. (B) HT22 cells were co-transfected with
GFP–RNF5, Akt1 and/or C106A DJ-1 for 24 h followed by fluorescent microscopy analysis.
Tom20, translocase of outer mitochondrial membrane 20 homologue.
c The Authors Journal compilation c 2012 Biochemical Society
Q. Zhang and others
Figure S7
Working model of DJ-1/RNF5/Fis1 signalling
DJ-1 promotes RNF5-mediated Fis1 proteasomal degradation through the activation of PI3K/Akt
signalling and inhibits Fis1-induced mitochondrial fragmentation and neuronal cell death.
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Received 11 April 2012/6 August 2012; accepted 8 August 2012
Published as BJ Immediate Publication 8 August 2012, doi:10.1042/BJ20120598
c The Authors Journal compilation c 2012 Biochemical Society