650?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 119? ? ? Number 3? ? ? March 2009
Parkin, PINK1, and DJ-1 form a
ubiquitin E3 ligase complex promoting
unfolded protein degradation
Hui Xiong,1 Danling Wang,1,2 Linan Chen,3 Yeun Su Choo,1 Hong Ma,1 Chengyuan Tang,1,2
Kun Xia,2 Wei Jiang,1 Ze’ev Ronai,1 Xiaoxi Zhuang,3 and Zhuohua Zhang1,2
1Burnham Institute for Medical Research, La Jolla, California, USA. 2State Key Laboratory of Medical Genetics, Central South University,
Changsha, Hunan, People’s Republic of China. 3Department of Molecular Pharmacology, University of Chicago School of Medicine, Chicago, Illinois, USA.
Mutations?in?PARKIN,?pten-induced putative kinase 1?(PINK1),?and?DJ-1?are?individually?linked?to?autosomal?
Parkinson disease (PD) is a progressive neurodegenerative movement
disorder affecting approximately 2% of the population over 65 years
of age. It is pathologically characterized by pronounced loss of dopa-
minergic neurons in the substantia nigra of the midbrain and for-
mation of ubiquitin-positive Lewy body aggregates (1, 2). Although
administration of L-DOPA temporarily relieves parkinsonism, no
treatment is currently available to prevent disease progression and
neurodegeneration. Understanding the molecular basis of PD is like-
ly to facilitate development of effective therapies of the disease.
Most PD cases are sporadic. Nevertheless, mutations in at least
7 genes are linked to early onset familial form PD, including
mutations in a-synuclein, ubiquitin carboxyl-terminal esterase L1
(uchL1), and leucine-rich repeat kinase 2 (LRRK2) for autosomal
dominant cases and mutations in Parkin, pten-induced putative
kinase 1 (PINK1), DJ-1, and ATPase type 13A2 (ATP13A2) for auto-
somal recessive cases (1–6). Characterization of these genes has
provided important insights into PD pathogenesis. For example,
α-synuclein is a major structural component of Lewy bodies in PD
(7). PD-associated α-synuclein mutant proteins show increased
propensity to self-aggregate to form oligomeric species and Lewy
body–like fibrils compared with WT α-synuclein, directly linking
disease-associated α-synuclein mutant proteins to PD pathology
(8, 9). However, the functional relationships of these genes and
how their respective disease-associated mutations cause selective
neuronal loss and Lewy body formation remains largely unknown,
even though mutations of these genes cause the same disease.
Among the genes linked to the autosomal recessive early onset
familial form of PD, Parkin encodes a protein with E3 ligase activity,
mediating ubiquitination and degradation of multiple proteins (5,
10–12). Sequence analysis strongly suggests that PINK1 is a putative
kinase with 2 identified substrates (13–15). DJ-1 protects cells from
oxidative stress both in transfected cells and in Drosophila (16–19).
Inactivation of Parkin or Dj-1 in mouse and PINK1 in Drosophila
results in mitochondrial dysfunction and increased sensitivity to
oxidative stress (20–25). Together, Parkin, PINK1, and DJ-1 likely
protect against oxidative stress in cells via a common mechanism,
suggesting potential functional interactions among the 3 proteins.
In this study, we aimed to investigate the functional relationship
of Parkin, PINK1, and DJ-1. Our results demonstrate that Parkin,
PINK1, and DJ-1 form what we believe is a novel E3 complex that
promotes ubiquitination and degradation of aberrantly expressed
and heat shock–induced Parkin substrates, Parkin and Synphilin-1.
Pathogenic PINK1 or Parkin mutants showed impaired ability to
degrade Parkin and Synphilin-1. These results suggest that the
Parkin/PINK1/DJ-1 (PPD) complex plays an important role in
degradation of un-/misfolded Parkin substrates.
Complex formation of Parkin, PINK1, and DJ-1 in vitro and in vivo. To
examine whether proteins encoded by PD-associated Parkin, PINK1,
or DJ-1 regulate a common functional pathway via complex forma-
tion, we coexpressed vesicular stomatitis virus glycoprotein–tagged
(VSVG-tagged) Parkin (ParkinWT), flag-tagged PINK1 (PINK1WT),
and myc-tagged DJ-1 (DJ-1WT) in various combinations in SH-SY5Y
neuroblastoma cells (Figure 1) and HEK293 cells (data not shown).
Immunoprecipitation of cell lysates using corresponding anti-tag
Authorship?note:?Hui Xiong and Danling Wang contributed equally to this work.
Conflict?of?interest:?The authors have declared that no conflict of interest exists.
Nonstandard?abbreviations?used: GST, glutathione-S-transferase; PD, Parkinson
disease; PINK1, pten-induced putative kinase 1; PPD, Parkin/PINK1/DJ-1 (complex);
UPS, ubiquitin-proteasome system; VSVG, vesicular stomatitis virus glycoprotein.
Citation?for?this?article: J. Clin. Invest. 119:650–660 (2009). doi:10.1172/JCI37617.
Related Commentary, page 442
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
antibodies revealed that Parkin, PINK1, and DJ-1 were specifically
coprecipitated in any combination of 2 or all 3 proteins (Figure 1,
A–F). We next performed in vitro interaction assays using purified
flag-myc dual-tagged Parkin, flag-VSVG dual-tagged PINK1, and
glutathione-S-transferase–VSVG (GST-VSVG) dual-tagged DJ-1
recombinant proteins (Supplemental Figure 1; supplemental mate-
rial available online with this article; doi:10.1172/JCI37617DS1).
Consistent with the coimmunoprecipitation results, Parkin bound
to PINK1, DJ-1, or both (Figure 1G). Likewise, DJ-1 interacted with
Parkin, PINK1, or both (Figure 1H). In contrast, none of the 3 pro-
teins bound to control fusion protein KChIP1 (data not shown).
These results suggest that Parkin, PINK1, and DJ-1 physically
associate. Gel filtration analysis of the complex assembled in vitro
suggested that Parkin, PINK1, and DJ-1 form an approximately
200-kDa complex (Supplemental Figure 2). Deletion studies indi-
cated that Parkin, PINK1, and DJ-1 proteins interact with each other
via distinct domains (Supplemental Figure 3), further supporting
the idea that a stable complex of the 3 proteins forms.
We next examined interaction of endogenous Parkin, PINK1, and
DJ-1 in human brain lysates. Immunoprecipitation of endogenous
Parkin with an anti-Parkin monoclonal antibody, but not an unre-
lated control mouse IgG, coprecipitated endogenous PINK1 and
DJ-1 (Figure 1I), suggesting that Parkin, PINK1, and DJ-1 form a
complex in vivo. We were not able to perform immunoprecipitation
of endogenous PINK1 because of unavailability of anti-PINK1 anti-
bodies for immunoprecipitation. To provide further evidence for
complex formation in vivo, we immunoprecipitated PINK1 from
SH-SY5Y cells stably expressing a low level of flag-tagged PINK1,
then determined coprecipitated proteins using mass spectrometry.
Three functional classes of proteins were found to interact with
PINK1 in cells expressing PINK1 but not in cells transfected with
vector alone. These included heat shock protein chaperones, mito-
chondrial proteins, and proteins involved in the ubiquitin-protea-
some pathway (data not shown). In addition, endogenous Parkin
and DJ-1 were identified (Supplemental Table 1). Together, Parkin,
PINK1, and DJ-1 formed a complex in vitro and in vivo that we
henceforth refer to as the PPD complex (Figure 1J).
Intracellular localization of the PPD complex. PINK1 is suggested to be
a mitochondrial protein and to localize to mitochondrion. We next
determined localization of the PPD complex by immunostaining
and cellular fractionation. Immunostaining of SH-SY5Y neuro-
blastoma cells (data not shown) and cultured primary human neu-
rons showed colocalization of Parkin, PINK1, and DJ-1 largely in
the cytoplasm (Supplemental Figure 4). Fractionation analysis of
SH-SY5Y cells stably expressing Parkin, PINK1, and DJ-1 detected
all 3 proteins in both the mitochondrial and the cytosolic fractions
(Figure 2A). Consistent with the immunostaining results, all 3
proteins were more abundant in the cytosolic fraction than in the
mitochondrial fraction. Immunoprecipitation of fractionated cell
lysates revealed that the majority of PPD complex was present in the
cytosolic fraction, while only a minor amount of the PPD complex
was detected in the mitochondrial fraction (Figure 2B). Interesting-
ly, the full-length PINK1 (64-kDa fragment) was detected mostly in
the mitochondrial fraction, while the proteolytic processed PINK1
fragments (55 kDa and 48 kDa) were mainly found in the cytosolic
fraction (Figure 2A and Supplemental Figure 5). Furthermore, Par-
kin coprecipitated with only the 55-kDa PINK1 fragment. Detec-
tion of the processed PINK1 mainly in the cytosol indicated that
the cytosolic PINK1 was not likely the result of increased expres-
sion of exogenous protein. These results suggest that the 55-kDa
PINK1 proteolytic fragment was likely the active PINK1 in the PPD
complex (Figure 2B). Thus, the PPD complex is mainly localized in
cytosolic fraction of the cell.
PPD promotes degradation of Parkin and Synphilin-1 via the ubiquitin-
proteasome system. Parkin functions as an E3 ubiquitin ligase in the
ubiquitin-proteasome system (UPS) (10–12). We next examined
whether PINK1 regulates degradation of the previously defined
Parkin substrates, Parkin and Synphilin-1 (11, 26). Expression of
PINK1 in SH-SY5Y (Figure 3, A and B) and HEK293 cells (data
not shown) remarkably reduced the steady-state level of Parkin or
Synphilin-1 compared with control transfectants. PINK1-induced
reduction in Parkin and Synphilin-1 levels was largely rescued by
treatment with the proteasome inhibitors MG132 and lactocys-
tin (data not shown) but only slightly inhibited by treatment with
the protease inhibitor leupeptin (Figure 3, A and B). Pulse chase
analysis showed that Parkin stability was remarkably reduced with
PINK1 expression. In the presence of MG132 (5 μM), PINK1-pro-
moted Parkin degradation was inhibited, resulting in accumula-
tion of both monomeric and ubiquitinated forms of Parkin (Fig-
ure 3, C and D). Thus, PINK1 promotes Parkin and Synphilin-1
degradation primarily via the UPS.
We next investigated ubiquitination of Parkin and Synphilin-1
in the presence of PINK1. Parkin or Synphilin-1 was coexpressed
with either PINK1WT or a pathogenic PINK1G309D mutant in
SH-SY5Y cells. As anticipated, Parkin levels were remarkably
reduced, while ubiquitination of Parkin was substantially increased
in cells expressing PINK1WT compared with control transfectants
(Figure 4, A and B). Increased Parkin ubiquitination was deemed
notable because the level of Parkin in cells expressing PINK1WT
was particularly low compared with that seen in cells expressing
Parkin alone. PINK1-dependent ubiquitination of Parkin was
further supported by reduced detection of monomeric ubiquitin
(Figure 4D). Likewise, remarkably reduced levels of Synphilin-1
protein (Figure 4H) and monomeric ubiquitin (Figure 4J) were
seen in cells expressing Synphilin-1 and PINK1 compared with
cells expressing Synphilin-1 alone. Nevertheless, in cells expressing
PINK1WT, Synphilin-1 ubiquitination appeared to be less exten-
sive than that seen in control cells, likely due to marked degrada-
tion of Synphilin-1 and the fact that little protein was available
for immunoprecipitation. These results suggest that PINK1 pro-
motes degradation of Parkin substrates primarily by promoting
ubiquitination of Parkin substrates. PINK1 promotes Synphilin-1
degradation in the absence of overexpressed Parkin (Figure 4, G
and H), most likely through the activity of endogenous Parkin in
SH-SY5Y cells. Coexpression of Parkin increased ubiquitination
and the steady-state level of PINK1 (Figure 4, E, F, K, and L). Thus,
PINK1 is unlikely a Parkin substrate for UPS degradation.
Roles of PINK1 in promoting Parkin ubiquitination were further
verified by an in vitro Parkin auto-ubiquitination assay using affin-
ity-purified Parkin and PINK1 fusion proteins. In the presence of
E1 or E2 alone, little ubiquitinated Parkin was detected (Figure 5).
Consistent with a previous report that auto-ubiquitinated Parkin
is limited in vitro (12), ubiquitination of Parkin was not substan-
tially increased when both E1 and E2 were included in the assay.
In contrast, Parkin ubiquitination was remarkably enhanced by
adding PINK1WT. Increased Parkin ubiquitination was likely spe-
cifically induced by PINK1 because the pathogenic PINK1G309D
protein had a much smaller effect than PINK1WT. PINK1WT pro-
moted ubiquitination of a pathogenic Parkin mutant (R42P) with
notably lower potency than it did for ParkinWT (Figure 5). Consis-
652?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
tent with this observation, PINK1G309D did not promote ubiqui-
tination and degradation of Parkin and Synphilin-1 in transfected
cells (Figure 4, B and H). The results suggest that PD-pathogenic
PINK1 and Parkin mutants impair PPD complex activity.
To determine the potential role of DJ-1 in the PPD complex, WT
DJ-1 (DJ-1WT) or a pathogenic loss-of-function mutant DJ-1L166P
was included in some of the experiments shown in Figure 4. Parkin
ubiquitination was consistently slightly increased in the presence of
Complex formation of Parkin, PINK1, and DJ-1. (A–F) Association of Parkin, PINK1, and DJ-1 in transfected cells. Parkin-VSVG, PINK1-flag,
and DJ-1–myc were expressed in various combinations and immunoprecipitated with antibodies to the corresponding tag, followed by detection
of coprecipitation of PINK1 and DJ-1 (A), Parkin and DJ-1 (B), and Parkin and PINK1 (C), respectively. (D–F) Inputs of Parkin (D), PINK1 (E),
and DJ-1 (F). Note that cotransfection of PINK1 significantly reduced Parkin levels in lysates. Tub, cytosolic marker tubulin. (G and H) In vitro
assembly of the PPD complex. Affinity-purified Parkin-myc-flag (Parkin), PINK1-VSVG-flag (PINK1), and GST–DJ-1–VSVG (DJ-1GST) were
incubated in various combinations, followed by precipitation with either anti-myc agarose (G) or GST agarose (H). Precipitates were detected with
an anti-VSVG antibody to detect both PINK1 and DJ-1–GST (G), an anti-Parkin antibody to detect Parkin (H), an anti-PINK1 antibody to detect
PINK1 (H), or an anti–DJ-1 antibody to detect DJ-1 (H). (I) Association of Parkin, PINK1, and DJ-1 in vivo. Lysates of human brain cortex from
2 unrelated individuals (lanes 1 and 2 for one individual, lanes 3 and 4 for the other) were immunoprecipitated with an anti-Parkin monoclonal
antibody (αParkin) or control mouse IgG (mIgG), followed by immunoblotting with a polyclonal anti-Parkin antibody, a polyclonal anti-PINK1
antibody, or a monoclonal anti–DJ-1 antibody. Multiple endogenous PINK1 proteolytic bands were detected (arrows). (J) A schematic illustration
of interaction among PPD complex components. IBR, in between RING fingers; MTS, mitochondrial targeting sequence; UBL, ubiquitin-like.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
DJ-1WT despite the fact that steady-state levels of Parkin were not
obviously altered (Figure 4A). Similar results were obtained with
Synphilin-1 (data not shown). In agreement with our recent finding
that PINK1 levels are stabilized by DJ-1WT but not by PD-associ-
ated DJ-1A39S (27), the steady-state level of PINK1 was consistently
increased in cells expressing DJ-1WT (Figure 4F). Therefore, DJ-1
likely modulates PINK1 in the PPD complex.
Accumulation of aberrantly expressed Parkin in PINK1- or DJ-1–defi-
cient cells. Parkin can act as a substrate of itself. We examined roles
of endogenous PINK1 and DJ-1 in promoting degradation and
ubiquitination of Parkin using PINK1- or DJ-1–deficient mouse
brains and cells generated from mouse embryos in which PINK1 or
DJ-1 was genetically deleted (28). Ablation of either PINK1 or DJ-1
had little effect on steady-state level of endogenous Parkin (data
not shown). We next determined ubiquitination of endogenous
Parkin using mouse brain slice cultures that were generated from
either WT or PINK1-null mice. Endogenous Parkin ubiquitination
was detected by immunoprecipitation of Parkin followed by immu-
noblotting detection of ubiquitin. In control WT mouse brain
slices, polyubiquitination of Parkin was detected as high molecular
weight–smeared protein species (Figure 6). In contrast, polyubiqui-
tinated Parkin in PINK1-null brain slices was remarkably reduced.
Heat shock treatment enhanced Parkin ubiquitination in WT
mouse brain slices but not in PINK1-null mouse brain slices. We
did not observe increased accumulation of endogenous Parkin in
PINK1-null brain slice cultures. One possible explanation is that an
alternative pathway is involved in degradation of non-ubiquitinated
Parkin. Moreover, treatment of HEK293 cells expressing Synphilin-1
with a mixture of Parkin-specific siRNA oligos inhibited Parkin
expression, rescued PINK1-induced Synphilin-1 degradation, and
resulted in accumulation of Synphilin-1 (Supplemental Figure 6, A
and C). Thus, PINK1 promotes Synphilin-1 degradation via a Par-
kin-mediated mechanism. These results suggest that endogenous
PINK1 plays an important role in Parkin E3 ligase–mediated pro-
tein ubiquitination under both normal and stress conditions.
Since heat shock stress induces protein un-/misfolding and Par-
kin-mediated protein ubiquitination (Figure 6), we further exam-
ined the hypothesis that the PPD complex mediates ubiquitina-
tion and degradation of un-/misfolded proteins. Overexpression
of human Parkin in PINK1-null cells resulted in very significant
accumulation of Parkin compared with that seen in control WT
cells (Figure 7B). Likewise, increased Parkin detection was seen
in DJ-1–null cells overexpressing Parkin compared with matched
control cells (Figure 7C). Nevertheless, level of Parkin in DJ-1–null
cells was consistently lower than that seen in PINK1-null cells.
Pulse chase analysis revealed that Parkin was more stable in both
PINK1-null cells (half-life of 4 h) and DJ-1–null cells (half life of
2–3 h) than in respective WT controls (half-life of <1 h) (Figure 7,
C–G). These results suggest that the PPD complex is involved in
degradation of aberrantly expressed un-/misfolded proteins.
Together, these results suggest that Parkin, PINK1, and DJ-1 are
essential components of the PPD E3 ligase activity and that the
complex likely plays an important role in degradation of un-/mis-
folded Parkin substrates.
The PPD complex promotes degradation of un-/misfolded Parkin
induced by heat shock stress. To further determine the role of the
PPD complex in promoting degradation of un-/misfolded pro-
teins, we examined Parkin degradation after heat shock stress. In
SH-SY5Y cells expressing Parkin alone, heat shock stress resulted
in increased ubiquitination and accumulation of Parkin, likely
un-/misfolded Parkin (Figure 8, A and B). The increased detec-
tion of Parkin ubiquitination was likely due to increased Parkin
accumulation. In contrast, PINK1 expression not only abolished
the Parkin accumulation induced by heat shock but also further
promoted Parkin degradation (Figure 8B). These findings are
consistent with increased endogenous Parkin ubiquitination by
heat shock treatment (Figure 6) and provide further evidence
that the PPD complex functions to promote degradation of
PD-pathogenic Parkin and PINK1 mutants impair substrate degra-
dation. We next examined the effect of PD-pathogenic mutants
of PINK1 and Parkin on the function of the PPD complex.
PINK1G309D and ParkinΔE4 were able to form a complex with
ParkinWT and PINK1WT, respectively (Figure 9, A and B). Com-
pared with PINK1WT-promoted degradation of ParkinWT,
PINK1WT poorly promoted degradation of pathogenic Parkin-
Detection of the PPD com-
plex in both mitochondria
and cytosolic fractions.
Cells expressing Parkin
alone or a combination
of Parkin, PINK1, and
DJ-1 were fractionated
to mitochondrial (Mito)
and cytosolic (Cyto) frac-
tions. Left: Expression of
Parkin, PINK1, DJ-1, mito-
chondria marker complex I
(Cplx I), and cytosolic
marker tubulin. Right:
of Parkin with PINK1 and
DJ-1 in mitochondria and
654?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
R42P, -T240W, and -ΔE4 (Figure 9, C and E; P < 0.001). Likewise,
pathogenic PINK1G309D, PINK1T313M, and PINK1P399L
had less of an effect on the steady-state levels (Figure 9, D and F;
P < 0.001) or on ubiquitination (data not shown) of ParkinWT
and Synphilin-1 compared with PINK1WT. Consistent with a pre-
vious study, expression of some pathogenic Parkin and PINK1
mutants was low compared with their WT counterparts in trans-
fected cells (29). Nevertheless, low levels of PD-pathogenic PINK1
mutant proteins unlikely accounted for their reduced ability to
promote degradation of Parkin substrates. In in vitro ubiquitina-
tion assays, amounts of PINK1G309D mutant protein equivalent
to those of WT protein failed to promote comparable Parkin
ubiquitination (Figure 5B). Thus, pathogenic Parkin and PINK1
mutant proteins impair substrate degradation likely through
reduced PPD complex activity.
We have identified in this study what we believe is a novel function-
al E3 ligase complex designated as the PPD complex, consisting of
3 PD-associated proteins, Parkin, PINK1, and DJ-1. We further
demonstrated that the PPD E3 ligase complex plays an important
role in promoting degradation of un-/misfolded Parkin substrates.
PD-pathogenic mutants of Parkin and PINK1 impaired E3 ligase
activity of the PPD complex. Our results suggest multiple PD-linked
genes function in PD pathogenesis via a common mechanism.
The significance of this study is the finding that 3 PD-relat-
ed proteins physically interact and form a functional E3 ligase
complex to regulate UPS-mediated protein degradation. The
findings suggest a function of PINK1 and DJ-1 that is indepen-
dent of maintaining mitochondrial homeostasis. In our investi-
gation we used 4 different methods to determine complex for-
mation by Parkin, PINK1, and DJ-1 in vitro and in vivo. These
include coimmunoprecipitation in cells overexpressing the 3
proteins, in vitro complex assembly using purified recombinant
proteins, coimmunoprecipitation in human brain lysates, and
immunoprecipitation followed by mass spectrometry analysis.
Our findings provide biochemical and mechanistic evidence
to support previous reports of genetic interaction of Parkin
and PINK1 in Drosophila (22, 23, 25) and association of digenic
mutations of PINK1 and DJ-1 with early onset familial PD cases
(27). Nevertheless, the PPD complex is not necessarily the only
important one involving PINK1, given that mass spectrometry
analysis identifies multiple PINK1 interacting proteins. The E3
ligase activity of the PPD complex was further shown in trans-
PINK1 promotes proteasomal degradation of Parkin and Synphilin-1. (A and B) SH-SY5Y cells coexpressing PINK1-flag and Parkin-VSVG (A)
or PINK1-flag and Synphilin-1–EGFP (Syn-1; B) were treated with either MG132 or leupeptin (Leu). Steady-state levels of Parkin, Synphilin-1,
PINK1, and actin (loading control) are shown. (C and D) Expression of PINK1 reduced Parkin stability via the proteasomal pathway. Cells trans-
fected with Parkin alone (C; top panel), Parkin and PINK1 (C; middle panel), or Parkin and PINK1 with MG132 treatment (C; bottom panel) were
pulse-labeled, followed by chasing for the indicated time intervals. Levels of Parkin were detected by immunoprecipitation. Results from a repre-
sentative experiment (C) and quantitation of 3 independent experiments are shown (D). Multiple Parkin bands likely representing ubiquitinated
Parkin were detected in the presence of PINK1 (arrows). (D) Diamonds indicate Parkin alone, squares indicate Parkin and PINK1, and triangles
indicate Parkin and PINK1 with MG132 treatment.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
fected cells and an in vitro ubiquitination assay using previously
defined (10, 11, 26) Parkin substrates, Synphilin-1, and Parkin
itself. Our results suggest that Parkin likely has very limited activ-
ity in the absence of PINK1. However, Parkin E3 ligase activity is
remarkably potentiated by expression of PINK1. Thus, PINK1
plays an essential role in maintaining high E3 ligase activity of
Parkin. These findings also provide an explanation for inconsis-
tent reports of Parkin E3 ligase activity in previous studies (12).
Another importance of this study is to identify the PPD complex
as what we believe is a novel E3 ligase. Three classes of E3 ligase
have been previously described, including HECT domain ligases,
RING finger ligases, and U-box domain ligases (30). RING finger
E3 ligases can be either a single protein, such as CBL (31), or a
multiple protein complex, such as the skip/cullin/F-box/Rbx1/2
(SCF) complex (32). In the PPD complex, Parkin is a RING finger
protein binding to E2 and substrate, while PINK1 and DJ-1 act as
regulatory components. PINK1, a serine/threonine kinase, makes
the PPD complex, to our knowledge, the first E3 ligase complex
with a kinase subunit. The role of serine/threonine kinase activi-
ty of PINK1 in this complex remains unknown. One possibility is
that PINK1 regulates protein ubiquitination by phosphorylating
either the complex components or substrates. DJ-1 facilitates but
is not required for E3 ligase activity of the complex. One poten-
tial role of DJ-1 in this complex is to stabilize PINK1 (27).
PINK1 regulates ubiquitination of Parkin and Synphilin-1. Parkin (left) or Synphilin-1 (right) was cotransfected into SH-SY5Y cells with PINK1WT,
a PD-pathogenic PINK1G309D mutant (PINK1m), DJ-1WT, or a PD-pathogenic DJ-1L166P mutant (DJ-1m) in the presence of ubiquitin (Ub) in
various combinations. In a Synphilin-1 degradation experiment, a PD-pathogenic ParkinR42P mutant (Parkinm) was included. Parkin (A and B)
or Synphilin-1 (G and H) were immunoprecipitated from equal amounts of cell lysates, followed by detection of ubiquitin (A and G), Parkin (B),
or Synphilin-1 (H). Expression levels of DJ-1WT (C), DJ-1L166P (C), and ubiquitin (D and J) were shown by direct immunoblotting. Ubiquitina-
tion and steady-state levels of PINK1 variants were observed by immunoprecipitation of PINK1, followed by immunoblotting of either ubiquitin
(E and K) or PINK1 (F and L).
656?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
The UPS plays critical roles in PD pathogenesis (5, 33, 34). Our
finding of PD-pathogenic mutants of PINK1 and Parkin to impair
the PPD E3 ligase activity supports a role of the PPD E3 ligase in PD
pathogenesis. Consistent with this notion, we have previous identi-
fied digenic mutations of PINK1 and DJ-1 that are associated with
early onset familial PD cases (27). Since the identification of Parkin
as an E3 ligase, one uncertainty is the identity of its substrates that
are also the substrates of the PPD complex. Parkin ubiquitinates
a number of proteins in vitro. Nevertheless, few of these in vitro
substrates accumulate in Parkin-deficient cells (20). In this study,
we have also found that levels of Parkin and Synphilin-1 are not
altered in either PINK1- or DJ-1–deficient cells. These results sug-
gest that Parkin substrates can be degraded by alternative mecha-
nisms under physiological conditions. However, overexpression
of Parkin substrates or heat shock treatment resulted in Parkin
substrate accumulation in PINK1-deficient cells. One explanation
is that the PPD complex functions as a quality control system to
degrade unfolded and misfolded Parkin substrates induced by oxi-
dative stress in neurons. Pathogenic mutations that reduce PPD
activity likely disrupt the UPS, leading to increased susceptibility
to oxidative stress and accumulation of un-/misfolded proteins in
cells, therefore eventual neuronal death and Lewy body formation.
PINK1 promotes Parkin auto-ubiquitination in vitro. Affinity-puri-
fied ParkinWT, the PD-associated ParkinR42P mutant, PINK1WT,
and PD-associated PINK1G309D mutant proteins in various com-
binations were assayed for in vitro ubiquitination in the presence
of recombinant E1, E2 (Ubc7), and HA-tagged ubiquitin. Proteins
were separated on SDS-PAGE and immunoblotted with an anti-
Parkin antibody to detect Parkin monomers (Parkin) and ubiqui-
tinated Parkin [Parkin-poly(Ub)], an anti-VSVG antibody to detect
PINK1, or an anti-HA antibody to detect ubiquitin. Ubiquitinated
Parkin appeared as a smear in the top panel.
Ubiquitination of endogenous Parkin in mouse brains with
PINK1 and DJ-1 ablation. Top: Brain slices from WT and
PINK1-deficient (KO) mice were immunoprecipitated with
either a monoclonal anti-Parkin antibody or a control mouse
IgG, followed by immunoblotting with an anti-ubiquitin anti-
body. Cells overexpressing exogenous Parkin were used as
a positive control. The experiments were done with or without
heat shock treatment (HS). Bottom: Immunoprecipitated Par-
kin protein was detected by anti-Parkin polyclonal antibody.
The 55-kDa band shown in control precipitations were IgG
heavy chain. Note that Parkin ubiquitination was remarkably
reduced in PINK1-null brain slices.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
WT Parkin, but not PD-pathogenic mutant Parkin, has been previ-
ously shown to protect cells against dopamine-induced oxidative
stress via a UPS-mediated mechanism (35). Parkin, PINK1, and
DJ-1 are also individually implicated in oxidative stress protection
(2, 18, 19, 21, 36–39). Furthermore, Parkin mediates polyubiqui-
tination, and subsequently degradation, of the highly aggregative
ER transmembrane protein Pael-R (12, 40). In this study, the aber-
rantly expressed proteins and heat shock–induced misfolded Par-
kin accumulated in the absence of PINK1.
This study connects 3 PD-linked genes in a single functional
pathway that further highlights a common mechanism of PD
pathogenesis. Parkin has been shown to ubiquitinate Synphilin-1
and a rare O-glycosylated form of α-synuclein (26, 41), suggesting
that these genes functionally interact to regulate PD pathogenesis.
A recent study indicates a role for Parkin in modulating ubiqui-
tination of LRRK2, a newly identified gene linked to autosomal
dominant familial PD (42). Together, this study should facilitate
further investigation of PD-pathogenic mechanisms and the
design of novel PD treatment strategies.
Mice, cell lines, plasmids, and siRNAs. The DJ-1–deficient mouse line has been
described previously (28). The Pink1-deficient mice were generated by replac-
ing a 5.6-kb genomic region in the Pink1 locus, including exons 4–7 and the
coding portion of exon 8, with FRT sequences flanked PGK-neo-polyA selec-
tion cassette. Both the 3.5-kb and 4.8-kb homologous arms were amplified
by PCR using genomic DNA isolated from E14Tg2A.4 embryonic stem cells
(BayGenomics) as a template. E14Tg2A.4 embryonic stem cells were electro-
Genetic ablation of mouse Pink1 or Dj-1 results in increased stability of aberrantly expressed Parkin. (A) Expression of Parkin, PINK1, and DJ-1
in PINK1- or DJ-1–deficient mouse fibroblasts. RT-PCR detection of Parkin, PINK1, and DJ-1 in PINK1 WT, PINK1 KO, DJ-1 WT, and DJ-1 KO
cells. Control, no cDNA template added. (B and C) Increased accumulation of aberrantly expressed Parkin in PINK1 KO and DJ-1 KO cells. Cells
transfected with control plasmid or plasmid encoding Parkin showed increased Parkin detection in PINK1 KO cells (B) and DJ-1 KO cells (C).
(C) Tubulin was used as a control. Lack of DJ-1 protein in DJ-1 KO cells was shown by immunoblotting. (D–G) Increased stability of Parkin in
PINK1 KO and DJ-1 KO cells. PINK1 KO, PINK1 WT, DJ-1 KO, and DJ-1 WT cells were transfected with Parkin, followed pulse chase analysis
of Parkin stability for the time frames indicated. Representative results of PINK1 (D) and DJ-1 (E) are shown. Quantitation was obtained from
PINK1 KO cells generated from 2 independent PINK1 KO mice (F) and DJ-1 KO cells generated from multiple DJ-1 KO mice (G).
658? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
porated (800 V and 3 μF) with 30 μg of linearized targeting construct. G418-
resistant clones were screened by Southern blotting for homologous recom-
bination with a 5′ external probe. Positive cells were injected into C57BL6/J
blastocysts to generate chimeras, which were then mated with C57BL6/J
WT mice to generate heterozygotes. Heterozygous mice on a 129 × C57BL/6
mixed background were bred to generate Pink1-null mice and their WT lit-
termate controls for experiments. Mice were genotyped by multiplex PCR
on genomic DNA extracted from tail snips: the first primer pair amplified
part of intron 6 of Pink1 (present in all mice), the second primer pair ampli-
fied part of neor (absent in WT mice), and the third primer pair amplified
intron 6 of PINK1 (absent in homozygous mutants). The absence of PINK1
expression was verified by RT-PCR and in situ hybridization (data not
shown). All animal procedures were approved by the Institutional Ani-
mal Care and Usage Committees (IACUC) of the University of Chi-
cago and the Burnham Institute for Medical Research.
Mouse embryonic fibroblasts were generated from PINK1- and DJ-1–
deficient mouse embryos and their WT control littermates. Cells
were immortalized with a retrovirus expressing SV40 large T antigen.
SH-SY5Y neuroblastoma cells and HEK293 cells were purchased
from ATCC. cDNAs encoding myc-tagged, flag-tagged, or VSVG-
tagged Parkin variants (WT, ΔE4, R42P, and T240W), PINK1 vari-
ants (WT, G309D, T313M, L339P) and DJ-1 variants (WT and L166P)
were PCR generated and subcloned into pcDNA3.1(–) (Invitrogen).
A SMARTpool of siRNAs for human Parkin was purchased from
Dharmacon. Plasmids encoding HA-ubiquitin and myc-ubiquitin
were previously described (43). GFP-HA–tagged Synphilin-1 was a
gift of Stuart Lipton at the Burnham Institute for Medical Research.
All constructs were confirmed by sequencing.
Antibodies and immunoassays. A monoclonal antibody against DJ-1
(E2.19) was purchased from Signet Laboratories. Polyclonal anti-Parkin
antibody was from Cell Signaling Technology. Anti-Parkin monoclonal
antibody, anti-myc–tagged monoclonal antibody (9E10.2), agarose con-
jugated with a polyclonal anti-myc antibody, monoclonal antibodies
against VSVG (P4D5) and FLAG (M2) tags and their conjugated agarose
were from Sigma-Aldrich. A polyclonal anti-PINK1 antibody was from
Imgenex. Immunoblotting and immunoprecipitation were performed
as previously described (44). Treatment with MG132 (5–10 μM)
and leupeptin (50 μM) was performed at 37°C overnight. Heat shock
was performed at 42°C for 2 h followed by recovery at 37°C.
Generation of Parkin, PINK1, and DJ-1 fusion proteins. cDNAs encod-
ing tagged Parkin (Parkin-flag-myc) and PINK1 (PINK1-flag-
VSVG) were cloned into pFastBac1 vectors and transformed into
DH10Bac competent cells. Individual recombinant bacmid DNAs
were then transfected into Sf9 insect cells using Superfect transfec-
tion reagent (Qiagen). Recombinant baculovirus was then trans-
ferred and amplified in Sf9 cells. Cell pellets were collected after
48 h followed by affinity purification using anti-flag antibody
(M2) conjugated to agarose and eluted with flag peptides accord-
ing to the manufacturer’s instructions (Sigma-Aldrich).
To generate DJ-1 fusion proteins, cDNA encoding DJ-1–VSVG was
subcloned into pGEX4T2. Protein expression and purification were
done according to the manufacturer’s instructions (GE).
Parkin, PINK1, and DJ-1 complex assembly in vitro. Affinity-purified
Parkin-myc-flag (0.1 μg), PINK1-VSVG-flag (0.1 μg), and GST–DJ-1–
VSVG (0.2 μg) fusion proteins were added to 400 μl binding buffer
(10 mM HEPES, pH 7.5, 142.4 mM KCl, 5 mM MgCl2, 1 mM EGTA,
1% NP-40, 5% BSA, and 10% glycerol) in various combinations. Anti-
myc beads (15 μl) and 15 μl GST beads were included in the binding
assay to precipitate Parkin and DJ-1, respectively. As an internal con-
trol, 0.2 μg of his-KChIP1 fusion protein was added to each assay.
Mixtures were rotated at room temperature for 3 h. Beads were washed
5 times with 1 ml of BSA-free binding buffer. Proteins binding to beads
were separated on 4%–20% Tris-Glycine gels and detected by immunoblot-
ting with corresponding antibodies.
Mitochondrial and cytosolic fractionation. Isolation of mitochondria and
cytosolic fraction from cells expressing Parkin, PINK1, and DJ-1 was done
as previously described (45).
Metabolic labeling and pulse-chase analysis. Cells were metabolically labeled
with 35S-protein labeling mix for 15–25 min, followed by a chase with
DMEM/10% FBS/3 mM unlabeled methionine for various time periods.
For MG132 treatment, cells were pretreated (5 μM) for 3 h followed by a
PINK1 promotes degradation of Parkin that has accumulated as a result of
heat shock treatment. Cells expressing Parkin alone (lanes 2–4); Parkin and
ubiquitin (lanes 5–7); and Parkin, ubiquitin, and PINK1 (lanes 8–10) without
heat shock treatment (lanes 1, 2, 5, 8), with heat shock treatment (lanes 3,
6, 9), or with heat shock treatment followed by a 4-h recovery time (RT) at
37°C (lanes 4, 7, 10). The cells were lysed and immunoprecipitated with an
anti-Parkin antibody, followed by immunoblotting with either an anti-ubiquitin
antibody (A) or an anti-Parkin antibody (B). Expression of PINK1 (C) and
tubulin (D) were shown with immunoblotting. Note that heat shock treatment
increased accumulation of Parkin protein (B; lanes 6–7). PINK1 promoted
degradation of Parkin even with heat shock treatment (B; lanes 9–10).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
chase in medium containing 5 μM MG132. Cells were lysed in 1 ml RIPA
buffer. Cell lysates were immunoprecipitated with respective antibodies.
The results were quantified using a phosphorimager.
Protein ubiquitination in transfected cells. Ubiquitination was analyzed
as previously described (46). Briefly, cells transfected with various plas-
mid combinations were lysed in 2% SDS buffer (2% SDS, 150 mM NaCl,
10 mM Tris-HCl, pH 8.0, 2 mM sodium orthovanadate, 50 mM sodium
fluoride, 1× protease inhibitors) and boiled for 10 min followed by soni-
cation. Lysates were diluted 1:10 in dilution buffer (10 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100), incubated at
4°C for 1 h with rotation, and centrifuged at 20,800 g for 30 min. Equal
amounts (1–1.5 mg) of protein were used for immunoprecipitation.
Immunoprecipitated proteins were washed with washing buffer (10 mM
Tris-HCl, pH 8.0, 1 M NaCl, 1 mM EDTA, 1% NP-40), boiled in SDS
sample buffer, and separated on SDS-PAGE. Ubiquitin and precipitated
proteins were immunodetected with antibodies.
In vitro ubiquitination assay. Affinity-purified Parkin-flag-myc (0.2 μg),
PINK1-flag-VSVG (0.2 μg), 10 ng recombinant mouse E1 (10 ng) and
GST-ubc7 (500 ng) were added to 40 μl ubiquitination reactions (50 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 10 nM okadaic acid, 0.6 mM DTT, 2 mM
NaF, 2 mM ATP, 0.03 U/μl creatine phosphokinase, 30 mM creatine phos-
phate) and 100 ng/μl bovine ubiquitin (Sigma-Aldrich) in various combina-
tions. Reactions proceeded for 1 h at 37°C and were stopped by addition of
SDS-PAGE sample buffer. Proteins were separated on a 4%–20% SDS-PAGE
gel and immunoblotted with anti-Parkin or anti-VSVG antibodies.
Mouse brain slice culture. Mesencephalon brain slice culture was prepared from
brains of P4 or P5 PINK1-null mice and their control littermates as previously
described with minor modifications (47). Briefly, 300-μm-thick coronal mes-
PD-pathogenic Parkin or PINK1 mutants impair Parkin degradation. (A) Interaction of PINK1WT with PD-pathogenic ParkinΔE4. Cells express-
ing PINK1WT alone (Control), PINK1WT and ParkinΔE4 (ΔE4), or PINK1WT and ParkinWT (Parkin) were immunoprecipitated with an anti-flag
antibody (to precipitate PINK1), followed by immunoblotting with an anti-myc antibody (to detect Parkin variants). (B) Interaction of ParkinWT
with PINK1G309D. Cells expressing ParkinWT alone (control), ParkinWT and PINK1G309D (G309D), or ParkinWT and PINK1WT (PINK1) were
immunoprecipitated with anti-myc antibody (to precipitate Parkin), followed by immunoblotting with an anti-flag antibody (to detect PINK1 vari-
ants). (C) PINK1 promoted degradation of PD-pathogenic Parkin mutants. ParkinWT (WT) and PD-pathogenic ParkinR42P, -T240W, and -ΔE4
were cotransfected with or without PINK1. Steady-state levels of Parkin, PINK1, and tubulin were analyzed by immunoblotting. PINK1 promoted
significant degradation of ParkinWT but not ParkinR42P, -T240W, or -ΔE4. (D) PD-pathogenic PINK1 mutants were impaired in promoting Parkin
degradation. PINK1WT (WT) and PD-pathogenic PINK1G309D, -T313M, and -P399L were cotransfected with or without Parkin. Steady-state
levels of Parkin, PINK1, and tubulin were detected by immunoblotting. PD-pathogenic mutants showed little or reduced ability to promote Parkin
degradation. (E and F) Quantitation of Parkin degradation affected by pathogenic Parkin (E) or PINK1 (F) mutations. The data were from 3
independent experiments. Relative Parkin levels were normalized to either the level of Parkin variants without PINK1 expression (E, black bars)
or the level of ParkinWT without PINK1 expression (Ctrl; F) in the same experiment.
research article Download full-text
660? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 119 Number 3 March 2009
encephalonal sections were prepared using a Mcllwain tissue chopper (Mickle
Laboratory Engineering Co.) and separated in ice-cold slice culture medium
consisting of 50% minimum essential medium, 25% HBSS, 25% heat-inacti-
vated horse serum supplemented with 36 mM glucose, 25 mM HEPES, 4 mM
NaHCO3, 1% fungizone, 100 U/ml penicillin, and 100 μg/ml streptomycin,
pH 7.2. Slices were placed on Millicell culture inserts and cultured in 6-well culture
plates. Organotypic cultures were maintained in an incubator gasified with a 5%
CO2/95% O2 atmosphere at 37°C, and the medium was changed 3 times/week.
Seven days after the cultures were established, half of the slices were treated
with heat shock in a 42°C incubator for 2 h followed by recovery at 37°C for an
additional 4 h. Endogenous Parkin was immunoprecipitated with a monoclo-
nal anti-Parkin antibody (Sigma-Aldrich). Endogenous ubiquitin was detected
with a rabbit monoclonal anti-ubiquitin antibody (Epitomics).
Statistics. Protein expression levels were quantified using ImageJ. All
quantitative data were analyzed with the 2-tailed Student’s t test using
InStat version 3 (GraphPad). All error bars indicate SEM.
This work was supported by NIH grants RO1 DC006497, RO1
NS057289, and PO1 ES016738 (to Z. Zhang); California Institute
for Regenerative Medicine grants RS1-00331-1 and RL1-00682-1
(to Z. Zhang); Chinese Natural Science Foundation grants (to Z.
Zhang); the American Parkinson Disease Association (to Z. Zhang
and Y. S. Choo); and the Michael J. Fox Foundation for Parkinson’s
Research (to X. Zhuang and Z. Zhang).
Received for publication October 1, 2008, and accepted in revised
form January 7, 2009.
Address correspondence to: Zhuohua Zhang, Burnham Institute
for Medical Research, 10901 N. Torrey Pines Road, La Jolla, Cali-
fornia 92037, USA. Phone: (858) 795-5286; Fax: (858) 646-3198;
1. Dawson, T.M., and Dawson, V.L. 2003. Molecular path-
ways of neurodegeneration in Parkinson’s disease.
2. Valente, E.M., et al. 2004. Hereditary early-onset
Parkinson’s disease caused by mutations in PINK1.
3. Paisan-Ruiz, C., et al. 2004. Cloning of the gene
containing mutations that cause PARK8-linked
Parkinson’s disease. Neuron. 44:595–600.
4. Zimprich, A., et al. 2004. Mutations in LRRK2
cause autosomal-dominant parkinsonism with
pleomorphic pathology. Neuron. 44:601–607.
5. Cookson, M.R. 2005. The biochemistry of Parkin-
son’s disease. Annu. Rev. Biochem. 74:29–52.
6. Ramirez, A., et al. 2006. Hereditary parkinsonism
with dementia is caused by mutations in ATP13A2,
encoding a lysosomal type 5 P-type ATPase. Nat.
7. Spillantini, M.G., et al. 1997. Alpha-synuclein in
Lewy bodies. Nature. 388:839–840.
8. Conway, K.A., et al. 2000. Acceleration of oligomer-
ization, not fibrillization, is a shared property of
both alpha-synuclein mutations linked to early-
onset Parkinson’s disease: implications for patho-
genesis and therapy. Proc. Natl. Acad. Sci. U. S. A.
9. Conway, K.A., Harper, J.D., and Lansbury, P.T.
1998. Accelerated in vitro fibril formation by a
mutant alpha-synuclein linked to early-onset Par-
kinson disease. Nat. Med. 4:1318–1320.
10. Shimura, H., et al. 2000. Familial Parkinson disease
gene product, parkin, is a ubiquitin-protein ligase.
Nat. Genet. 25:302–305.
11. Zhang, Y., et al. 2000. Parkin functions as an E2-
dependent ubiquitin- protein ligase and promotes
the degradation of the synaptic vesicle-associ-
ated protein, CDCrel-1. Proc. Natl. Acad. Sci. U. S. A.
12. Imai, Y., Soda, M., and Takahashi, R. 2000. Parkin
suppresses unfolded protein stress-induced cell
death through its E3 ubiquitin-protein ligase activity.
J. Biol. Chem. 275:35661–35664.
13. Valente, E.M., et al. 2004. PINK1 mutations are
associated with sporadic early-onset parkinsonism.
Ann. Neurol. 56:336–341.
14. Plun-Favreau, H., et al. 2007. The mitochondrial
protease HtrA2 is regulated by Parkinson’s disease-
associated kinase PINK1. Nat. Cell Biol. 9:1243–1252.
15. Pridgeon, J.W., Olzmann, J.A., Chin, L.S., and Li,
L. 2007. PINK1 protects against oxidative stress by
phosphorylating mitochondrial chaperone TRAP1.
PLoS Biol. 5:e172.
16. Bonifati, V., et al. 2003. Mutations in the DJ-1 gene
associated with autosomal recessive early-onset
parkinsonism. Science. 299:256–259.
17. Taira, T., et al. 2004. DJ-1 has a role in antioxidative
stress to prevent cell death. EMBO Rep. 5:213–218.
18. Menzies, F.M., Yenisetti, S.C., and Min, K.T. 2005.
Roles of Drosophila DJ-1 in survival of dopami-
nergic neurons and oxidative stress. Curr. Biol.
19. Meulener, M., et al. 2005. Drosophila DJ-1 mutants
are selectively sensitive to environmental toxins
associated with Parkinson’s disease. Curr. Biol.
20. Goldberg, M.S., et al. 2003. Parkin-deficient mice
exhibit nigrostriatal deficits but not loss of dopa-
minergic neurons. J. Biol. Chem. 278:43628–43635.
21. Palacino, J.J., et al. 2004. Mitochondrial dysfunc-
tion and oxidative damage in parkin-deficient mice.
J. Biol. Chem. 279:18614–18622.
22. Clark, I.E., et al. 2006. Drosophila pink1 is required
for mitochondrial function and interacts geneti-
cally with parkin. Nature. 441:1162–1166.
23. Park, J., et al. 2006. Mitochondrial dysfunction in
Drosophila PINK1 mutants is complemented by
parkin. Nature. 441:1157–1161.
24. Wang, D., et al. 2006. Antioxidants protect PINK1-
dependent dopaminergic neurons in Drosophila.
Proc. Natl. Acad. Sci. U. S. A. 103:13520–13525.
25. Yang, Y., et al. 2006. Mitochondrial pathology
and muscle and dopaminergic neuron degenera-
tion caused by inactivation of Drosophila Pink1
is rescued by Parkin. Proc. Natl. Acad. Sci. U. S. A.
26. Chung, K.K., et al. 2001. Parkin ubiquitinates the
alpha-synuclein-interacting protein, synphilin-1:
implications for Lewy-body formation in Parkin-
son disease. Nat. Med. 7:1144–1150.
27. Tang, B., et al. 2006. Association of PINK1 and DJ-1
confers digenic inheritance of early-onset Parkin-
son’s disease. Hum. Mol. Genet. 15:1816–1825.
28. Chen, L., et al. 2005. Age-dependent motor deficits
and dopaminergic dysfunction in DJ-1 null mice.
J. Biol. Chem. 280:21418–21426.
29. Beilina, A., et al. 2005. Mutations in PTEN-induced
putative kinase 1 associated with recessive parkin-
sonism have differential effects on protein stability.
Proc. Natl. Acad. Sci. U. S. A. 102:5703–5708.
30. Glickman, M.H., and Ciechanover, A. 2002.
The ubiquitin-proteasome proteolytic pathway:
destruction for the sake of construction. Physiol.
31. Joazeiro, C.A., et al. 1999. The tyrosine kinase nega-
tive regulator c-Cbl as a RING-type, E2-dependent
ubiquitin-protein ligase. Science. 286:309–312.
32. Petroski, M.D., and Deshaies, R.J. 2005. Function
and regulation of cullin-RING ubiquitin ligases.
Nat. Rev. Mol. Cell Biol. 6:9–20.
33. Hattori, N., and Mizuno, Y. 2004. Pathogenetic
mechanisms of parkin in Parkinson’s disease. Lancet.
34. Moore, D.J., West, A.B., Dawson, V.L., and Dawson,
T.M. 2005. Molecular pathophysiology of Parkin-
son’s disease. Annu. Rev. Neurosci. 28:57–87.
35. Jiang, H., Ren, Y., Zhao, J., and Feng, J. 2004. Parkin
protects human dopaminergic neuroblastoma cells
against dopamine-induced apoptosis. Hum. Mol.
36. Greene, J.C., et al. 2003. Mitochondrial pathol-
ogy and apoptotic muscle degeneration in Dro-
sophila parkin mutants. Proc. Natl. Acad. Sci. U. S. A.
37. Pesah, Y., et al. 2004. Drosophila parkin mutants
have decreased mass and cell size and increased
sensitivity to oxygen radical stress. Development.
38. Kim, R.H., et al. 2005. Hypersensitivity of DJ-1-
deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetra-
hydropyrindine (MPTP) and oxidative stress. Proc.
Natl. Acad. Sci. U. S. A. 102:5215–5220.
39. Moore, D.J., et al. 2005. Association of DJ-1 and
parkin mediated by pathogenic DJ-1 mutations
and oxidative stress. Hum. Mol. Genet. 14:71–84.
40. Imai, Y., et al. 2002. CHIP is associated with Parkin,
a gene responsible for familial Parkinson’s disease,
and enhances its ubiquitin ligase activity. Mol. Cell.
41. Shimura, H., et al. 2001. Ubiquitination of a new
form of alpha-synuclein by parkin from human
brain: implications for Parkinson’s disease. Science.
42. Smith, W.W., et al. 2005. Leucine-rich repeat kinase 2
(LRRK2) interacts with parkin, and mutant LRRK2
induces neuronal degeneration. Proc. Natl. Acad. Sci.
U. S. A. 102:18676–18681.
43. Habelhah, H., et al. 2004. Ubiquitination and
translocation of TRAF2 is required for activation
of JNK but not of p38 or NF-kappaB. EMBO J.
44. Zhang, Z., et al. 1998. Destabilization of beta-
catenin by mutations in presenilin-1 potentiates
neuronal apoptosis. Nature. 395:698–702.
45. Choo, Y.S., Johnson, G.V., MacDonald, M., Detloff,
P.J., and Lesort, M. 2004. Mutant huntingtin direct-
ly increases susceptibility of mitochondria to the
calcium-induced permeability transition and cyto-
chrome c release. Hum. Mol. Genet. 13:1407–1420.
46. Didier, C., et al. 2003. RNF5, a RING finger protein
that regulates cell motility by targeting paxillin
ubiquitination and altered localization. Mol. Cell.
47. Stoppini, L., Buchs, P.A., and Muller, D. 1991. A
simple method for organotypic cultures of nervous
tissue. J. Neurosci. Methods. 37:173–182.