From the Novartis Pharma AG, Nervous System Research, CH-4002 Basel, Switzerland
Received for publication, February 4, 2003, and in revised form, March 5, 2003
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ABSTRACT |
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Parkin is a ubiquitin-protein isopeptide
ligase (E3) involved in ubiquitin/proteasome-mediated protein
degradation. Mutations in the parkin gene cause a
loss-of-function and/or alter protein levels of parkin. As a result,
the toxic build-up of parkin substrates is thought to lead to autosomal
recessive juvenile Parkinsonism. To identify a role for the
ubiquitin-like domain (ULD) of parkin, we created a number of
hemagglutinin (HA)-tagged parkin constructs using mutational and
structural information. Western blotting and immunocytochemistry showed
a much stronger expression level for HA-parkin residues 77-465
(without ULD) than HA-parkin full-length (with ULD). The deletion of
ULD in Drosophila parkin also caused a sharp increase in
expression of the truncated form, suggesting that the function of the
ULD of parkin is conserved across species. By progressive deletion
analysis of parkin ULD, we found that residues 1-6 of human parkin
play a crucial role in controlling the expression levels of this gene.
HA-parkin residues 77-465 showed ubiquitination in vivo,
demonstrating that the ULD is not critical for parkin
auto-ubiquitination; ubiquitination seemed to cluster on the central
domain of parkin (residues 77-313). These effects were specific for
the ULD of parkin and not transfection-, toxic-, epitope tag-, and/or
vector-dependent. Taken together, these data suggest that
the 76 most NH2-terminal residues (ULD) dramatically
regulate the protein levels of parkin.
Dopaminergic neurons found in the substantia nigra pars
compacta are progressively lost in the crippling neurodegenerative movement disorder, Parkinson's disease (1). Autosomal recessive juvenile Parkinson's
(ARJP),1 which recapitulates
the classical parkinsonian symptoms of tremor, rigidity and
bradykinesia, is characterized by an early age of onset, and it is
linked to loss-of-function mutations in the parkin gene and
has a recessive mode of inheritance where both alleles of parkin are
mutated (2-7). Parkin has been suggested to be an E3 ligase and, in
combination with its E1 and E2 enzyme partners, may be involved in the
polyubiquitination of substrate proteins (8-11). Parkin is thought not
to directly ubiquitinate substrate proteins but rather act as a
"docking station" for the coming together of its substrates and
appropriate E2 enzymes allowing the transfer of ubiquitin from the E2
enzyme to the target protein. Polyubiquitination of proteins is a
priming event for protein degradation via the ubiquitin/proteosome
pathway (12, 13). The lack of parkin function (i.e. mutation
in both alleles) is thought to lead to the progressive accumulation of
it substrates leading to cell stress, degeneration, and eventually
death of dopaminergic neurons (7).
Human parkin is 465 amino acids in length (~52 kDa) with its residues
1-76 forming a ubiquitin homology-like domain (ULD) that shares 62%
homology to ubiquitin and may be involved in target protein recognition
(14): residues 145-232 forming a central domain with unknown function,
residues 237-449 making a carboxyl-terminal RING(R1)-IBR-RING(R2)
domain involved in substrate and E2 interaction (2, 10, 15-17), and
the extreme three carboxyl-terminal residues creating a PDZ binding
motif (18). Recently a number of proteins have been shown to interact
with parkin (for reviews, see Refs. 8 and 19). These include the E2
enzymes UbcH7 and UbcH8 (10, 11, 20), a septin GTPase named CDCrel-1
(11), parkin-associated endothelin receptor-like (Pael) (9, 20),
O-glycosylated In addition to ubiquitination of its substrates, parkin is also known
to catalyze its own ubiquitination and proteasomal-mediated degradation, thus regulating its own cellular levels (11, 26). The E2
enzymes involved in parkin self-ubiquitination are currently unknown.
Nevertheless, a set of studies have shown that E2 enzymes, UbcH7 and
UbcH8, interact with the RING-IBR-RING domain parkin and are involved
in the ubiquitination of parkin substrates (9-11, 14, 20, 26). More
specifically, in one report both UbcH7 and UbcH8 were shown to bind to
parkin with equal affinities (20). Another group have isolated a
parkin-UbcH7 complex from human brain (10) and showed that UbcH8 weakly
binds parkin (14). A third report suggested that parkin only interacts
with UbcH8 and that the low expression level of UbcH7 in brain makes it
less physiological relevant for interaction with parkin (11). Finally, in yeast two-hybrid studies UbcH7 has been shown to interact with a
human homologue of Drosophila ariadne (HHARI) and
UbcH7-associated protein 1 (H7-AP1) (16, 17). Both HHARI and H7-AP1
contain a RING-IBR-RING domain and together with parkin constitute a
family of parkin/aridine like ubiquitin ligases (PAULs) (7). In these studies, the weak interaction between parkin-UbcH8 and HHARI-UbcH8 was
suggested not to be physiologically relevant (17).
The structural domains within parkin that may regulate its expression
and/or self-ubiquitination have not been clearly investigated. In
addition, the E2 enzymes involved in parkin ubiquitination and control
its degradation rate also remain unclear. Our present study
investigates some of the factors that may control cellular protein
levels of parkin. Here, we demonstrate that the ULD of parkin,
specifically its first six residues, is important for regulating
protein levels of parkin in the cell. These data suggest that residues
77-313 of parkin play an important role in self-ubiquitination.
Plasmids Construction and Molecular Biology--
cDNA
fragments of parkin (human clone AB009973; Drosophila clone
AE003593) corresponding to the residues indicated in Fig. 1 were
amplified using PCR and subcloned into the mammalian expression vector
pCI (Promega, Madison, WI) or into the Sf9/baculovirus GST tag
expression vector pGEX-PreScission (Amersham Biosciences) by
standard cloning procedures. Amino acid residue mutations and deletions
were introduced in human parkin cDNA using QuikChangeTM
kit (Stratagene, Amsterdam, Netherlands). A hemagglutinin (HA) or a Myc
epitope tag was added to the NH2 terminus of human parkin or Drosophila parkin, respectively. Similar procedures were
used for the creation of Myc-UbcH7 (human clone X92962) and
Myc-UbcH8 (human clone AF031141) mammalian expression
constructs. Isolation of all DNA from transformed Escherichia
coli was performed using Qiagen plasmid kits (Qiagen, Basel,
Switzerland). The integrity of constructs was verified by DNA
sequencing as described by manufactures (Applied
Biosystems-PerkinElmer Life Sciences, Rotkreuz, Switzerland).
Cell Culture and Biochemistry--
HEK-293 cells were grown in
80-cm2 flasks using Dulbecco's modified Eagle's
medium/Ham's F-12 culture media (3.151 g/liter glucose, with 0.524 g/liter L-alanyl-L-glutamine (Invitrogen, Basel, Switzerland) supplemented with 10% dialyzed fetal calf serum
(Amimed, Basel, Switzerland) at 37 °C in a 5% CO2
incubator. HEK-293 cells were transfected as described previously (27) with 0.5-1 µg each plasmid DNA in the presence of Opti-MEM using LipofectAMINE/PLUS reagent (Invitrogen). The cells were used ~48 h
after transfection. For expression analysis the cells were scraped from
multiwell plates, the cell pellets solubilized in 50-100 µl of
radioimmune precipitation assay buffer, and the samples (10-20 µl)
denatured. Electrophoretic separation was carried out on 10 or 12%
SDS-polyacrylamide gels (BioWhittaker, Allschwill, Switzerland) as
described previously (27). The blots were developed either by
incubating in alkaline phosphatase buffer (Promega), and
immunoreactivity was visualized as a blue-purple color or by incubating
in enhanced chemiluminescence (ECL) supersignal west femto substrate
reagent (Pierce, Bonn, Germany) and then exposed to film and developed.
For immunocytochemistry, transfected HEK-293 cells were grown on
coverslips and immunocytochemistry performed as described previously
(27).
Antibodies--
Polyclonal rabibit primary antibodies include
anti-parkin (AbCam, Cambridge, UK), anti-HA (Santa Cruz Biotechnology,
Santa Cruz, CA), and anti-ubiquitin (Calbiochem, Schwalbach, Germany). Monoclonal mouse primary antibodies include anti-Myc (Oncogen, Boston, MA) and anti-HA (Santa Cruz Biotechnology). The secondary antibodies that were alkaline phosphatase-conjugated were goat anti-rabbit IgG (Promega) and goat anti-mouse IgG (Promega). The secondary antibodies that were horseradish peroxidase-conjugated were
goat anti-rabbit IgG (Sigma) or goat anti-mouse IgG (Sigma). The
secondary antibodies used in immunocytochemistry were Texas Red-X goat
anti-rabbit IgG (Molecular Probes, Eugene, OR) or Texas Red-X goat
anti-mouse IgG (Molecular Probes). All antibodies were used at
dilutions recommended by the manufacturer. Protein concentrations were
determined using the Bio-Rad protein assay kit using serum bovine
albumin as a standard (Bio-Rad, Munchen, Germany).
The ULD of Parkin Regulates Parkin Expression but Not Its
Ubiquitination--
The parkin gene (PARK2) was
identified by positional cloning from genetic materials donated by
Japanese families affected by ARJP (2). It was shown that ARJP has a
recessive mode of inheritance where both alleles of parkin are mutated
(4). To date several mutations in parkin have been linked to ARJP
(2-7). We generated a number of parkin constructs to examine the
function of the parkin domains and the effects of disease-causing
mutations on parkin expression (Fig.
1A). When equivalent amounts
of cDNA were transfected we found that cellular levels of
NH2-terminal HA-tagged full-length parkin, HA-parkin-FL,
proteins (either wild type or mutants) were too low for detection by
Western blot analysis (Fig. 1B). In contrast, all the
constructs where residues 1-76 (ULD) were removed, HA-parkin-77
deletion mutants, showed high levels of expression in HEK-293 cells
(Fig. 1B). None of the point mutations tested were found to
significantly alter the expression levels of HA-parkin-FL or
HA-parkin-77.
The self-ubiquitination of parkin is an event that controls parkin
degradation rates (14, 24). In some reports, overexpressed parkin has
been found ubiquitinated in vivo (20), while in other studies parkin was not ubiquitinated (14). To explain these results, it
has been suggested that the higher expression levels of parkin lead to
the better detection of ubiquitinated parkin (20). The molecular
mass of full-length parkin is ~52 kDa, the mass of
HA-parkin-77 is ~43 kDa, and the molecular mass of ubiquitin is
~8.5 kDa. In our study, HA-parkin-77-expressed proteins showed band
sizes corresponding to the molecular masses of native (~43 kDa) and
monoubiquitinated (~51 kDa) HA-parkin-77 (Fig. 1B). We also detected band smears for some (but not all) HA-parkin-77 proteins,
indicating these band patterns are specific for particular forms of
HA-parkin-77 (Fig. 1B). Of interest, when
HA-parkin-77-transfected cells were resuspended, sonicated, and rotated
in PT×E buffer (phosphate-buffered saline, 1% Triton X-100, 0.1 mM EDTA, pH 7.4; see Ref. 27), these band patterns were
found predominantly in the cell pellet fraction as compared with the
cell sonicate. As reported previously, we suggest these high molecular
weight band patterns to represent polyubiquitinated parkin (20).
Specifically, we found HA-parkin constructs encoding only the central
domain and RING1 (residues 77-313) were mono- and polyubiquitinated, whereas those encoding the central domain alone (residues 77-237) were
mono- but not polyubiquitinated (Fig. 1B). Taken
together, this data indicate that residues 1-76 (the ULD) of parkin
regulate its protein levels, and as a result the high protein levels of HA-parkin-77 lead to elevated ubiquitination. Furthermore, the removal
of the ULD of parkin does not significantly alter parkin ubiquitination
and that parkin polyubiquitination requires the presence of central and
RING1 domains but not the ULD.
To examine the effects of UbcH7 and UbcH8 on the protein levels and
ubiquitination of parkin we co-expressed them with UbcH7 or UbcH8.
Overexpression of UbcH7 or UbcH8 appeared neither to increase parkin
ubiquitination nor to regulate the expression of HA-parkin-FL or
HA-parkin-77 (data not shown). Interestingly, the proteasome inhibitor
MG132 also showed no significant effects on the expression levels of
HA-parkin-FL or HA-parkin-77 (data not shown). These results suggest
that the ubiquitination of parkin may be associated with a regulation
of its function rather than proteolysis-associated ubiquitination or
that proteolysis-associated ubiquitination of overexpressed
HA-parkin-FL or HA-parkin-77 may not be the (only) mechanism
controlling their cellular protein levels. Next, we performed a number
of in vitro ubiquitination assays to identify E2 enzymes
involved in the auto-ubiquitination of HA-parkin-77. Despite observing
the ubiquitination of p27 (as control), we did not observe any evidence
of parkin self-ubiquitination using the E2 enzyme UbcH7 or other E2
enzymes UbcH2, -3, -5a, -5b, -6, -7, and -10 (data not shown). These
results are in agreement with a previous report suggesting that UbcH7,
and perhaps UbcH8, are involved in parkin-substrate ubiquitination, but
these two enzymes do not directly affect parkin self-ubiquitination
(20). We also attempted to clarify conflicting data from previous
studies suggesting that parkin interacts with only UbcH7, or UbcH8, or both (11, 14, 20). In yeast two-hybrid assays (and
co-immunoprecipitation studies) we were unable to observe any specific
interaction between parkin and UbcH7 or UbcH8, despite identifying
novel interacting proteins for full-length parkin (parkin-FL) in a
large-scale yeast two-hybrid
study.2 Interestingly, in
yeast two-hybrid studies, we found that the NH2-terminal
deletion mutant parkin 77-465 (but not parkin-FL or parkin 1-76)
showed a strong activation of Parkin Expression Is Not Transfection-, Toxic-, Epitope Tag-,
and/or Vector-dependent--
Numerous
investigators have successfully expressed full-length wild type and
mutant parkin using different tags in HEK-293 cells and neuroblastoma
cells (18, 20, 21). Within these studies, some differences have been
noted in terms of E2 enzymes that interact with parkin and the
ubiquitination of overexpressed parkin (compare Refs. 10-11, 14, and
20). These differences have previously been linked, in part, to unknown
cell type-specific factor(s), possibly E2 enzymes, co-factors, and/or
associated proteins (20). Although reasons for the poor expression of
HA-parkin-FL is presently unclear, our current studies have enabled to
detect differences between HA-parkin-FL and HA-parkin-77 and thus
determine the effects of ULD on parkin expression. To further validate
our hypothesis that the ULD of parkin plays a role in regulating its cellular protein levels, we have performed a number of control experiments.
First, as control, we observed a single, specific, and antibody
concentration-dependent band for native full-length parkin in a variety of cell types and brain tissue regions (Fig.
2A). Of note, we did not find
endogenous full-length parkin to be ubiquitinated (Fig. 2A).
Second, to determine whether the differences in HA-parkin-77 and
HA-parkin-FL protein expression were due to differences in transfection
methods, we tested a number of transfection protocols (Fig.
2B). LipofectAMINE and NeuroPorter transfection complex mixtures gave similar results showing that HA-parkin-77 was highly expressed compared with that of HA-parkin-FL and that these results were not due to differences in transfection methods (Fig.
2B). Third, to exclude the possibility that lack of
parkin-FL expression is not epitope tag- and/or vector
type-dependent, we tested the role of ULD in a
Myc-tagged Drosophila parkin. In HEK-293 cells, we
found that Drosophila Myc-parkin-FL showed no
expression whereas Drosophila Myc-parkin-93 (lacking
the ULD, residues 1-92) showed high expression levels (Fig.
2B). Finally, to confirm that the lack of parkin-FL
expression was not due to detection problems caused by our parkin gene
sequence or by antibody recognition, we used a large batch
non-mammalian cellular expression system. Using Sf9/baculovirus
expression, we still found that GST-parkin-FL was poorly expressed as
compared with GST-parkin-77 (Fig. 2C). However, by using
large culture volumes and affinity purification we were able to observe
small amounts of GST-parkin-FL, further indicating that the differences
in GST-parkin-77 and GST-parkin-FL are not due to the epitope tag
and/or vector type used (Fig. 2C). Taken together, these
results indicate that the ULD of parkin is responsible for controlling
not only the protein levels of human parkin but also
Drosophila parkin and that these effects are not dependent
on the epitope tags and/or vectors used.
To further examine the role of the ULD of parkin, we performed
immunocytochemistry to observe the distribution patterns of parkin
77-465 on a single cell level and to determine any low level
expression of parkin-FL. In HEK cells, the distribution pattern of
endogenous parkin was found to be diffuse and cytoplasmic (Fig.
2D). No oddities (for example clusters, endoplasmic
reticulum retention, and/or nuclear build up) were found in the
distribution patterns of transfected HA-tagged human parkin constructs
(Fig. 2D). In agreement with Western blotting results, in
these studies, we found low protein levels of HA-parkin-FL as compared
with the high expression levels of HA-parkin-77. Similar results were
found using Myc-tagged Drosophila parkin, where
full-length Myc-parkin showed a lower expression level as
compared with Myc-parkin-93 (Fig. 2D). As control,
the E2 enzymes showed similar expression levels as human HA-parkin-77
and Drosophila Myc-parkin-93, indicating that
expression was not non-specifically dependent on the used vector or
epitope tag (Fig. 2D).
To test for differences in toxicity between HA-parkin-FL and
HA-parkin-77, we also determined whether transient expression of
HA-parkin-FL and HA-parkin-77 altered cell viability. HA-parkin constructs, together with a luciferase reporter plasmid, were transiently transfected into HEK-293 cells. Cellular viability was
quantified in terms of luciferase activity. When compared with empty
pCI vector-transfected cells (taken as 100%), HA-parkin-FL (138 ± 12%) and HA-parkin-77 (134 ± 13%) had no drastic effects on
cellular viability and in some cases showed a slight protective effect
as suggested previously by other reports (9, 20, 22, 29). In contrast
the transfection of Bax cDNA (15 ± 2%) and caspase cDNA
(2 ± 0) into HEK-293 cells resulted in cellular death. These
results indicate that differences in HA-parkin-FL and HA-parkin-77 expression cannot be explained by some intrinsic cytotoxicity related
to these expression constructs (data not shown).
Residues 1-6 of the ULD of Parkin Regulate Protein
Expression--
To further characterize the exact site within the ULD
of parkin responsible for controlling its protein levels, a series of progressive deletion mutants within the ULD of parkin were created and
tested for expression (Fig.
3A). Using the same
concentrations of cDNA for transfections, we found that removal of
the first six residues, namely MIVFVR, of parkin (see deletion
constructs
In summary, in this study we identify two structural/functional domains
within parkin, namely the ULD (residues 1-76, specifically the MIVFVR
epitope) as a site that controls parkin expression and the central + RING1 domain (residues 77-313) as a site that plays a role in parkin
ubiquitination. The molecular mechanism regulating parkin protein
levels is not well understood. We propose that the ULD of parkin could
play a role in protein-protein interaction events where this domain is
recognized either directly or indirectly by an associated protein that
targets parkin for proteolysis. Under normal conditions,
polyubiqutination of parkin could alter proteins interacting with
parkin, modulate parkin function, and/or regulate the rate parkin
proteolysis. In disease or under experimental conditions an altered ULD
may result in parkin no longer being recognized by accessory protein(s)
and/or being degraded and thus leading to changes of parkin protein
levels in the cell.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-synuclein (10), synphilin-1 (21),
calcium/ calmodulin-dependent serine protein kinase
(CASK/Lin2) (18), and more recently HSP-70 and CHIP1 (22). Parkin
has also been shown to interact with actin filaments but not
microtublues in COS1 cells (23), to bind actin and the actin-binding
protein, filamin (24), and be associated with synaptic vesicles
(25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
A, Alignment of disease-linked mutations
of parkin. Schematic representation of parkin domains and
constructs made is shown (comparative sizes of domains are not to
scale). Based on previous information on disease-causing mutations,
various constructs of truncated and mutated fragments of HA-tagged
human parkin were made. Circles indicate site of point
mutation along the parkin protein. B, in vivo
ubiquitination of HA-parkin-77 in transiently transfected HEK-293
cells. Twenty-three different parkin constructs were independently
transfected into HEK-293 cells and the cell pellets used in Western
blots using a HA rabbit antibody. Parkin lacking residues 1-76 (the
ULD) showed high levels of expression compared with parkin containing
the ULD. Both mono- and polyubiquitinated bands were found for parkin
proteins containing the central and RING1 domains, but only
monoubiquitinated bands were found for parkin fragments containing only
the central domain.
-galactosidase, suggesting that
residues 1-76 of parkin may also play a negative role in gene
transcription (data not shown). In agreement, parkin has been suggested
to play a role in transcription and/or in ubiquitinating a nuclear
substrate (28).
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Fig. 2.
A, native parkin was observed in
a variety of cell types and brain tissue regions. Western blotting was
performed using parkin antibody. B, LipofectAMINE (see
"Experimental Procedures") or NeuroPorter (see the manufacturer's
instructions, Gene Therapy Systems Inc., San Diego, CA) transfection
methods were tested. Western blotting was performed using HA rabbit
antibody or Myc mouse antibody. The two methods gave similar results
for both human parkin and Drosophila parkin, indicating that
the ULD of parkin reduces parkin expression. C,
GST-(PreScission)-parkin was prepared using Sf9/baculovirus
expression. After large batch culture the GST-(PreScission)-parkin was
purified by standard GST affinity chromatography and treated with
PreScission (see the manufacturer's instructions, Amersham
Biosciences). The PreScission-cleaved parkin (parkin-FL or
parkin-77) was collected and processed for Western blotting using a
parkin antibody. The expression of parkin-FL was only observed after
longer exposure. D, single cell expression levels of parkin
in HEK-293 cells. Endogenous parkin was found in HEK-293 cells as
detected by parkin rabbit antibody followed by Texas Red-conjugated
secondary antibody. Transiently transfected HA-tagged parkin proteins
were detected by HA rabbit antibody followed by Texas Red-conjugated
secondary antibody. HA-tagged human parkin (wild type) showed a low
level of expression at the single cell level. On comparison parkin
residues 77-465 (no ULD) showed high expression levels. Similar
results were observed for Drosophila parkin. Myc-UbcH7 and
Myc-UbcH8 showed similar high expression levels as human and
Drosophila parkin without its ULD.
1 or
1a) resulted in its expression detectable by
Western blotting (Fig. 3B). In contrast, any parkin
construct encoding the first 6 residues showed no detectable expression
(Fig. 3B). These results suggest that the first 6 residues
of the ULD of mammalian parkin play an important role in regulating its
protein levels. Since the ULD of Drosophila parkin also
appears to regulate its expression levels similar to that seen for the
ULD of its human counterpart (Fig. 2B), we searched for this
motif in Drosophila. Although this linear epitope is
conserved in human, mouse, and rat sequences of parkin, it was not
found in the Drosophila parkin (residues 1-6 correspond to
MLELLQ). Interestingly, Drosophila parkin also lacks the PDZ
binding motif found at the extreme carboxyl terminus of human, mouse,
and rat sequences of parkin. This PDZ binding motif is thought to be
critical for parkin interaction with CASK (18). Therefore it appears
that Drosophila parkin has some (but not complete) identity
in its structural/functional domains as compared with mammalian parkin.
Taken together, we propose that the ULD (although variable in sequence)
is likely to have a conserved function through evolution in terms of
regulating parkin levels in the cell.
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Fig. 3.
Effects of residues 1-6 (MIVFVR) of the ULD
of parkin on protein levels. A, schematic
representation of a series of deletions in the UDL of parkin is shown
and was made to further investigate the site within the UDL that may
regulate parkin expression levels. B, only the deletion
mutant lacking the residues FAATMIVFVR (of which FAAT belong the linker
sequence and MIVFVR belong to parkin) and the mutant lacking MIVFVR
were expressed, suggesting that MIVFVR regulates the levels of
parkin.
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ACKNOWLEDGEMENTS |
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We are grateful to Andre Strauss for work with Sf9/baculovirus expression system and Bastian Hengerer for the isolation of the Substantia Nigra.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 41-61-324-29-42;
Fax: 41-61-324-38-11; E-mail:
kumlesh_k.dev@pharma.novartis.com.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.C300051200
2 K. K. Dev, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ARJP, autosomal recessive juvenile Parkinson's; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; ULD, ubiquitin-like domain; GST, glutathione S-transferase.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Hornykiewicz, O. (1998) Neurology 1998, S2-S9 |
2. | Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y., and Shimizu, N. (1998) Nature 392, 605-608[CrossRef][Medline] [Order article via Infotrieve] |
3. | Leroy, E., Anastasopoulos, D., Konitsiotis, S., Lavedan, C., and Polymeropoulos, M. H. (1998) Hum. Genet. 103, 424-427[CrossRef][Medline] [Order article via Infotrieve] |
4. | Mizuno, Y., Hattori, N., and Matsumine, H. (1998) J. Neurochem. 71, 893-902[Medline] [Order article via Infotrieve] |
5. |
Abbas, N.,
Lucking, C. B.,
Ricard, S.,
Durr, A.,
Bonifati, V.,
De Michele, G.,
Bouley, S.,
Vaughan, J. R.,
Gasser, T.,
Marconi, R.,
Broussolle, E.,
Brefel-Courbon, C.,
Harhangi, B. S.,
Oostra, B. A.,
Fabrizo, E.,
Bohme, G. A.,
Pradier, L.,
Wood, N. W.,
Filla, A.,
Meco, G.,
Denefle, P.,
Agid, Y.,
and Brice, A.
(1999)
Hum. Mol. Genet.
8,
567-574 |
6. |
Lucking, C. B.,
Durr, A.,
Bonifati, V.,
Vaughan, J.,
De Michele, G.,
Gasser, T.,
Harhangi, B. S.,
Pollak, P.,
Bonnet, A.-M.,
Nichol, D.,
Mari, M. D.,
Marconi, R.,
Broussolle, E.,
Rascol, O.,
Rosier, M.,
Arnould, I.,
Oostra, B. A.,
Breteler, M. M. B.,
Filla, A.,
Meco, G.,
Denefle, P.,
Wood, N. W.,
Agid, Y.,
and Brice, A.
(2000)
N. Engl. J. Med.
342,
1560-1567 |
7. | Kahle, P. J., Leimer, U., and Haass, C. (2000) Trends Biochem. Sci 25, 524-527[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Haass, C.,
and Kahle, P. J.
(2001)
Science
293,
224-225 |
9. | Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y., and Takahashi, R. (2001) Cell 105, 891-902[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Shimura, H.,
Schlossmacher, M. G.,
Hattori, N.,
Frosch, M. P.,
Trockenbacher, A.,
Schneider, R.,
Mizuno, Y.,
Kosik, K. S.,
and Selkoe, D. J.
(2001)
Science
293,
263-269 |
11. |
Zhang, Y.,
Gao, J.,
Chung, K. K.,
Huang, H.,
Dawson, V. L.,
and Dawson, T. M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13354-13359 |
12. | Hershko, A., and Ciechanover, A. (1998) Annu. Rev. Biochem. 67, 425-479[CrossRef][Medline] [Order article via Infotrieve] |
13. | Joazeiro, C. A., and Weissman, A. M. (2000) Cell 102, 549-552[Medline] [Order article via Infotrieve] |
14. | Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S., Shimizu, N., Iwai, K., Chiba, T., Tanaka, K., and Suzuki, T. (2000) Nat. Genet. 25, 302-305[CrossRef][Medline] [Order article via Infotrieve] |
15. | Morett, E., and Brok, P. (1999) Trends Biochem. Sci. 24, 229-231[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Moynihan, T. P.,
Ardley, H. C.,
Nuber, U.,
Rose, S. A.,
Jones, P. F.,
Markham, A. F.,
Scheffner, M.,
and Robinson, P. A.
(1999)
J. Biol. Chem.
274,
30963-30968 |
17. |
Ardley, H. C.,
Tan, N. G.,
Rose, S. A.,
Markham, A. F.,
and Robinson, P. A.
(2001)
J. Biol. Chem.
276,
19640-19647 |
18. |
Fallon, L.,
Moreau, F.,
Croft, B. G.,
Labib, N.,
Gu, W. J.,
and Fon, E. A.
(2002)
J. Biol. Chem.
277,
486-491 |
19. | Dev, K. K., van der Putten, H., Sommer, S., and Rovelli, G. (2003) Neuropharmacol, in press |
20. |
Imai, Y.,
Soda, M.,
and Takahashi, R.
(2000)
J. Biol. Chem.
275,
35661-35664 |
21. | Chung, K. K., Zhang, Y., Lim, K. L., Tanaka, Y., Huang, H., Gao, J., Ross, C. A., Dawson, V. L., and Dawson, T. M. (2001) Nat. Med. 10, 1144-1150[CrossRef] |
22. | Imai, Y., Soda, M., Hatakeyama, S., Akagi, T., Hashikawa, T., Nakayama, K., and Takahashi, R. (2002) Mol. Cell 10, 55-67[Medline] [Order article via Infotrieve] |
23. | Huynh, D. P., Scoles, D. R., Ho, T. H., Del Bigio, M. R., and Pulst, S. M. (2000) Ann. Neurol. 48, 737-744[CrossRef][Medline] [Order article via Infotrieve] |
24. | Choi, P., Passer, B., Farrer, M., D'Adamio, L., Sparkman, D., Lee, J. M., and Wolozin, B. (2000) Soc. Neurosci. Abstr. 26, 13.11 |
25. | Kubo, S. I., Kitami, T., Noda, S., Shimura, H., Uchiyama, Y., Asakawa, S., Minoshima, S., Shimizu, N., Mizuno, Y., and Hattori, N. (2001) J. Neurochem. 78, 42-54[CrossRef][Medline] [Order article via Infotrieve] |
26. | Choi, P., Ostrerova-Golts, N., Sparkman, D., Cochran, E., Lee, J. M., and Wolozin, B. (2000) Neuroreport 11, 2635-2638[Medline] [Order article via Infotrieve] |
27. | Dev, K. K., Nishimune, A., Henley, J. M., and Nakanishi, S. (1999) Neuropharmacol. 38, 635-644[CrossRef][Medline] [Order article via Infotrieve] |
28. | Zheng, N., Wang, P., Jeffrey, P. D., and Pavletich, N. P. (2000) Cell 102, 533-539[Medline] [Order article via Infotrieve] |
29. |
Mengesdorf, T.,
Jensen, P. H.,
Mies, G.,
Aufenberg, C.,
and Paschen, W.
(2002)
Proc. Natl. Acad. Sci. U. S. A.
99,
15042-15047 |