Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY 14214, USA
* Author for correspondence (e-mail: jianfeng{at}buffalo.edu)
Accepted 5 June 2003
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Summary |
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Key words: Parkin, Centrosome, Aggresome, -Tubulin, Parkinson's disease
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Introduction |
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Subsequent studies have found that parkin is a protein-ubiquitin E3 ligase
(Shimura et al., 2000) and
many of its mutations are clustered in domains critical for its E3 ligase
activity (Giasson and Lee,
2001
). It suggests that abrogation of parkin's E3 ligase activity
and ensuing accumulation of its substrates are critical for the selective
degeneration of DA neurons in SNpc. Several substrates of parkin have been
identified. They include CDCrel-1 (Zhang
et al., 2000
), a member of the septin family GTPases that binds to
syntaxin-1 (Beites et al.,
1999
), but appears to be dispensable in neurotransmitter release
(Peng et al., 2002
). Another
substrate of parkin is Pael receptor, a homolog of endothelin receptor type B,
whose accumulation in brains with parkin mutations apparently leads to
unfolded protein stress in endoplasmic reticulum and may cause selective
neuronal death (Imai et al.,
2001
). More recently, two proteins related to
-synuclein,
were found to be substrates of parkin. One of them is an O-glycosylated form
of
-synuclein (Shimura et al.,
2001
), the other is the
-synuclein-binding protein,
synphilin-1 (Chung et al.,
2001
).
Identification of these substrates is a key step towards understanding the
biological function of parkin. Another important issue is to determine the
subcellular location where ubiquitination and degradation of these substrates
occur. Previous studies have shown that accumulation of misfolded proteins,
either by inhibition of the 26 S proteasome or by mutation in the protein,
leads to the formation of a large aggregate in the centrosomal region of the
cell (Wigley et al., 1999;
Johnston et al., 1998
). In
mitotic cells at interphase, the centrosome is a perinuclear structure
containing a pair of centrioles, from which a radial array of microtubules is
extended. One of the key components of the centrosome is
-tubulin,
which is responsible for nucleating microtubules
(Joshi et al., 1992
).
-tubulin also exists in the cytosol in a large complex with several
proteins of unknown functions (Wiese and
Zheng, 1999
). Increasing evidence has shown that the centrosome
plays a significant role in the formation of protein aggregates in the cell,
in addition to its well-recognized function as the microtubule-organizing
center. Small aggregates of polyubiquitinated proteins are thought to be
transported to the centrosome by microtubule-associated motor proteins to form
a large inclusion body known as an `aggresome'
(Johnston et al., 1998
;
Kopito, 2000
), which also
contains chaperones and components of the 26 S proteasome
(Johnston et al., 1998
;
Wigley et al., 1999
;
Garcia-Mata et al., 1999
). The
formation of an aggresome is thought to provide the cell with a mechanism to
sequester toxic aggregates of misfolded proteins that cannot be efficiently
handled by the ubiquitin-dependent proteolysis system, and a staging ground
for subsequent fusion with lysosomes to degrade these protein aggregates by an
autophagic pathway (Kopito,
2000
).
One of the histological hallmarks of Parkinson's disease is the Lewy body,
which is often a large single intracellular inclusion that contains
ubiquitinated proteins (Lowe et al.,
1988; Galvin et al.,
1999
),
-synuclein
(Spillantini et al., 1997
) and
parkin (Schlossmacher et al.,
2002
). However, PD patients with parkin mutations almost
invariably lack any Lewy body in their brain tissue
(Kitada et al., 1998
),
suggesting that Lewy bodies cannot be formed effectively in the absence of
functional parkin proteins. To understand the role that parkin may play in the
formation of protein aggregates, we examined the subcellular location of
parkin when cells were treated with specific inhibitors of the 26 S
proteasome. Here we report the recruitment of parkin to the centrosome in
response to inhibition of proteasomes. The possible molecular mechanism
underlying this process and its functional implication are also discussed.
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Materials and Methods |
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Transfection, immunoprecipitation and western blot
HEK293, BE(2)-C, SH-SY5Y, COS7 and 3T3 cells were purchased from ATCC
(Manassas, VA). They were maintained in DMEM with 10% FCS and antibiotics.
Transfection of FLAG-tagged parkin in HEK293 cells was performed using Fugene
6 (Roche, Indianapolis, IN) according to the manufacturer's protocol. Sixty
hours after transfection, cells cultured in 10 cm dishes were lysed on ice in
cold lysis buffer (1% Triton X-100, 10 mM Tris pH 7.6, 50 mM NaCl, 30 mM
sodium pyrophosphate, 50 mM NaF, 5 mM EDTA and 0.1 mM
Na3VO4) for 20 minutes. Lysates were centrifuged at
4°C at 16,000 g and supernatant fractions were incubated
with anti-FLAG-conjugated agarose for 1 hour at 4°C. Immunoprecipitates
were washed three times with the lysis buffer, boiled in 2x SDS loading
buffer for 5 minutes, separated on SDS-polyacrylamide gels, and analyzed by
western blotting with anti--tubulin using the ECL method according to
the manufacturer's protocol (Amersham, Piscataway, NJ). For experiments using
rat brain homogenates, one whole brain was homogenized in 15 ml of ice-cold
lysis buffer on ice in a tissue grinder (Fisher Scientific, Pittsburgh, PA).
The homogenate was centrifuged at 16,000 g for 20 minutes and
ultracentrifuged at 338,000 g for 30 minutes. The supernatant
fractions were incubated with various antibodies for 1 hour at 4°C,
followed by incubation with protein A/G plus agarose beads (Santa Cruz
Biotechnology, Santa Cruz, CA) under the same conditions. Gel separation of
precipitated proteins and western blot analysis were the same as described
above.
Immunocytochemistry
SH-SY5Y, HEK293 and COS7 cells were cultured on collagen-coated coverslips
and treated with various drugs at different dosage and for different times as
indicated in the figure legends. Cells were fixed with cold methanol at
4°C for 20 minutes, and blocked with 3% of BSA for 1 hour. This was
followed by incubation in primary antibodies for 1 day at 4°C and
secondary antibodies for another day at 4°C. In some experiments, the
cells were also stained with the DNA-binding dye TO-PRO-3 (1:1000 in PBS,
Molecular Probes, Eugene, OR) for 5 minutes to visualize the nucleus.
Immunofluorescent images were acquired on a Bio-Rad confocal microscope.
Monochrome images (512x512 pixels) were pseudocolored and merged with
the software Confocal Assistant (freeware by Todd Clark Brelje). The size of
the parkin aggregate was measured using the SPOT software (Diagnostic
Instruments, Sterling Heights, MI) to calculate the area of a handdrawn circle
around the aggregate. Statistical analysis was performed with the software
Origin (Origin Lab, Northampton, MA).
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Results |
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When we stained SH-SY5Y with a mono-specific antibody against parkin
(Ren et al., 2003), a punctate
expression pattern was seen in the cell, with a major spot localized in the
perinuclear region (Fig. 1B).
After the cells were treated with lactacystin (10 µM for 12 hours), a
specific inhibitor of the 26 S proteasome
(Fenteany and Schreiber, 1998
),
we observed significant accumulation of parkin in the perinuclear region
(Fig. 1E). Our previous study
has shown that parkin puncta are localized along microtubules
(Ren et al., 2003
). As
microtubules are centered on the centrosome in mitotic cells at interphase, we
suspected that the major spot of parkin immunoreactivity in the perinuclear
region may be at the centrosome. To test this, we co-stained the cells with
antibodies against parkin and
-tubulin, a centrosome marker. The major
spot of parkin in the perinuclear region before lactacystin treatment
(Fig. 1C) and the large
accumulation of parkin after the treatment
(Fig. 1F) were all co-localized
with
-tubulin signals. When we added the parkin antigenic peptide in
the primary antibodies, no signal was observed in FITC channel, while
-tubulin fluorescence was intact (data not shown). We also obtained the
same results with another centrosome marker, pericentrin. In addition, similar
effects were found when we treated the cells with MG132, another specific
proteasome inhibitor (data not shown). Thus, inhibition of the 26 S proteasome
apparently leads to accumulation of endogenous parkin in the centrosome.
|
Next, we studied the kinetics of this recruitment process by measuring the average area of parkin accumulation in the centrosomal region in SH-SY5Y cells treated with 10 µM lactacystin for different time periods. As shown in Fig. 2, the accumulation of parkin became significant at 4 hours (P<0.05, compared to no treatment), and reached a plateau at 8 hours. We also studied the dose response of this effect. After the cells were treated with various concentration of lactacystin for 12 hours, the average area of parkin accumulation was measured and plotted against the dosage. The centrosomal recruitment of parkin was significant at 5 µM lactacystin (P<0.001, compared to no treatment), and continued to rise with increasing concentration of the drug (Fig. 2F). At doses above 10 µM, SH-SY5Y cells exhibited significant cell death. Similar results on the lactacystin-induced centrosomal recruitment of endogenous parkin were also obtained when we examined another dopaminergic neuroblastoma cell line BE(2)-C (data not shown).
|
Lactacystin does not induce non-selective accumulation of proteins in
the centrosome
To ascertain that the centrosomal recruitment of parkin is not due to an
indiscriminate process of protein accumulation in response to proteasomal
inhibition, we examined the subcellular localization of several other proteins
before and after lactacystin treatment. Two examples are shown in
Fig. 3, in which we co-stained
SH-SY5Y cells with antibodies against CREB and -tubulin
(Fig. 3A,B) or antibodies
against MAP kinase and
-tubulin
(Fig. 3C,D). Lactacystin
treatment (10 µM for 12 hours) did not cause significant accumulation of
either protein in the cell. We also examined the subcellular localization of
endogenous S6 kinase in HEK293 cells before and after lactacystin treatment.
It did not accumulate in the centrosome in response to the treatment (data not
shown). Thus, the recruitment of parkin to the centrosome appears to be
selective.
|
Lactacystin induces centrosomal recruitment of transfected parkin in
HEK293 cells
To provide more evidence on the generality of this effect, we examined
HEK293 cells transfected with FLAG-tagged parkin before and after lactacystin
treatment. HEK293 cells express very low level of parkin endogenously (see
untransfected cells in Fig.
4C). Transient expression of transfected parkin produced a
diffusely cytosolic localization for this protein
(Fig. 4C). After lactacystin
treatment (10 µM for 12 hours), 9.33±0.67% (mean±s.e.m.) of
the transfected cells showed centrosomal accumulation of parkin
(Fig. 4B,D,F). Unlike the
situation in SH-SY5Y cells where a small amount of endogenous parkin was
accumulated in the centrosome even before lactacystin treatment
(Fig. 1A-C), transfected parkin
in HEK293 cells was not enriched in the centrosome in the absence of
lactacystin (Fig. 4E). Thus, we
quantified the recruitment process by calculating the percentage of
transfected cells that had centrosomal accumulation of parkin. The
lactacystin-induced centrosomal recruitment of parkin in HEK293 cells was
dependent on the dose (Fig. 4G)
and duration (Fig. 4H) of the
treatment. Taken together, our results obtained from multiple cell lines on
endogenous or transfected parkin indicate that the specific recruitment of
this protein in response to inhibition of proteasomes is a general
phenomenon.
|
Parkin binds to -tubulin in the rat brain and HEK293
cells
In untreated SH-SY5Y cells, the biggest parkin spots always seemed to be
co-localized with -tubulin in the centrosome
(Fig. 1A-C). With lactacystin
treatment, parkin was recruited to the centrosome, so was
-tubulin
(Fig. 2A,D). These observations
suggest that parkin may be physically bound to
-tubulin. To test this
idea, we performed co-immunoprecipitation experiments in rat brain homogenate,
which was ultracentrifuged at 4°C to obtain supernatant fractions with no
significant amount of microtubules. The lysates were treated with or without
the microtubule depolymerizing drug colchicine (25 µM for 15 minutes at
4°C) and immunoprecipitated with pre-immune serum, anti-parkin, or
anti-parkin pre-incubated with its antigenic peptide. As shown in
Fig. 5A,
-tubulin was
strongly co-immunoprecipitated with parkin, and this interaction was not
affected by colchicine treatment, which ensured the dissociation of any
residual microtubules in the ultracentrifuged lysates and the existence of
free
-tubulin.
|
As our previous studies have shown that parkin also binds to /ß
tubulin heterodimers very strongly (Ren et
al., 2003
), we wanted to test whether the binding between parkin
and
-tubulin is a direct interaction or an indirect one mediated by the
association of
-tubulin with
/ß heterodimers.
Ultracentrifuged rat brain lysates were treated with or without colchicine,
and immunoprecipitated with an irrelevant antibody (anti-neurabin) or the
-tubulin antibody. Western blot analysis of the immunoprecipitates
showed that only a very small fraction of
-tubulin was associated with
-tubulin. In contrast, the binding between
- and ß-tubulin
was much stronger, consistent with existence of
/ß-tubulin
heteodimers (Fig. 5B).
Colchicine treatment had no effect on the co-immunoprecipitation, indicating
that the ultracentrifuged brain lysate did not contain any measurable amount
of microtubules. These results suggest that the strong binding between parkin
and
-tubulin cannot be entirely attributed to an indirect interaction
mediated by the relatively weak association between
-tubulin and
/ß heterodimers. It seems likely that a certain fraction of
-tubulin may bind to parkin directly and another fraction may do so via
association with
/ß heterodimers.
To confirm the observation in rat brain homogenate, we performed similar
experiment in HEK293 cells. After we transfected HEK293 cells with or without
FLAG-tagged parkin, the lysates were immunoprecipitated with
anti-FLAG-conjugated agarose to see whether exogenously expressed parkin was
bound to -tubulin. As shown in Fig.
5C,
-tubulin was indeed co-immunoprecipitated with
parkin.
Centrosomal recruitment of parkin is dependent on intact microtubule
networks
Our previous study had shown that parkin binds to microtubules and
/ß tubulin heterodimers (Ren
et al., 2003
). The interaction between parkin and
-tubulin,
and the fact that a fraction of
-tubulin is associate with microtubules
at the centrosome, suggest that the centrosomal recruitment of the
parkin/
-tubulin complex may be dependent on microtubules. To test this,
we treated SH-SY5Y cells with the microtubuledepolymerizing drug colchicine or
the microtubule-stabilizing drug taxol. As shown in
Fig. 6B, colchicine treatment
(5 µM for 12 hours) changed the punctate localization of parkin
(Fig. 1B) into a more diffusely
cytosolic pattern. The existence of parkin in the centrosome was much less
apparent than in untreated cells (compare
Fig. 6C with
Fig. 1C). After the cells were
treated with colchicine and lactacystin (both at 5 µM for 12 hours), no
significant accumulation of parkin was seen in the centrosome
(Fig. 6D-F). In the presence of
colchicine, parkin exhibited a similar pattern of subcellular localization
with or without lactacystin treatment (compare
Fig. 6E with B). In contrast to
the situation with a single treatment of lactacystin at 10 µM, a
significant degree of cell death was observed when SH-SY5Y cells were treated
with 10 µM lactacystin and colchicine at the same time. To test whether 10
µM lactacystin would induce centrosomal recruitment of parkin in the
presence of colchicine, we chose COS7 cells, which exhibited very little cell
death when treated with both drugs at 10 µM. No significant accumulation of
parkin in the centrosome was observed under this condition (data not shown),
which is consistent with the results from SH-SY5Y cells.
|
We also treated SH-SY5Y cells with taxol (5 µM for 12 hours). The treatment increased the intensity of parkin staining along microtubules, however, parkin was not observed in the centrosome (Fig. 6G-I). After the cells were incubated with taxol and lactacystin (both at 5 µM for 12 hours), parkin was still absent in the centrosome (Fig. 6J-L). In addition, there was no accumulation of parkin elsewhere in the cell, compared to cells treated with taxol alone (Fig. 6K and H). Similar results were obtained in COS7 cells treated with taxol alone (10 µM for 12 hours) or together with lactacystin (both at 10 µM for 12 hours) (data not shown). Thus, the centrosomal recruitment of parkin appears to be dependent on intact microtubules. Both the disruption of microtubules (by colchicine) and their overt stabilization (by taxol) blocked the movement of parkin towards the centrosome.
The expression level of parkin is not significantly affected by
lactacystin, colchicine or taxol
As the fluorescence intensity of parkin appears to differ in cells treated
with vehicle, lactacystin, colchicine or taxol (Figs
1 and
6), we performed western
blotting on lysates from SH-SY5Y cells after various treatment (5 µM of
each drug for 12 hours) to examine the expression level of parkin under these
conditions. As shown in Fig. 7,
almost all parkin was in the supernatant fraction in untreated cells, and the
amount was not significantly changed by treatment with lactacystin, colchicine
or a combination of the two. However, when the cells were treated with taxol
or taxol plus lactacystin, the amount of parkin was significantly reduced in
the supernatant fraction and increased commensurately in the pellet fraction.
As a control for equal loading, we examined the level of p38 MAP kinase, a
cytosolic protein that has no known interaction with microtubules. The amount
of p38 in the supernatant fraction was not changed by these treatments, and it
did not go into the pellet fraction after taxol treatment. Similar results
were obtained from COS7 and 3T3 cells with regard to the expression level of
parkin in response to these drugs (10 µM of each drug for 12 hours, data
not shown).
|
Thus, these data showed that the total amount of parkin was not
significantly affected by lactacystin, colchicine or taxol treatment, either
alone or in combination. However, taxol-induced microtubule polymerization
brought down a portion of parkin from the supernatant to the pellet fraction.
This is consistent with the strong interaction between parkin and microtubules
(Ren et al., 2003). Taken
together, our results showed that the centrosomal recruitment of parkin in
response to lactacystin was caused by change of localization, rather than
alteration of expression.
Lactacystin induces centrosomal accumulation of the parkin substrate
CDCrel-1 and ubiquitinated proteins
As parkin is a protein-ubiquitin E3 ligase, we hypothesize that its
recruitment to the centrosome may be linked to ubiquitination of misfolded
proteins accumulated there. We transfected HEK293 cells with myc-tagged
CDCrel-1, a known substrate of parkin
(Zhang et al., 2000), and
treated the cells with or without lactacystin (10 µM for 12 hours). When we
co-stained the cells with anti-
-tubulin and anti-myc tag, it was clear
that lactacystin induced the centrosomal accumulation of transfected CDCrel-1
(compare Fig. 8E with B, and F with
C). The antibody against myc tag specifically recognized
transfected myc-CDCrel-1 in the cell (Fig.
8M). We also co-stained these cells with antibodies against
-tubulin and ubiquitin. Prior to lactacystin treatment, ubiquitin
staining was diffuse in the cell, with a large portion in the cytosol
(Fig. 8H). Most of the staining
represented ubiquitinated proteins, rather than free ubiquitin, as evidenced
by western blots of total cell lysates with anti-ubiquitin
(Fig. 8N). After lactacystin
treatment, ubiquitinated proteins were accumulated in the centrosome
(Fig. 8K,L), which is
consistent with previous reports on the recruitment of proteasome components
and ubiquitinated proteins to the centrosome
(Wigley et al., 1999
;
Garcia-Mata et al., 1999
). The
presence of CDCrel-1, parkin and ubiquitinated proteins simultaneously in the
centrosome after lactacystin treatment suggests that parkin is recruited to
the centrosome to ubiquitinate its substrates, such as CDCrel-1, which is
accumulated there.
|
![]() |
Discussion |
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The potential mechanism for the centrosomal recruitment of parkin may be
the binding between parkin and -tubulin, a protein that shuttles
between a cytosolic pool and a centrosomal pool
(Schiebel, 2000
;
Wiese and Zheng, 1999
).
Accumulation of misfolded proteins leads to significant increase of
-tubulin in the centrosome
(Johnston et al., 1998
;
Wigley et al., 1999
;
Garcia-Mata et al., 1999
).
Although the detailed mechanism for the centrosomal recruitment of
-tubulin is unclear, it appears to be dependent on transport proteins
on microtubules (Johnston et al.,
1998
; Garcia-Mata et al.,
1999
), whose minus ends are anchored on
-tubulin ring
complexes in the centrosome (Schiebel,
2000
; Wiese and Zheng,
1999
). The binding between parkin and
-tubulin appears to
be independent of microtubules, as colchicine added to rat brain lysate did
not disrupt the co-immunoprecipitation of parkin and
-tubulin
(Fig. 5A). Thus, it seems that
parkin/
-tubulin complexes are normally present in the cytosol in the
absence of lactacystin treatment. Inhibition of proteasomes triggers a
response that recruits the complex to the centrosome.
This recruitment process is apparently dependent on intact microtubule
networks in the cell. When microtubules were depolymerized by colchicine,
recruitment of parkin did not occur (Fig.
6A-F). However, when microtubules were rendered overtly stable by
taxol, parkin became more concentrated on bundled microtubules
(Fig. 6H,K), and never moved to
the centrosome (Fig. 6I,L). A
large portion of parkin became so strongly associated with microtubules that
it entered the pellet fraction with them
(Fig. 7). Even the portion of
parkin that was normally in the centrosome
(Fig. 1B,C) was no longer
present when SH-SY5Y cells were treated with taxol
(Fig. 6H,I). As parkin bound to
-tubulin (Fig. 5), as
well as
/ß tubulin (Ren et
al., 2003
), the data suggest that taxol could perhaps change the
dynamic partition of parkin between
-tubulin and
/ß
tubulin. With taxol treatment, parkin-
/ß tubulin complexes are
polymerized into microtubules, leaving very little parkin to interact with
-tubulin, which may prevent the movement of parkin toward the
centrosome. The other possibility is that taxol treatment may cause
centrosome-independent nucleation of microtubules, which would prevent the
centrosomal recruitment of parkin even if its trafficking is not affected by
taxol. Further studies are necessary to elucidate the mechanism(s) by which
taxol blocks the accumulation of parkin at the centrosome.
Although the lactacystin-induced centrosomal recruitment of parkin was
observed in several cell lines, the effect was not seen in cultured neurons.
The subcellular localization of parkin was punctate, decorating along
microtubules (Ren et al.,
2003), but exhibited no significant accumulation anywhere in the
neuron with or without lactacystin treatment (data not shown). The expression
level of
-tubulin was very low in neurons, compared to mitotic cells.
Almost all
-tubulin proteins are localized in the centrosome in neurons
(Baas and Joshi, 1992
),
however, microtubules are not anchored on the centrosome in these postmitotic
cells (Yu et al., 1993
). It is
believed that microtubules are still nucleated from
-tubulin in the
centrosome, but soon after their formation, they are released into the
cytosol, probably with the help of katanin
(Baas, 1999
). The lack of
microtubules anchored on the centrosome perhaps makes it impossible for
protein aggregates to be transported along the microtubules to the centrosome,
which in turn may cause the formation of inclusion bodies at a different site.
Regardless of this, it is still thermodynamically favorable for small
aggregates to coalesce into a single large inclusion body, primarily because
of gains in entropy, as misfolded proteins expose large amounts of their
hydrophobic surface to water. The presence of parkin
(Schlossmacher et al., 2002
),
its substrate
-synuclein
(Spillantini et al., 1997
;
Shimura et al., 2001
) and many
ubiquitinated proteins (Lowe et al.,
1988
; Galvin et al.,
1999
) in the Lewy body suggests that parkin may be involved in the
formation of this cytoplasmic inclusion. This is corroborated by the lack of
Lewy bodies in PD patients with parkin mutations
(Kitada et al., 1998
). It
appears that functional parkin may be required for the formation of Lewy
bodies. Further studies using human postmortem tissues are necessary to find
out whether
-tubulin or the centrosome is involved in the formation of
Lewy bodies in neurons, and if so, whether the interaction between
-tubulin and parkin plays a role in the process.
The lactacystin-induced centrosomal accumulation of parkin (Figs
1,
4), its substrate such as
CDCrel-1 (Fig. 8),
ubiquitinated proteins (Fig. 8)
(Johnston et al., 1998),
chaperons (Garcia-Mata et al.,
1999
), and proteasomes (Wigley
et al., 1999
) appears to be a concerted response to an
overwhelming increase of misfolded proteins caused by inhibition of their
degradation. However, our data on MAP kinase, CREB
(Fig. 3) and S6 kinase (data
not shown) clearly showed that these proteins did not go to the centrosome
when their degradation was inhibited. It is possible that they represent
another category of proteins that do not easily misfold and aggregate.
Proteins that are known to form aggresomes tend to be transmembrane proteins
(e.g. CFTR, PS1) or membrane-associated proteins (e.g. CDCrel-1) that are
prone to misfold in the cytosol (Johnston
et al., 1998
). These misfolded proteins may expose large patches
of hydrophobic surface that would bind to and inactivate many cellular
proteins non-specifically (Taylor et al.,
2002
). Their aggregation at the centrosome would minimize the
toxic effect and expedite their degradation en masse by fusion with lysosomes
in an autophagic manner (Klionsky and Emr,
2000
).
In summary, our results demonstrate that parkin, an E3 ligases linked to
PD, was recruited to the centrosome in response to inhibition of the 26 S
proteasome. The recruitment is accompanied by the centrosomal accumulation of
-tubulin, to which it binds, as well as its substrates CDCrel-1, which
it ubiquitinates (Zhang et al.,
2000
). Thus, the centrosomal accumulation of parkin may enhance
the ubiquitination of its substrates and facilitate their aggregation in the
centrosome so that they can be efficiently degraded later. This novel function
of parkin in mitotic cells may also play a role in the formation of inclusion
bodies in neurons, which is an important process in neurodegenerative
disorders including Parkinson's disease.
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Acknowledgments |
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