From the Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS, INSERM,
Université Louis Pasteur, B.P. 10142, Illkirch C.U. de
Strasbourg, 67404 cedex, France and the ¶ Department of
Immunology, University College London Medical School, Windeyer
Bldg., 46 Cleveland St., London W1P 6DB, United Kingdom
Received for publication, November 26, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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Polyglutamine diseases, including Huntington's
disease, designate a group of nine neurodegenerative disorders
characterized by the presence of a toxic polyglutamine expansion in
specific target proteins. Using cell and mouse models, we have shown
that expanded polyglutamine led to activation of the stress kinase JNK
and the transcription factor AP-1, which are implicated in neuronal death. Polyglutamine expansion-induced stress shared common
features with protein-damaging stress such as heat shock, because
activation of JNK involved inhibition of JNK phosphatase activities.
Indeed, expanded polyglutamine impaired the solubility of the
dual-specificity JNK phosphatase M3/6. Aggregation of M3/6 by
polyglutamine expansion appeared to be indirect, because M3/6 was not
recruited into polyglutamine inclusions. The heat shock protein HSP70,
which is known to inhibit JNK during the heat shock response,
suppressed polyglutamine-mediated aggregation of M3/6 and activation of
JNK. Interestingly, levels of HSP70 were down-regulated by
polyglutamine expansion. We suggest that reduction of HSP70 by expanded
polyglutamine is implicated in aggregation and inhibition of
M3/6 and in activation of JNK and AP-1.
Nine neurodegenerative disorders, including Huntington's disease
and spinocerebellar ataxia type 7 (SCA7)1 are caused by
expansion of a CAG trinucleotide repeat coding for a polyglutamine
tract in unrelated target proteins (1). Progressive neuronal loss
characterizes all polyglutamine diseases, although the affected brain
regions are variable between disorders (2). An inverse correlation
between age of onset of the disease and the length of the expansion is observed.
Polyglutamine expansion (polyQ) confers a gain of toxic function to the
respective proteins, which results in neurodegeneration (3, 4). Why and
how polyQ is toxic has still to be determined, although recent findings
have shown that it impairs different critical cellular processes. PolyQ
is highly prone to aggregation, as shown in vitro (5) and
in vivo, where it forms nuclear inclusions (NIs) (6). NIs
are believed to impair major cellular processes through sequestration
of chaperones, proteasomal components, and transcription factors (7,
8). Indeed, overexpression of chaperones in Drosophila and
mice is able to rescue the phenotypes induced by expression of polyQ
(9, 10), and there is evidence showing that protein degradation (11)
and transcription (12) are impaired by polyQ. However, other studies
suggest that NIs are not required to initiate neurodegeneration (13)
and that soluble polyQ can directly interferes with cellular processes such as transcription (14, 15).
Protein aggregation is also induced by protein-damaging stress such as
heat shock (16). In contrast to UV irradiation, osmotic stress and
certain cytokines that activate the c-Jun N-terminal Kinase (JNK) via a
signal transduction pathway that involves small GTP-binding proteins
(17) and a cascade of protein kinases (18, 19), protein-damaging
stresses, including heat shock and arsenite, lead to activation of JNK
primarily through inhibition of JNK phosphatase(s) (16, 20). Recent
findings suggest that the dual-specificity JNK phosphatase M3/6 is
inactivated by protein-damaging stress (21, 22). M3/6 solubility
properties would change after heat shock, resulting in inhibition of
phosphatase activity. The heat shock protein HSP70 negatively regulates
JNK activity by promoting JNK dephosphorylation (16, 23). It is
believed that redistribution of HSP70 at the sites of damaged proteins
affects its JNK regulatory function (16).
Members of the JNK family, which are encoded by three different genes,
phosphorylate and activate the transcription factor c-Jun (24). C-Jun
homo- or heterodimerizes with proteins of the Fos family, forming AP-1
complexes. AP-1 activity is implicated in numerous functions, including
proliferation, survival, and programmed cell death (25). In the nervous
system, excitotoxic stress such as stimulation with kainic acid
induces apoptosis in a manner dependent on both JNK3 (the
brain-specific isoform) and c-Jun (26, 27). In addition, withdrawal of
NGF in sympathetic neurons triggers a cell death program that is
inhibited by dominant negative mutants of c-Jun (28).
As discussed above, there is evidence that the toxicity of polyQ
results in part from its propensity to misfold, a situation that might
be equivalent to the one induced by a protein-damaging stress. Thus, to
get insight into polyQ toxicity, we have investigated the possibility
that polyQ could lead to a cellular stress comparable to a
protein-damaging stress. Here we have shown that polyQ expression resulted in activation of JNK and c-Jun, through inhibition of JNK
phosphatase activities. In particular, the solubility of the JNK
phosphatase M3/6 was impaired by polyQ. M3/6 was not recruited into
NIs, suggesting that its aggregation was indirectly induced by polyQ.
Expression of HSP70 suppressed polyQ-mediated aggregation of M3/6 and
activation of JNK, suggesting that HSP70 is a critical regulator of
polyQ-induced stress. In addition, expression of polyQ in cells
resulted in down-regulation of HSP70 levels and in its recruitment into
NIs. Altogether, our results indicate that impairment of HSP70 by polyQ
is responsible for inactivation of JNK phosphatase(s). As a result,
activation of JNK and c-Jun, which are involved in neuronal death,
might be critical in the triggering of polyQ-mediated neurodegeneration.
Antibodies and Reagents--
Monoclonal 1C2 and 2A10 antibodies
and polyclonal 1261 and M3/6 antibodies have been previously described
(21, 29, 30). Anti-phosphorylated SEK-1, JNK, p38, ERK, and c-Jun
(ser63) antibodies were from Cell Signaling. Anti-JNK3, anti-c-Jun
(sc-45), anti-ERK (K23), and anti-CBP (A22) were from Santa Cruz
Biotechnology. Anti-FLAG (M2), anti-actin, and anti- Transient Transfection and Treatments of COS-1
Cells--
pTL1-based expression vectors encoding the first 133 amino
acids of the human huntingtin protein with either 15 or 125 glutamines and FLAG-tagged at the N-terminal extremity have been
previously described (29). The expression vector encoding murine
c-Jun and reporter constructs were a gift from P. Sassone-Corsi.
Expression vector encoding murine M3/6 has been described previously
(31). A pcDNA-based expression vector encoding HSP70 was
generated from a pH2.1 plasmid containing the sequence of a human
hsp70 gene, which was given by R. I. Morimoto.
Transient transfections were performed by the phosphate calcium method
as follows: COS-1 cells were cultured in DMEM/5% fetal calf serum
(FCS) and plated at 30% confluence before transfection. After
transfection (15 h), medium was changed and cells were grown for
additional 48 h.
Cell Culture and Differentiation of NG108 Cell
Lines--
Generation, culture, and differentiation of reverse
tetracycline-inducible NG108 cell lines expressing an N-terminal
FLAG-tagged version of the first 80 amino acids of the human huntingtin
protein with either 15 or 125Q have been previously described (32). Briefly, clones were maintained in DMEM/10% FCS, antibiotics, 0.5 mg/ml G418, and 250 µg/ml hygromycin B. For neuronal differentiation and induction of expression, medium was replaced by DMEM/1% FCS, antibiotics, G418, hygromycin B, 10 µM forskolin, 100 µM isobutylmethylxanthine, and 1 µg/ml doxycycline.
Transient transfections with CAT- and lacZ-reporter constructs were
performed as followed. Cells maintained in the DMEM/10% FCS medium
were plated at 30% confluence and transfected with the phosphate
calcium method. After transfection (15 h), medium was replaced by the
differentiation and induction medium for the indicated period.
Proteins: Extraction, Fractionation, and
Immunoprecipitation--
COS-1 and NG108 cell extracts were prepared
as follows: cells were washed twice in ice-cold PBS and directly lysed
in boiling Laemmli buffer. Mice retinas were dissected and homogenized
in TGEK buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 5 mM EDTA, 150 mM KCl, 1 mM
phenylmethylsulfonyl fluoride, a mixture of protease inhibitors, 1 mM sodium orthovanadate, and 20 mM sodium
fluoride). They were incubated for 15 min on ice, sonicated, and
centrifuged for 20 min at 13,000 rpm and 4 °C. Supernatants were collected.
For fractionation experiments, proteins were prepared as described
previously (33). Briefly, the soluble SN1 fraction of cells or retinas
was recovered in TGEK buffer, as described above. After centrifugation,
the pellet was conserved and solubilized in an SDS buffer (final
concentration of 2% SDS, 5%
For immunoprecipitation experiments, protein extracts were prepared as
described above, by lysing cells or retinas in TGEK buffer. Equal
amounts of whole cell extracts were incubated with anti-M3/6 antibody
overnight at 4 °C, and with protein A-Sepharose for additional 30 min. Immunoprecipitates were washed three times and resuspended in TGEK buffer.
Western Blotting--
Protein extracts were resolved by standard
SDS-PAGE. Samples were electroblotted onto Protan nitrocellulose
(Schleicher and Schuell). Membranes were incubated in PBS/5% low fat
milk overnight at 4 °C with non-phosphospecific antibodies, and in
TBS/5% bovine serum albumin with phosphospecific antibodies.
Immunocomplexes were revealed by chemiluminescence with anti-mouse or
anti-rabbit antibodies.
CAT ELISA Assays--
Quantitative determination of
chloramphenicol acetyltransferase (CAT) in transfected cells was
performed using a CAT ELISA kit (Roche Molecular Biochemicals).
Briefly, cells were lysed and proteins extracts were prepared according
to the manufacturer's instructions. The extracts were incubated on
ELISA wells, which were precoated with a polyclonal antibody to CAT. A
sandwich ELISA was then realized, by incubating an anti-CAT antibody
coupled to digoxigenin, followed by an anti-digoxigenin antibody
coupled to peroxidase. A colorimetric substrate was added, and the
reaction was quantitated by measuring the optical density at 405 nm.
Results were normalized with respect to protein concentration and
transfection efficiency. Cells were cotransfected with a lacZ-reporter
construct and the AP-1 Binding Assays--
AP-1 binding assays were realized using
the kit Trans-AM AP-1 (Active Motif) and following the instructions
given by the manufacturer. Briefly, nuclear extracts from NG108 cells
or mice retinas were prepared by standard methods using the kit
reagents. 10 µg of nuclear extracts was incubated on ELISA wells,
where oligonucleotides containing the TRE had been precoated. Binding
of AP-1 complexes to TRE was determined using a phospho c-Jun antibody,
followed by a secondary antibody coupled to peroxidase. A colorimetric substrate was then incubated, and the reaction was quantified by
measuring the optical density at 450 nm.
Immunofluorescence--
Fixed eyes were incubated
overnight in PBS/30% sucrose and snap frozen on dry ice. 50-µm
free-floating sections were blocked in PBS/0,3% Triton X-100/5%
bovine serum albumin, washed in PBS, and incubated with primary and
secondary antibodies (Cy3- or Oregon Green-conjugated) diluted in
PBS/5% bovine serum albumin. M3/6, HSP70, 1261, and 1C2 antibodies
were diluted to 1/200 each.
Expression of Expanded Polyglutamine Results in Moderate but
Sustained Hyperphosphorylation of JNK and c-Jun--
We asked whether
expression of polyQ could result in a toxic cellular stress. To
investigate this hypothesis, we tested whether stress-activated protein
kinases (SAPKs) were activated in different polyQ models. COS cells
were transiently transfected with expression vectors encoding a
FLAG-tagged version of the first 133 amino acids of huntingtin
protein (htt133) with either a normal (15) or mutated (125) number of
glutamines. Western blot analysis performed on whole cell extracts with
anti-activated JNK antibody (P-JNK) revealed that JNK was moderately
phosphorylated in presence of mutated htt (Fig.
1A). The activation was
specific, because basal phosphorylation levels of other related
kinases, p38/SAPK and ERK/MAPK, were not affected (Fig. 1A).
The downstream target of JNK, c-Jun transcription factor, was also
hyperphosphorylated in cells expressing mutated htt, as detected with
an anti-phospho c-Jun antibody (P-c-Jun), which reveals N-terminal
phosphorylation of c-Jun (Fig. 1A).
We then wished to extend these results to a more physiological cell
system. For this purpose, we expressed a FLAG-tagged truncated version
of mutated htt (V125Q) in stable and inducible NG108 cell lines, which
we differentiated into neurons for various days (32). Whole cell
extracts analyzed by Western blot showed that both JNK and c-Jun were
hyperphosphorylated in NG108 V125 cells (Fig. 1B). In
contrast, in parental cells and in NG108 cells expressing the same
truncated form of htt, but with 15Q, JNK and c-Jun were not
hyperphosphorylated (Fig. 1B and not shown). Interestingly, JNK and c-Jun activation was concomitant to aggregation of mutated htt,
which was detectable 5 days after induction (Fig. 1B). From day 5, aggregated htt remained in the stacking part of the SDS-PAGE gel.
We then asked whether expression of polyQ could also lead to sustained
activation of JNK in vivo. To address this, we analyzed R6/1
transgenic mice, which express expanded-exon 1 of htt in brain (4). To
establish whether the molecular process of JNK activation could be
common to other polyQ diseases, we included in the study transgenic
mice expressing mutated ataxin-7, the protein involved in SCA7. The R7E
mice we used express mutated ataxin-7 specifically in photoreceptors
and have been shown to represent a good model of SCA7 retinal pathology
(30). Interestingly, we found that mutated exon 1 of htt is also
expressed in photoreceptors of R6/1 animals (34). Taking advantage of
this observation and to compare both pathologies at the molecular
level, we analyzed the phosphorylation status of JNK and c-Jun in
retinas of R6/1 and R7E animals. R6/1 mice were sacrificed at 8 months, i.e. a few weeks before death, and R7E mice, for
which disease progression is more severe, were sacrificed at both early
and late time points after the onset of retinopathy (1 and 3 months).
Control wild-type littermates as well as transgenic mice expressing the
normal ataxin-7 (with 10Q, referred to as R7N) were included in the
study (30). Expression of mutated htt or ataxin-7 was controlled with
the 1C2 antibody, which recognizes specifically expanded polyglutamine (29). JNK was hyperphosphorylated in retinas expressing mutated htt
exon 1 and ataxin-7 (Fig. 1, C and D). Migration
of part of the neuronal-specific JNK3 isoform was shifted to higher
molecular weights, suggesting that JNK3 was itself a target of
activation (Fig. 1D). c-Jun was also found
hyperphosphorylated in R6/1 and R7E transgenic mice (Fig. 1,
C and E). Hyperphosphorylation of c-Jun was
already detectable in R7E mice of 1 month, which corresponds to the
beginning of the pathology. This suggested that activation of the JNK
pathway by polyQ is an early and persistent event. In conclusion,
polyglutamine-expanded htt and ataxin-7 both induce activation of JNK
and c-Jun proteins, indicating that the polyQ tract is sufficient to
mediate this effect. In addition, we show that activation of JNK by
polyQ occurs in vivo in animal models. Thus, these data
extend previous observations showing that overexpression of mutated htt
in PC12 cells differentiated into neurons results in a cellular stress
that leads to activation of JNK (35).
Hyperphosphorylation of c-Jun by Polyglutamine Expansion Leads to
Activation of AP-1-dependent Gene
Expression--
N-terminal phosphorylation of c-Jun regulates AP-1
activity (25) and is involved in neuronal cell death (27, 28, 36). Thus, we asked whether expression of polyQ in our different models would result in enhancement of AP-1 transcriptional activity. To test
this, we coexpressed in cells mutated htt, c-Jun, and a reporter gene
under the control of a promoter containing the AP-1 element (also named
the 12-O-tetradecanoylphorbol-13-acetate-responsive element or TRE). Expression of mutated htt in COS cells induced a
4-fold activation of the TRE reporter (Fig.
2A). Similarly, stable
expression of mutated htt in NG108 V125 cells differentiated for 3.5 and 6.5 days resulted in modest but progressive activation of the
expression of the CAT gene (up to 2-fold, Fig. 2B).
PolyQ did not increase transcription from other responsive elements, such as the serum-responsive element (not shown).
Binding assays that allow testing the amount of active (i.e.
phosphorylated) c-Jun bound to the TRE further supported these results.
The principle of the new and sensitive technology we used is based on
an ELISA (see "Experimental Procedures"). Nuclear extracts prepared
from NG108 V125 cells differentiated for 7 days into neurons were
tested by this method. Binding of AP-1 to TRE was significantly
increased (about 3-fold) in extracts prepared from cells induced for
expression of htt V125Q, compared with non-induced cells (Fig.
2C). The binding was specific because the presence of free
TRE-oligonucleotides abolished the ELISA signal when incubated with
nuclear extracts (not shown). Next, we performed nuclear preparations
from retinas of R6/1 mice of 8 months. Fig. 2D shows that
binding of AP-1 to TRE was significantly augmented when extracts were
from transgenic, versus normal mice. We also measured the
binding capacity of AP-1 complexes derived from retinas of R7E and
wild-type mice of 1 and 3 months (Fig. 2E). AP-1 binding was
significantly increased in transgenic mice of 3 months. Thus, binding
of active AP-1 to TRE is increased by polyQ. In conclusion, these
results suggest that polyQ induces activation of
AP-1-dependent gene expression.
Activation of JNK by Polyglutamine Expansion Involves Inactivation
of the Dual-specificity JNK Phosphatase M3/6--
JNK is
activated through phosphorylation by upstream kinases, including
SEK-1/MKK4 and MKK7 (18, 19). The phosphorylation of JNK is a
reversible event, involving the action of JNK phosphatases. Whereas
stresses such as UV irradiation or osmotic stress induce strong
activation of kinases upstream of JNK (37), protein-damaging stress
such as heat shock induces an activation of JNK that is mainly mediated
by inhibition of JNK phosphatases (16). We wished to investigate which
mechanism was primarily implicated in polyQ-mediated activation of JNK.
To test this, we examined the phosphorylation status of the JNK
activator SEK-1, using an anti-phospho SEK-1 antibody. Expression of
mutated htt in COS cells slightly induced the phosphorylation of SEK-1
(Fig. 3A). In retinas of R7E
mice at 7 months of age, which corresponds to a very late pathological stage, SEK-1 was also moderately hyperphosphorylated (Fig.
3D). However, the phosphorylation levels of SEK-1 were
normal in neuronal NG108 V125 cells and in R6/1 and R7E mice of,
respectively, 8 and 3 months (Fig. 3, B-D). These data
suggest that early activation of JNK by polyQ mainly results from
inactivation of JNK phosphatases and that a secondary event might
further activate JNK through upstream kinases.
To further investigate this hypothesis, we asked whether we could
identify a JNK phosphatase that would be inactivated by polyQ. We
focused on the mouse M3/6 dual-specificity JNK phosphatase, whose
orthologue hVH5 in the human is highly expressed in the nervous system
(38). In addition, upon protein-damaging stress, M3/6 solubility is
decreased, which leads to its inactivation (21, 22). Thus, we wished to
test whether M3/6 solubility was impaired in cells expressing polyQ.
COS cells were cotransfected with expression vectors encoding M3/6 and
mutated or normal htt. Fractionation experiments were conducted as
described (33) to isolate insoluble proteins from cell extracts.
Fraction SN1 contained soluble proteins, which represent most of the
proteins. Indeed, Western blot analysis shows that ERK proteins are
primarily localized in this cell fraction (Fig.
4A). Insoluble material was
recovered in two different fractions referred to as SN2 and P, which
correspond respectively to SDS-soluble and SDS-resistant but formic
acid-soluble material (33). Thus, fraction P contained the most
insoluble material. As shown in Fig. 4A, mutated htt, which
aggregates in cells, was significantly detected in this fraction with
1C2 antibody. The M3/6 content present in the three fractions was
analyzed by Western blot analysis, using an anti-M3/6 antibody (21).
Fig. 4A shows that this antibody detects the full-length
90-kDa M3/6 protein as well as two other major breakdown products.
Remarkably, in cells that were left untreated or transfected with
normal htt133, large amounts of M3/6 were detected in fractions SN2
(mainly as a full-length protein) and P (as a breakdown product). This
indicated that M3/6 might be prone to aggregation. As shown on Fig.
4A, expression of mutated but not of normal htt increased
this intrinsic tendency. Indeed, the content of M3/6 was significantly
decreased in fraction SN1 and increased in fraction P.
We then performed the same kind of experiments with neuronal NG108 V125
cells, expressing or not mutated htt. The content of the different
fractions was controlled with anti-ERK and 1C2 antibodies, as in COS
cells (Fig. 4B). The complete htt V125Q as well as a cleaved
form were detected in fraction P. In cells that did not express mutated
htt, endogenous M3/6 was barely visible in SN1 but was present in SN2
as a full-length protein and as a major breakdown product.
Interestingly, fraction P of cells expressing mutated htt was clearly
enriched in the full-length M3/6 protein and the breakdown product.
We then examined the solubility of M3/6 in retinas of R7E and wild-type
mice at 2.5 months of age. In fraction SN1 of wild-type animals, we
detected two bands at 90 kDa (Fig. 4C), possibly
corresponding to phosphorylated and dephosphorylated forms of M3/6
protein (31). SN2 and P fractions contained only a breakdown product of
the phosphatase. In R7E mice, the upper band of fraction SN1
disappeared, and fraction P was enriched in the cleaved form of M3/6.
1C2 antibody, which was used as a control, revealed the presence of the
full-length mutated ataxin-7 in fractions SN1 and SN2, but only of a
fragment of mutated ataxin-7 in fraction P (Fig. 4C), as
previously described (30).
Altogether, these data show that M3/6 is intrinsically prone to
degradation and aggregation, indicating that these two processes are
essential for the regulation of the phosphatase activity. We also
provide evidence that polyglutamine-expanded proteins promote
aggregation and degradation of M3/6. Thus, although our results do not
exclude that other JNK phosphatases than M3/6 might be affected by
polyQ, they show that M3/6 at least is impaired in cells expressing
polyQ.
Aggregation of M3/6 by PolyQ Is Not Direct--
Many
proteins, including chaperones, proteasomal components, and
transcription factors have been shown recruited into NIs (7, 8). Thus,
we asked whether polyQ-induced aggregation of M3/6 could be the
consequence of the sequestration of the phosphatase by NIs. To test
this, we performed immunofluorescence-based experiments. Fixed retinas
of R7E animals of 2.5 months were double-labeled with the anti-ataxin-7
2A10 and anti-M3/6 antibodies using confocal laser microscopy and
counterstaining with 4',6-diamidino-2-phenylindole. As described before
(30) and shown Fig. 5A
(upper panel), the immunoreactivity of mutated ataxin-7 was
primarily concentrated in large nuclear inclusions (NIs), corresponding
to aggregated ataxin-7. M3/6 immunoreactivity was cytoplasmic and never
colocalized with that of ataxin-7. Similar results were obtained with
NG108 V125 cells (not shown). This indicates that polyQ does not
sequester M3/6 into NIs and thus indirectly affects the solubility of
M3/6.
HSP70 Suppresses PolyQ-induced Aggregation of M3/6 and
Activation of JNK--
As discussed above, our data show that
polyQ-mediated and protein-damaging stresses have similar molecular
features. The heat shock protein HSP70 was shown to suppress the
activation of JNK caused by protein-damaging stresses by reverting the
inhibition of JNK dephosphorylation (16). One possible mechanism
linking HSP70 to JNK involves the control of JNK phosphatases
activities by HSP70. We investigated the possibility that HSP70 could
suppress polyQ-induced activation of JNK, through inhibition of the
M3/6 JNK phosphatase. COS cells were transfected with expression
vectors encoding HSP70 together with mutated or normal htt. The
solubility of M3/6, which likely reflects its activity, was examined by
fractionation experiments. The fractionation procedure was controlled
with the 1C2 antibody (Fig. 6). As
expected, overexpression of HSP70 in cells improved the solubility of
polyQ aggregates. Fig. 6 shows that the amount of polyQ is decreased by
HSP70 in fraction P, which contains the most insoluble materials, and
increased in fraction SN2. Endogenous M3/6 was detected in the
SN1-soluble fraction mainly as full-length proteins. In contrast,
fractions SN2 and P contained mostly breakdown products. Interestingly, similar fragments were generated from both ectopic and endogenous M3/6
(Figs. 4A and 6). In response to polyQ-induced stress,
full-length form and breakdown products of M3/6 accumulated in fraction
P of both conditions. As shown in Fig. 6, the solubility of M3/6 was
clearly improved in cells expressing mutated htt together with HSP70.
Indeed, expression of ectopic HSP70 resulted in the reduction of M3/6
the immunoreactivity of M3/6 in fraction P. Conversely, the content of
M3/6 in fraction SN2 was enriched upon expression of ectopic HSP70. In
addition, overexpression of HSP70 led to the appearance of an
additional form of full-length M3/6, characterized by a higher mobility
shift on Western blot, which presumably corresponds to the
phosphorylated form of M3/6 (31). We then asked whether HSP70-mediated
solubilization of M3/6 was correlated with the suppression of JNK
signaling, in polyQ-expressing cells. Fig. 6 shows that the
phosphorylation of JNK is significantly reduced by HSP70 in cells
expressing mutated htt. Altogether, our results suggest that HSP70
promotes the stabilization of a soluble form of the JNK phosphatase
M3/6, which is phosphorylated and active. Thus, HSP70 appears to be a
critical regulator of polyQ-induced activation of JNK.
HSP70 Protein Levels Are Down-regulated by Polyglutamine
Expansion--
As discussed above, our data show that polyQ-mediated
and protein-damaging stresses have similar molecular features.
Redistribution of the heat shock protein HSP70 at the sites of damaged
proteins is believed to prevent its capacity to activate JNK
dephosphorylation (16). Thus, we asked whether such an effect could
lead to polyQ-induced aggregation of M3/6. Indeed, heat shock proteins
are recruited into NIs of polyQ models (7), and in particular, NIs of
R7E mice are positive for HSP40/HDJ-2 proteins (30). Therefore, we
asked whether HSP70 was also recruited into NIs of R7E mice. Fixed
retinas of mice at 3 months of age were double-labeled with the
anti-ataxin-7 1261 and anti-HSP70 antibodies and analyzed by confocal
microscopy (Fig. 5A, lower panel). Most NIs, if
not all, were labeled with HSP70. This suggested that recruitment of
HSP70 into NIs could be responsible for M3/6 aggregation. To test this
idea, we asked whether HSP70 was depleted from the soluble cell
fraction. Surprisingly, HSP70 proteins expressed in R7E mice were
completely recovered in the soluble SN1 fraction (Fig. 4C). No HSP70 was detected in the insoluble SN2 and P fractions. However, HSP70 total protein levels were clearly reduced in transgenic, compared
with wild-type mice (Figs. 4C and 7C). These
results were further confirmed in R6/1 mice and in neuronal NG108 V125 cells (Fig. 7, A and
B). Reduction of HSP70 by polyQ appeared to be specific,
because the constitutive heat shock 70 chaperone, HSC70, was not
affected (Fig. 7, A and C). To conclude, these data show that polyQ leads to down-regulation of HSP70 proteins and
suggest that this effect, enhanced by the recruitment of HSP70 into
NIs, might be implicated in the inactivation of the JNK phosphatase M3/6.
PolyQ results in neuronal dysfunction and death. Thus, deciphering
the molecular events implicated in polyQ toxicity is critical for the
design of therapeutic routes. Here we show that polyQ induces a
protein-damaging stress that triggers activation of JNK and AP-1
transcription factor, through inhibition of JNK phosphatase(s). Our
data show that the JNK phosphatase M3/6 is a target for JNK activation
by polyQ and that HSP70, which promotes JNK dephosphorylation, might be
involved in this process. Indeed, we show that polyQ impairs HSP70
levels and localization, and M3/6 solubility. The sequence of events we
propose is schematized in Fig.
8A.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tubulin
antibodies were from Sigma. Anti-HSP70 and anti-HSC70 antibodies were
from Stressgen. Secondary antibodies (goat anti-mouse or goat
anti-rabbit immunoglobulins) conjugated to peroxidase or CY3 or Oregon
Green were from Jackson Laboratories.
-mercaptoethanol, 15% glycerol) by
boiling for 10 min. The preparation was centrifuged for 20 min at
13,000 rpm and room temperature, and the supernatant was recovered,
giving the SN2 fraction. The pellet (fraction P) was solubilized by
incubation in pure formic acid at 37 °C for 30 min then lyophilized
overnight and resuspended in SDS buffer.
-galactosidase activity was dosed to assess
the efficacy of transfection.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of polyglutamine expansion results
in hyperphosphorylation of JNK and c-Jun. A, JNK and
c-Jun are hyperphosphorylated in COS cells expressing ectopic
htt133Q125. COS cells were transfected either with htt133Q15 or
htt133Q125 constructs. Whole cell extracts were analyzed by Western
blot with the different anti-phospho-MAPK antibodies, including
anti-P-JNK, anti-P-p38, and anti-P-ERK antibodies. Membranes were also
revealed with anti-P-c Jun and anti-c-Jun antibodies. Anti-FLAG
antibody was used to control transfection efficiencies. B,
JNK and c-Jun hyperphosphorylation is concomitant to aggregation of
mutated htt in the inducible neuronal NG108 V125 cell line (V125Q).
Control cells (WT) are clones that only express the
tetracycline-inducible transactivator. A time course over 8 days was
performed after differentiation and induction of mutated htt
expression. Whole cell extracts were prepared at the indicated periods
and analyzed by Western blot with anti-P-JNK and anti-P-c Jun
antibodies. Anti-FLAG and anti- -tubulin antibodies were used to
control htt expression and total protein levels, respectively. The
arrows indicate soluble (in the resolving) and aggregated
(in the stacking) htt V125Q. C, basal phosphorylation levels
of JNK and c-Jun are increased in retinas of R6/1 mice. Retinas of R6/1
and wild-type littermate mice aged 8 months were dissected, and total
protein extracts were prepared and analyzed by Western blot. Expression
of expanded exon 1 of htt and total protein levels were controlled with
1C2 and anti-actin antibodies, respectively. D and
E, JNK3 and c-Jun are hyperphosphorylated in retinas of R7E
mice. Retinas of R7E, R7N, and wild-type littermate mice at different
ages (1, 2.5, 3, and 7 months) were dissected, and total proteins were
extracted. Western blot analysis was performed subsequently with
anti-P-JNK and anti-JNK3 and antibodies (D) and with
anti-P-c-Jun (E). 1C2 and anti-CBP antibodies were used to
control expression of mutated ataxin-7 and total protein levels,
respectively.
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Fig. 2.
Expression of polyglutamine expansion results
in transcriptional activation of AP-1 complex. A,
ectopic expression of htt133Q125 in COS cells induces a 4-fold increase
in AP-1-dependent gene expression of CAT. COS cells were
transfected with both TRE-CAT and lacZ reporter plasmids together with
either the empty expression vector, htt133Q15, or htt133Q125. Protein
extracts were prepared 65 h post-transfection, and the CAT amount
present in the extracts was assessed using a CAT ELISA kit (Roche
Molecular Biochemicals). Results were normalized with respect to
-galactosidase activities and are expressed as arbitrary units. A
value of 1 was assigned to the sample corresponding to cells
cotransfected with empty pTL1 and TRE-CAT plasmids. Experiments were
repeated three times, and for each repetition samples were duplicated.
B, induction of htt V125Q expression in neuronal NG108 V125
cells results in activation of AP-1-dependent expression of
CAT. NG108 V125 cells were transfected with the TRE-CAT construct or
not (no reporter). Cells were then differentiated and, concomitantly,
expression of htt V125Q was induced or not with doxycycline. Protein
extracts were prepared at the indicated periods, and the levels of CAT
within the extracts were determined using the CAT ELISA method. Results
were normalized with respect to
-galactosidase activities and are
expressed as arbitrary units. Values obtained at 3.5 and 6.5 days
cannot be compared, because measurements were not performed the same
day. Values of 1 were given for the samples transfected with the
TRE-CAT plasmid, but not treated with doxycycline. The experiment was
repeated twice, and each repetition duplicates were performed.
C, binding of AP-1 complexes to TRE is increased in NG108
cells expressing htt V125Q. NG108 V125 cells were differentiated for 7 days and, concomitantly, expression of htt V125Q was induced or not.
Nuclear extracts were prepared and tested for binding to the AP-1
element, using the Trans-AM AP-1 kit (Active Motif). Results are
expressed as optical density values. Two independent experiments were
realized, with duplicates in each of it. The asterisk
indicates that the difference between both values is significant with a
p < 0.05 according to Student's t test.
D, binding of AP-1 complexes to TRE is increased in R6/1
mice. Retinas of R6/1 and wild-type littermate mice of 8 months were
dissected and nuclear proteins were prepared. The extracts were tested
for binding to the AP-1 element as in C. The experiment was
repeated twice, and for each repetition, duplicates corresponding to
two different mice were performed. Results are significant at
p < 0.05. E, binding of AP-1 proteins to
TRE is augmented in R7E mice. Retinas of R7E and wild-type littermate
mice of both 1 and 3 months were dissected and nuclear proteins were
extracted. Protein extracts were tested for binding to AP-1 as in
C. Experiments were done twice, and values are duplicated as
described for D, i.e. two R7E and two WT mice
were used for each age, in each experiment. The data are significant at
p < 0.05 for mice of 3 months of age.
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Fig. 3.
Hyperphosphorylation of JNK by
polyglutamine expansion results from inactivation of JNK
phosphatases. A, SEK-1 is slightly hyperphosphorylated
in COS cells expressing ectopic htt133Q125. COS cells were
transfected with htt133Q15 or htt133Q125. Phosphorylation
levels of SEK-1 were detected with P-SEK-1 antibody. B,
SEK-1 is not hyperphosphorylated in neuronal NG108 V125 cells
expressing htt V125Q. NG108 V125 cells were differentiated and induced
for 8 days. The parental cell line was used as control, as in Fig.
1B. The P-SEK-1 antibody was used to check phosphorylation
levels of SEK-1, as in A. In C and D
phosphorylation levels of SEK-1 are normal in retinas of R6/1 and R7E
mice. Protein extracts from retinas of R6/1 mice of 8 months and of R7E
mice of 3 and 7 months were analyzed with the P-SEK-1 antibody.
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Fig. 4.
The solubility of the JNK phosphatase M3/6 is
impaired by polyglutamine expansion. A, expression of
expanded htt in COS cells promotes aggregation of ectopic M3/6. COS
cells were transfected with M3/6 together with htt133Q15 or htt133Q125.
Soluble and insoluble proteins were prepared and recovered in fractions
SN1 (soluble), SN2 (rather insoluble), and P (very insoluble). Analysis
of the content of M3/6 in the different fractions was realized by
Western blot. 1C2 and anti-ERK antibodies were used to control the
fractionation procedure. The full-length M3/6 is indicated by the
arrow. B, aggregation of endogenous M3/6 is
increased in neuronal NG108 V125 cells expressing htt V125Q. NG108 V125
cells were differentiated for 7 days and at the same time induced or
not with doxycycline. Proteins were submitted to the fractionation
protocol to recover soluble and insoluble materials. The anti-M3/6
antibody was used to analyze the different fractions. C,
expression of mutated ataxin-7 augments the aggregation potential of
M3/6 in R7E mice. Retinas of R7E and wild-type littermate animals of
2.5 months were dissected and subjected to fractionation. Western blot
analysis of the different fractions was performed as for (A)
and (B) with anti-M3/6 antibody. Detection with 1C2 antibody
revealed the presence of the full-length mutated ataxin-7 in SN1 and
SN2 and of a fragment of about 40 kDa in P. In between, cross-reacting
bands were detected (not shown).
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Fig. 5.
NIs do not recruit M3/6 phosphatase but
HSP70. HSP70, but not M3/6, is recruited into NIs in the
R7E model. Colocalization studies of M3/6 and ataxin-7 (upper
panel) and of HSP70 and ataxin-7 (lower panel) were
performed by double-immunofluorescent confocal analysis. Sections of
retinas from R7E animals of 2.5 months were stained with anti-ataxin-7
antibodies (monoclonal 2A10 in the upper panel and
polyclonal 1261 in the lower panel) and anti-M3/6 or
anti-HSP70 antibodies. Colocalization in NIs was systematically
observed for HSP70, but never for M3/6.
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Fig. 6.
Overexpression of HSP70 inhibits
polyQ-mediated aggregation of M3/6 and activation of JNK. COS
cells were transfected with htt133Q15 or htt133Q125 together or not
with HSP70. Fractionation experiments were performed to recover soluble
and insoluble proteins, as SN1, SN2, and P fractions. The content of
the different fractions was analyzed by Western blotting, using
anti-M3/6 and 1C2 antibody. The soluble SN1 fraction was controlled
with anti-ERK, anti-HSP70, and anti-FLAG antibody. The P-JNK antibody
was also used to test the activation level of JNK in this
fraction.
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Fig. 7.
HSP70 protein levels are down-regulated by
polyglutamine expansion. A, HSP70 is reduced in
neuronal NG108 V125 cells expressing mutated htt. NG108 V125 cells were
differentiated for 7 days and induced or not with doxycycline. Whole
cell extracts were prepared and, because HSP70 is expressed at low
levels in NG108 cells, immunoprecipitated with anti-HSP70 antibody.
Detection of HSP70 was then performed with anti-HSP70 antibody. Protein
levels were controlled with an anti-HSC70 antibody, before
immunoprecipitation. B and C, HSP70 is
down-regulated in R6/1 and R7E mice. Retinas of transgenic and
wild-type animals of 8 months (for R6/1) and of 1 and 3 months (for
R7E) were dissected. Proteins were extracted and directly analyzed with
the anti-HSP70 antibody by Western blot. Indeed, the constitutive
expression level of HSP70 is rather elevated in mouse retinas.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Model for the JNK-dependent
toxicity of polyglutamine expansion. A, proposed model
of the sequence of events, by which polyQ might trigger activation of
JNK and AP-1. In the absence of polyQ and stress, HSP70 might repress
JNK dephosphorylation, through activation of phosphatases such as M3/6.
Kinases upstream of JNK, such as SEK-1, have a low basal
phosphorylation level. In consequence, the basal activation levels of
JNK and c-Jun are very low. When polyQ are expressed, HSP70 is both
down-regulated and recruited into NIs, which might result in the
aggregation of M3/6 and in the increase of the basal phosphorylation
and activation levels of JNK and c-Jun. In neurons, prolonged
activation of c-Jun has been shown to trigger activation of toxic or
death transcriptional programs. B, schematic representation
of the different pathways that might converge to activation of JNK by
polyQ. See text for details.
Whether aggregation of polyQ causes activation of JNK is an important outcome, because the toxicity of NIs is still debated. We observe that both events are concomitant, suggesting they are correlated (Fig. 1B). We also show that polyQ-induced stress and protein-damaging stress such as heat shock, which causes locally protein aggregation, have common features, as both induce activation of JNK through inhibition of JNK phosphatase(s). However, in response to heat shock, hsp70 gene expression is induced, resulting in a secondary inactivation of JNK (16). Strikingly, polyQ-induced stress does not lead to activation of such an auto-regulatory feedback loop. In contrast, we observe a significant and sustained reduction of HSP70 total proteins amounts, indicating that polyQ impairs HSP70 either at the level of gene expression or at the level of protein turnover. This might suggest that soluble polyQ affects some critical cellular processes implicated in the production and maintenance of HSP70 and that down-regulation of HSP70 is not caused by polyQ aggregation. According to this view, reduction of HSP70 proteins by soluble polyQ could be a primary event resulting independently in activation of JNK, via inhibition of its JNK-inactivating function, and in polyQ aggregation, as a consequence of the lack of HSP70-chaperoning activity (Fig. 7B). Indeed, HSP70 is a molecular chaperone essential for de novo protein folding and prevention of protein aggregation (39). Thus, reduction of HSP70 protein levels might enhance the intrinsic tendency to aggregation of polyQ-containing proteins as well as other proteins such as M3/6 for instance. Aggregated-polyQ might recruit HSP70 in a secondary step, thus enhancing repression of JNK dephosphorylation (Fig. 8B) (40).
Down-regulation of HSP70 by polyQ might also trigger activation of JNK by impairing processes involved in intracellular protein breakdown. Aggregated proteins that cannot be refolded by chaperones are targeted to the degradation machinery, which is mainly represented by the proteasome in eukaryotes (41). Chaperones such as HSP70 can promote this situation, by linking physically aggregated proteins and proteasomal components such as the ubiquitin-ligase CHIP or the ubiquitin-related BAG-1 (42, 43). As a result, down-regulation of HSP70 by polyQ might also trigger proteasomal dysfunction and impair degradation of polyQ-containing proteins, which are ubiquitinated (6), as well as other proteins targeted to the proteasome. Interestingly, very recently, polyQ has been shown to trigger endoplasmic reticulum (ER) stress and activation of JNK through proteasomal dysfunction (44, 45). PolyQ-mediated ER stress involves activation of ASK-1, a kinase upstream of SEK-1. In conclusion, reduction of HSP70 proteins by polyQ might induce both protein-damaging and ER stresses. Aggregation of polyQ could enhance these stresses by recruiting chaperones and proteasomal components (Fig. 8B) (7).
In response to stress, activation of JNK and AP-1 results in regulation of pathways of both cell life and death (25). Interestingly, prolonged activation of JNK3 and c-Jun has been associated with neuronal cell death. Indeed, jnk3-deficient mice and c-junala63/73 mice, which express a version of c-Jun that cannot be phosphorylated by JNK, are protected against the neuronal death triggered by excitotoxic stress such as stimulation with kainic acid (26, 27). We show here that polyQ induces prolonged activation of JNK and c-Jun/AP-1 in neurons. Thus polyQ-mediated neuronal toxicity might critically involve activation of c-Jun/AP-1-dependent programs. Although AP-1 complexes have been extensively studied, target genes involved in neurodegeneration are not well defined. C-Jun-dependent induction of pro-apoptotic molecules such as Bim or FasL has, however, been documented (28, 36).
Down-regulation of HSP70 is also susceptible to facilitate induction of apoptosis via JNK-independent mechanisms. HSP70 exerts a cytoprotective function by inhibiting the action of key components of the apoptotic machinery. HSP70 directly interacts with Apaf-1, thus preventing the recruitment of procaspase 9 to the apoptosome (46, 47). The action of apoptosis-inducing factor, a caspase-independent death effector, is also antagonized by HSP70 (48). In addition, depletion of HSP70 has been shown to trigger cell death of tumor cells through activation of apoptotic pathways (49). To what extent this can be true in neurons is now being investigated.
In conclusion, by revealing the deficiency of HSP70 proteins in various
polyQ models, this study gives clues to understanding polyQ toxicity
and confirms the relevance to develop therapeutic protocols aimed to
activate chaperone expression. In addition, our findings underscore the
importance of JNK and AP-1 in polyQ diseases, which offers new
therapeutic perspectives. Consequently, it might be of benefit to
develop strategies for JNK and AP-1 inhibition.
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ACKNOWLEDGEMENTS |
---|
We thank J.-L. Mandel and P. Sassone-Corsi for fruitful discussions and careful reading of the manuscript; J.-L. Vonesh and M. Boeglin for microscopy imaging; M. Lemeur, E. Metzger, and the staff at the Institut de Génétique et de Biologie Moléculaire et Cellulaire animal facility for mouse care; and C Weber for technical assistance. We also thank R. I. Morimoto for the pH2.1-hsp70 plasmid.
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FOOTNOTES |
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* This work was supported by funds from the Centre National de Recherche Scientifique and the Institut National de la Recherche Médicale.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 may be addressed. Tel.: 33-388-653-413; Fax: 33-388-653-246; E-mail: merienne@igbmc.u-strasbg.fr (K. M.) and yvon{at}igbmc.u-strasbg.fr (Y. T.).
Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M212049200
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ABBREVIATIONS |
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The abbreviations used are: SCA7, spinocerebellar ataxia type 7; TRE, TPA-responsive element; polyQ, polyglutamine expansion; NIs, nuclear inclusions; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CAT, chloramphenicol acetyltransferase; SAPK, stress-activated protein kinase; P-JNK, anti-activated JNK antibody; MAPK, mitogen-activated protein kinase; P-c-Jun, anti-phospho c-Jun antibody; ER, endoplasmic reticulum; SEK, SAPK/ERK kinase; MKK, MAP kinase kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Zoghbi, H. Y., and Orr, H. T. (2000) Annu. Rev. Neurosci. 23, 217-247[CrossRef][Medline] [Order article via Infotrieve] |
2. | Ross, C. (1995) Neuron 15, 493-496[Medline] [Order article via Infotrieve] |
3. | Burright, E. N., Clark, H. B., Servadio, A., Matilla, T., Feddersen, R. M., Yunis, W. S., Duvick, L. A., Zoghbi, H. Y., and Orr, H. T. (1995) Cell 82, 937-948[Medline] [Order article via Infotrieve] |
4. | Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W., and Bates, G. P. (1996) Cell 87, 493-506[Medline] [Order article via Infotrieve] |
5. | Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G. P., Davies, S. W., Lehrach, H., and Wanker, E. E. (1997) Cell 90, 549-558[Medline] [Order article via Infotrieve] |
6. | Ross, C. A. (1997) Neuron 19, 1147-1150[Medline] [Order article via Infotrieve] |
7. | Cummings, C. J., Mancini, M. A., Antalffy, B., DeFranco, D. B., Orr, H. T., and Zoghbi, H. Y. (1998) Nat. Genet. 19, 148-154[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Nucifora, F. C., Jr.,
Sasaki, M.,
Peters, M. F.,
Huang, H.,
Cooper, J. K.,
Yamada, M.,
Takahashi, H.,
Tsuji, S.,
Troncoso, J.,
Dawson, V. L.,
Dawson, T. M.,
and Ross, C. A.
(2001)
Science
291,
2423-2428 |
9. | Warrick, J. M., Chan, H. Y. E., Gray-Board, G. L., Chai, Y., Paulson, H. L., and Bonini, N. M. (1999) Nat. Genet. 23, 425-428[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Cummings, C. J.,
Sun, Y.,
Opal, P.,
Antalffy, B.,
Mestril, R.,
Orr, H. T.,
Dillmann, W. H.,
and Zoghbi, H. Y.
(2001)
Hum. Mol. Genet.
10,
1511-1518 |
11. |
Bence, N. F.,
Sampat, R. M.,
and Kopito, R. R.
(2001)
Science
292,
1552-1555 |
12. |
Luthi-Carter, R.,
Strand, A.,
Peters, N. L.,
Solano, S. M.,
Hollingsworth, Z. R.,
Menon, A. S.,
Frey, A. S.,
Spektor, B. S.,
Penney, E. B.,
Schilling, G.,
Ross, C. A.,
Borchelt, D. R.,
Tapscott, S. J.,
Young, A. B.,
Cha, J. H.,
and Olson, J. M.
(2000)
Hum. Mol. Genet.
9,
1259-1271 |
13. | Klement, I. A., Skinner, P. J., Kaytor, M. D., Yi, H., Hersch, S. M., Clark, H. B., Zoghbi, H. Y., and Orr, H. T. (1998) Cell 95, 41-53[Medline] [Order article via Infotrieve] |
14. |
McCampbell, A.,
Taye, A. A.,
Whitty, L.,
Penney, E.,
Steffan, J. S.,
and Fischbeck, K. H.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
15179-15184 |
15. | Steffan, J. S., Bodai, L., Pallos, J., Poelman, M., McCampbell, A., Apostol, B. L., Kazantsev, A., Schmidt, E., Zhu, Y. Z., Greenwald, M., Kurokawa, R., Housman, D. E., Jackson, G. R., Marsh, J. L., and Thompson, L. M. (2001) Nature 413, 739-743[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Meriin, A. B.,
Yaglom, J. A.,
Gabai, V. L.,
Zon, L.,
Ganiatsas, S.,
Mosser, D. D.,
and Sherman, M. Y.
(1999)
Mol. Cell. Biol.
19,
2547-2555 |
17. | Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037[Medline] [Order article via Infotrieve] |
18. | Yan, M., Dai, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800[Medline] [Order article via Infotrieve] |
19. |
Tournier, C.,
Whitmarsh, A. J.,
Cavanagh, J.,
Barrett, T.,
and Davis, R. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7337-7342 |
20. | Cavigelli, M., Li, W. W., Lin, A., Su, B., Yoshioka, K., and Karin, M. (1996) EMBO J. 15, 6269-6279[Abstract] |
21. | Palacios, C., Collins, M. K., and Perkins, G. R. (2001) Curr. Biol. 11, 1439-1443[CrossRef][Medline] [Order article via Infotrieve] |
22. | Theodosiou, A., and Ashworth, A. (2002) Oncogene 21, 2387-2397[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Park, H. S.,
Lee, J. S.,
Huh, S. H.,
Seo, J. S.,
and Choi, E. J.
(2001)
EMBO J.
20,
446-456 |
24. | Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, 2135-2148[Abstract] |
25. | Shaulian, E., and Karin, M. (2002) Nat. Cell Biol. 4, E131-E136[CrossRef][Medline] [Order article via Infotrieve] |
26. | Yang, D. D., Kuan, C. Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., Rakic, P., and Flavell, R. A. (1997) Nature 389, 865-870[CrossRef][Medline] [Order article via Infotrieve] |
27. | Behrens, A., Sibilia, M., and Wagner, E. F. (1999) Nat. Genet. 21, 326-329[CrossRef][Medline] [Order article via Infotrieve] |
28. | Whitfield, J., Neame, S. J., Paquet, L., Bernard, O., and Ham, J. (2001) Neuron 29, 629-643[Medline] [Order article via Infotrieve] |
29. | Trottier, Y., Devys, D., Imbert, G., Saudou, F., An, I., Lutz, Y., Weber, C., Agid, Y., Hirsch, E. C., and Mandel, J. L. (1995) Nat. Genet. 10, 104-110[Medline] [Order article via Infotrieve] |
30. |
Yvert, G.,
Lindenberg, K. S.,
Picaud, S.,
Landwehrmeyer, G. B.,
Sahel, J. A.,
and Mandel, J. L.
(2000)
Hum. Mol. Genet.
9,
2491-2506 |
31. |
Johnson, T. R.,
Biggs, J. R.,
Winbourn, S. E.,
and Kraft, A. S.
(2000)
J. Biol. Chem.
275,
31755-31762 |
32. |
Lunkes, A.,
and Mandel, J.-L.
(1998)
Hum. Mol. Genet.
7,
1355-1361 |
33. | Lunkes, A., Lindenberg, K. S., Ben-Haiem, L., Weber, C., Devys, D., Landwehrmeyer, G. B., Mandel, J. L., and Trottier, Y. (2002) Mol. Cell 10, 259-269[Medline] [Order article via Infotrieve] |
34. |
Helmlinger, D.,
Yvert, G.,
Picaud, S.,
Merienne, K.,
Sahel, J.,
and Devys, D.
(2002)
Hum. Mol. Genet.
11,
3351-3359 |
35. |
Liu, Y. F.
(1998)
J. Biol. Chem.
273,
28873-28877 |
36. |
Le-Niculescu, H.,
Bonfoco, E.,
Kasuya, Y.,
Claret, F. X.,
Green, D. R.,
and Karin, M.
(1999)
Mol. Cell. Biol.
19,
751-763 |
37. | Davis, R. J. (2000) Cell 103, 239-252[Medline] [Order article via Infotrieve] |
38. | Martell, K. J., Seasholtz, A. F., Kwak, S. P., Clemens, K. K., and Dixon, J. E. (1995) J. Neurochem. 65, 1823-1833[Medline] [Order article via Infotrieve] |
39. |
Hartl, F. U.,
and Hayer-Hartl, M.
(2002)
Science
295,
1852-1858 |
40. |
Gabai, V. L.,
Meriin, A. B.,
Mosser, D. D.,
Caron, A. W.,
Rits, S.,
Shifrin, V. I.,
and Sherman, M. Y.
(1997)
J. Biol. Chem.
272,
18033-18037 |
41. |
Hohfeld, J.,
Cyr, D. M.,
and Patterson, C.
(2001)
EMBO Rep.
2,
885-890 |
42. | Connell, P., Ballinger, C. A., Jiang, J., Wu, Y., Thompson, L. J., Hohfeld, J., and Patterson, C. (2001) Nat. Cell Biol. 3, 93-96[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Luders, J.,
Demand, J.,
and Hohfeld, J.
(2000)
J. Biol. Chem.
275,
4613-4617 |
44. |
Kouroku, Y.,
Fujita, E.,
Jimbo, A.,
Kikuchi, T.,
Yamagata, T.,
Momoi, M. Y.,
Kominami, E.,
Kuida, K.,
Sakamaki, K.,
Yonehara, S.,
and Momoi, T.
(2002)
Hum. Mol. Genet.
11,
1505-1515 |
45. |
Nishitoh, H.,
Matsuzawa, A.,
Tobiume, K.,
Saegusa, K.,
Takeda, K.,
Inoue, K.,
Hori, S.,
Kakizuka, A.,
and Ichijo, H.
(2002)
Genes Dev.
16,
1345-1355 |
46. | Beere, H. M., Wolf, B. B., Cain, K., Mosser, D. D., Mahboubi, A., Kuwana, T., Tailor, P., Morimoto, R. I., Cohen, G. M., and Green, D. R. (2000) Nat. Cell Biol. 2, 469-475[CrossRef][Medline] [Order article via Infotrieve] |
47. | Saleh, A., Srinivasula, S. M., Balkir, L., Robbins, P. D., and Alnemri, E. S. (2000) Nat. Cell Biol. 2, 476-483[CrossRef][Medline] [Order article via Infotrieve] |
48. | Ravagnan, L., Gurbuxani, S., Susin, S. A., Maisse, C., Daugas, E., Zamzami, N., Mak, T., Jaattela, M., Penninger, J. M., Garrido, C., and Kroemer, G. (2001) Nat. Cell Biol. 3, 839-843[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Nylandsted, J.,
Rohde, M.,
Brand, K.,
Bastholm, L.,
Elling, F.,
and Jaattela, M.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7871-7876 |