From the UMR144 CNRS/Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France and
INSERM U434
Centre d'Etude du Polymorphisme Humain, 75010 Paris, France
Received for publication, October 17, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Schwannomin (Sch) is the product of the
NF2 tumor suppressor gene. The NF2 gene is
mutated in patients affected by neurofibromatosis type 2, a syndrome
associated with multiple tumors of the nervous system. Here we found
that Sch, when its N-terminal FERM domain was misfolded by the
pathogenetic mutation Neurofibromatosis 2 is an inherited disorder that predisposes the
patient to the development of nervous system tumors such as schwannomas
and meningiomas. The NF2 tumor suppressor gene responsible
for this syndrome has been identified by positional cloning (1). The
NF2 gene is also implicated in sporadic schwannomas and
meningiomas as well as in mesotheliomas induced by asbestos. The
product of the NF2 gene, schwannomin
(Sch),1 also known as merlin,
is highly related to ERM (ezrin, radixin, moesin) proteins (2). These
proteins are about 600 amino acids long. They display a globular
N-terminal domain of about 300 amino acids called the FERM domain. The
highest homology between schwannomin and ERM proteins is in their FERM
domain (63% identity). ERM proteins have a well established role as
linkers between the plasma membrane and the actin cytoskeleton. Sch is
a regulator of cell growth and a mediator of contact
inhibition, probably through its ability to organize or sense the
attachment of actin to the plasma membrane (3-5).
For both Sch and ERM proteins, the FERM domain is responsible for their
localization at the cytoplasmic side of the plasma membrane (6, 7). The
FERM domain interacts with membrane proteins and with filamentous actin
(3, 8). The crystal structures of FERM domains from ERM proteins and
Sch have confirmed that they display a similar overall structure (9,
10). The C-terminal half of these molecules is not well conserved,
except in the last 100 amino acids of the tail, which contains a FERM binding site (11). ERM proteins contain in addition a filamentous actin
binding site in the tail. The interaction between the FERM domain and
the tail occurs intramolecularly or intermolecularly, giving rise to
closed monomers or oligomers, respectively (2). Oligomers also form
between ERM proteins and Sch (12). The interaction between the FERM
domain and the tail masks important functional sites for membrane
partners and actin binding. The activation of Sch and ERM proteins,
thus, involves a conformational change unmasking these sites.
Here we studied the effect of a pathogenetic mutation in the FERM
domain of schwannomin, Sch Cell, Drugs, and Antibodies--
LLC-PK1 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and maintained at 37 °C in 10% CO2. The following drugs
were used at the indicated final concentration: nocodazole (10 µg/ml,
Sigma), MG132 (50 µM, Calbiochem), clasto-lactacystin
cDNA Constructs and Transfection--
Ezrin/Sch chimeras
were designed according to a multiple alignment of ERM members and
NF2 gene products from Homo sapiens, Caenorhabditis elegans, and Drosophila
melanogaster to preserve the overall structure of this family of
proteins. These ezrin/Sch chimeras were derived from the previously
described Ezr
Transfection of the different cell lines was performed by
electroporation as described (16). Transiently transfected cells were
analyzed 20 h after transfection. Nocodazole was applied for
15 h, 5 h after replating transfected cells. Pools of stable transfectants were selected by 2-3 weeks of culture in medium containing 0.7 mg/ml G418 (Invitrogen).
Metabolic Labeling and Pulse-chase Analysis--
Pools of stable
LLC-PK1 transfectants were passaged in 6-cm dishes so as to reach
confluency the next morning. Metabolic labeling was achieved in
Dulbecco's modified Eagle's medium without Met and Cys complemented
with 250 µCi/ml 35S-labeled Met and Cys from Redivue
Promix (Amersham Biosciences). For the chymotrypsin digestion
experiment, cells were labeled for 1 h. For pulse-chase, cells
were labeled for 15 min and chased in standard Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum. After
immunoprecipitation and SDS-PAGE, signals were quantified using a STORM
860 PhosphorImager and ImageQuant software (Molecular Dynamics). Only
experiments where an exponential decay regression (y = ae Lysates, Immunoprecipitations, and Chymotryptic
Digestion--
To prepare total cellular lysates, cells were rinsed
once with cold PBS, extracted with 100 µl of boiling 1× SDS loading
buffer, and scraped. The lysates were then sonicated. For
immunoprecipitations, cells were extracted with 1 ml of cold RIPA
buffer (50 mM Hepes, 150 mM NaCl, 10 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1%
SDS, pH 7.4) supplemented with protease inhibitors (Sigma) for 2 min at
4 °C. The extracts were then clarified in a microcentrifuge at
20,000 × g for 10 min at 4 °C. Denatured lysates
were obtained by adding 500 µl of boiling 10 mM Hepes,
150 mM NaCl, 1% SDS, pH 7.4, and scraping. The lysates
were boiled for 2 min. RIPA was then reconstituted with 4.5 ml of cold
54.4 mM Hepes, 150 mM NaCl, 11 mM
EDTA, 1.1% Nonidet P-40, 0.56% sodium deoxycholate, pH 7.4. The
extracts were clarified by centrifugation at 4000 rpm for 10 min.
For immunoprecipitations from LLC-PK1 stable transfectants, lysates
were incubated with 10 µl of protein A-Sepharose (Amersham Biosciences) and 2 µg VSV G pAb for 2 h (or overnight for
volumes larger than 1 ml). Beads were washed 4 times with 1 ml of RIPA buffer. Precipitated proteins were eluted by boiling for 2 min in 20 µl of 1.5× SDS loading buffer. For chymotryptic digestion, after 3 RIPA washes, beads were washed with 1 ml of digestion buffer (20 mM MES, 300 mM KCl, 0.5 mM
dithiothreitol, pH 6.7) supplemented with 0.1% Triton X-100. Then,
beads were incubated in 10 µl of digestion buffer with or without 0.1 µg of chymotrypsin (Sigma) for 1 h at room temperature with
agitation. Digestion reactions were stopped by adding 10 µl of 3×
SDS loading buffer and boiling for 2 min.
The 35S signal was enhanced by incubating the gels in 1 M salicylate for 20 min. Dried gels were exposed to films
at Immunofluorescence--
Cells on glass coverslips were rinsed
twice with PBS, fixed with 100% methanol for 5 min at Electron Microscopy--
Cells on coverslips were fixed in 80 mM cacodylate, 0.05% CaCl2, 2.5%
glutaraldehyde, pH 7.4, for 1 h. After several washes in water,
post-fixation was performed with 1% osmium tetroxide, 1.5% potassium
ferrocyanate in water for 45 min at 4 °C. Cells were washed several
times in water and then en bloc-stained with 2% uranyl acetate in 40%
ethanol for 30 min. Dehydration was then achieved in a series of
ethanol baths, and the coverslips were processed for flat embedding in
Epon 812 resin (Taab Cie). Ultrathin sections were made using a
Reichert Ultracut-FCS ultramicrotome (Leica). Sections were contrasted
with ethanolic uranyl acetate and Reynolds lead citrate solution before
visualization at 80 kV in a Philips CM120 electron microscope.
Precipitated GST proteins were diluted in PBS. A 10-µl sample was
applied to carbon-coated grids and allowed to adsorb for a few minutes.
The grids were washed four times with water. Grids were subsequently
stained with 2% uranyl acetate in water for 2 min, rinsed once with
water, air-dried, and viewed in the electron microscope.
Sch
The juxtanuclear accumulation of Sch
Aggresome-forming proteins are usually short-lived proteins. In line
with this observation, we recently observed that mutant forms of Sch
are efficiently degraded by the ubiquitin-proteasome pathway (14). Here
we found that the Sch
To examine the ultrastructure of Sch The Delineation of a Sch Determinant for Aggresome Formation Using
Ezrin/Sch Chimeras--
To explain the different behaviors
of ezrin
We next sought to examine the expression of these misfolded chimeras.
We immunoprecipitated through the VSV G epitope the transfected
proteins from denatured extracts to solubilize the aggregated material
(see "Materials and Methods"). By VSV G immunoblotting of the
immunoprecipitates, we could detect all chimeras at their expected size
or at a slightly higher size when they contained the stretch of 7 prolines of ezrin (amino-acids 469-475; Fig. 4C). We
noticed a ladder of conjugates of Sch Aggresome Formation by Ezrin/Sch Chimeras Is Independent
from Their Degradation Rate--
The centrosome is associated with
active proteasomes, suggesting that this location is a privileged site
for degradation of ubiquitinated proteins (24, 25). In line with this,
it can be envisioned that the microtubule-dependent
transport of proteasomal substrates toward the centrosome, evidenced by
the formation of aggresomes, is a way to ensure a high rate of
degradation under normal conditions and that the aggresomes are the
result of inhibition or overwhelming of this "centralized"
degradation machinery. If this hypothesis is correct, it predicts that
the ability for a misfolded protein to form aggresomes upon
overexpression is related to a fast degradation rate when it does not
aggregate. In fact, in stable LLC-PK1 transfectants of Sch GFP-Sch535-595 Forms Aggresomes--
Having
determined that the C terminus of Sch induced a chimera containing a
misfolded FERM domain to form aggresomes, we next asked whether it
would form aggresomes when fused to a folded protein. For this purpose,
we constructed fusion proteins with GFP, which fluoresces in green when
properly folded (Fig. 6). The green fluorescent signal was detected in
large aggresomes upon transient transfections of LLC-PK1 cells with
GFP-Sch340-595, indicating that the C terminus of Sch can
aggregate, whereas the appended GFP moiety remains folded. Importantly,
the 61-amino acid sequence Sch535-595 fused to GFP also
formed aggresomes, although these were usually smaller than the one
formed by GFP-Sch340-595. Consistent with the chimera
analysis, GFP-Sch544-595 never formed an aggresome.
GFP-Sch535-580, which contains the common part between the
two isoforms of Sch, did not form an aggresome either. This might
reflect a difference of sensitivity between the chimera and the GFP
assays or a contribution of the alternative amino acids of Sch isoform
2 in the formation of aggresomes. Nonetheless, our results demonstrate
that the small Sch535-595 fragment is sufficient to induce
a folded protein such as GFP to form
aggresomes.
We then assessed the stability of these GFP fusion proteins by a
pulse-chase experiment. All GFP-Sch fusion proteins had a half-life of
about 10 h, which was lower than the one of GFP alone (>48h) but
longer than those of the misfolded ezrin/Sch chimeras. Importantly, the
aggresome-forming proteins, GFP-Sch340-595 and
GFP-Sch535-595, had a comparable turnover to
GFP-Sch544-595 and GFP-Sch535-580 that did
not form aggresomes. This result further confirmed that aggresome
formation and degradation are independent processes.
Sch535-595 Spontaneously Aggregates in Vitro--
To
study further the properties of the aggresome determinant, we purified
from Escherichia coli GST proteins fused to
Sch535-595 or to Sch544-595 as a negative
control. Purified GST-Sch535-595 and
GST-Sch544-595 in PBS/glycerol (50%) were first
centrifuged at 12,000 × g for 10 min to ensure they
were both soluble at the beginning of the experiment. When equal
amounts of GST-Sch535-595 and GST-Sch544-595
were then kept for 3 days at 4 °C, Sch535-595 but not
Sch544-595 formed a visible precipitate. The precipitated
material was pelleted by a 10-min centrifugation at 12,000 × g, washed in PBS, and resuspended in SDS loading buffer. The
amount of protein present in the supernatant and in the pellet was
analyzed by SDS-PAGE and Coomassie Blue staining (Fig.
7A). By band densitometry of
the amount of GST proteins in the total and supernatant fractions we
estimated that more than 50% of GST-Sch535-595 had
precipitated during the incubation. The pellet was resuspended in
1/5 the original volume of the suspension. When we examined the
precipitate-containing pellet by electron microscopy after negative
staining, numerous aggregates of GST-Sch535-595 were
observed (Fig. 7B). The aggregates were inhomogeneous in size but could be large, spanning several hundreds of nm. This experiment demonstrates that Sch535-595 aggregates in the
absence of cellular factors.
Here we showed that a mutant form of the NF2 tumor
suppressor, Sch Strikingly, in contrast to Sch Although Sch535-595 is one of the strongest aggresome
determinants, there are probably others distributed in Sch and at
least one in the misfolded FERM domain of Sch, as evidenced by
the behavior of the chimera SE. It is also possible that another
aggresome determinant resides in Sch340-534, since
Sch535-595 seems slightly less efficient in aggresome
formation than Sch340-595 when fused to GFP. The presence
of aggresome determinants in the C terminus of Sch was unexpected given
that wild type Sch does not form aggresomes upon overexpression. It is
likely that the major Sch535-595 aggresome determinant is
masked by the interaction with the folded FERM domain. It is also
possible that the binding of the folded FERM domain to the plasma
membrane counteracts the forces of microtubule-dependent transport if the wild type Sch were to aggregate. We showed that Sch535-595 is able to trigger folded protein like GFP or
GST to aggregate independently of the misfolded FERM domain.
Sch535-595 displays a significant homology to its
counterpart in ezrin (34% identical to ezrin526-585).
This homology probably reflects the conservation of the FERM binding
site rather than the aggresome determinant, since misfolded ezrin does
not form aggresomes in our assay. Sch535-595 is predicted
to form mostly Protein aggregation is a characteristic of many neurodegenerative
diseases (27). However, its involvement in the pathology of
neurofibromatosis type 2 remains to be documented. The question arises
then of the role of aggregation-prone sequences in Sch, which seem to
be absent from the related protein ezrin. Maybe part of the answer
resides in the fact that mutant forms of Sch with a misfolded FERM
domain were efficiently degraded by the ubiquitin-proteasome pathway
(14). Actually, tumor analysis has consistently displayed an absence of
mutant Sch, even in cases where the mutant alleles are expressed.
Presumably, without experimental overexpression or inhibition of
proteasomes, misfolded Sch does not have time to aggregate.
Aggresome-forming proteins are usually short-lived. When it has been
examined, aggresome-forming proteins were found to be substrates of the
proteasomes (27). Why such a relationship? It has been proposed that
centrosomes are a privileged degradation site by proteasomes.
Proteasomes and their activator complexes were found to be concentrated
at the centrosome (24, 25). The accumulation of proteasomal substrates
at the centrosome when degradation is inhibited can be interpreted as
if the centrosome was indeed the site of their degradation (28).
However, in the absence of the direct imaging of the degradation
process itself, this hypothesis is hard to formally prove or dismiss.
In the hypothesis where the degradation process is localized at the
centrosome, the microtubule-dependent transport of
misfolded proteins could be an efficient way to bring substrates to the degradation machinery. The correlation between fast degradation and
efficient aggresome formation when Sch Therefore, these results support the alternative scenario in which
degradation and aggregation represents two alternate fates for
misfolded proteins (27). These proteins are normally degraded. Aggresomes are formed only when the degradation machinery is inhibited or overwhelmed by overexpression. The ubiquitination of aggregated proteins in aggresomes simply reflects that these substrates were normally destined to be degraded but does not imply that ubiquitination triggers the microtubule-dependent transport. Actually,
some proteins were described to form aggresomes without being
ubiquitinated even though they were proteasomal substrates (20, 29).
Because we observed aggregation with the purified
GST-Sch535-595 in vitro in the absence of
cellular factors, aggregation is most likely the first step in the
formation of an aggresome.
Because degradation and aggregation are independent processes, the
frequent association of fast degradation and efficient aggresome
formation in many proteins remains to be explained. It is possible that
the inherent toxicity of aggregates (30) strongly counter-selected the
presence of aggresome determinants in long-lived proteins during
evolution. In particular, aggresomes have been found to inhibit the
ubiquitin-proteasome system, which is required for many important
cellular functions (31). In other words, aggresome formation might be a
property of short-lived proteins because in short-lived proteins,
degradation overcomes aggregation.
The mechanism and the role of aggresome formation by the cell remains
poorly understood. The microtubule-mediated transport for example
requires dynein (29), but how dynein recognizes protein aggregates is
not known. The isolation of an easily produced aggresome determinant
such as Sch535-595 opens up a new way to study the
mechanism of aggresome formation by reconstituting the steps after
aggregation in a cell-free system.
F118, formed aggresomes, i.e.
aggregates that cluster at the centrosome as a result of microtubule-dependent transport. Strikingly the related
protein ezrin affected by the same mutation did not form aggresomes
even though its FERM domain was similarly misfolded. By studying
ezrin/Sch chimeras, we delineated a sequence of 61 amino acids in the C terminus of Sch that determined the formation of aggresomes. Aggresome formation by these chimeras was independent from their rate of degradation. Sch535-595 was sufficient to induce
aggresomes of a green fluorescent fusion protein in vivo
and aggregates of a glutathione S-transferase fusion
protein in vitro. Taken together, these results suggest that aggresome formation is controlled primarily by aggresome determinants, which are distinct from degradation determinants, or from misfolding, through which aggresome determinants might be exposed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
F118. The deletion of the phenylalanine 118 codon has been described in two unrelated families affected by
neurofibromatosis type 2 as well as in a sporadic meningioma (see
references in Ref. 13). We recently found that the
F mutation impairs the proper folding of Sch FERM domain (8). Because the
phenylalanine affected by this deletion is conserved in ERM proteins,
we introduced the equivalent
F102 deletion into ezrin. Strikingly,
Sch
F, but not ezrin
F, formed aggresomes upon transient transfection. We mapped the principal determinant of this behavior with
ezrin/Sch chimeras.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-lactone (50 µM, Calbiochem), cycloheximide (10 µM, Sigma). The following antibodies were used: VSV G pAb (1 µg/ml), P5D4 VSV G mAb (1:2000), 20 S proteasome pAb (1:1000, Affiniti Research Products), GTU-88
-tubulin mAb (1:1000; Sigma), 7A3 vimentin mAb (1:100, gift of C. Maison, Institut Curie, Paris), N356
-tubulin mAb (1:2000, Amersham Biosciences), GFP pAb
(Clontech). The indicated concentrations were used
for immunofluorescence. For immunoblotting, 1 µg/ml of primary
antibodies was used.
F and Sch
F constructs in pCB6 (7, 14) and contained
at their C terminus the VSV G tag. Briefly, chimeras were obtained
as described in Table I
either by PCR amplification of one cDNA fragment bringing a
restriction site for the insertion into the other cDNA or by PCR-mediated recombination in which the fusion of the two cDNAs is obtained during PCR with two primers overlapping by 20 nucleotides. GFP fusion proteins were constructed in pEGFP-C1
(Clontech). Sch340-595 was inserted as
a XhoI-EcoRI fragment.
Sch535-595, Sch544-595,
Sch535-580 were PCR-amplified and inserted as
XhoI-BamHI fragments at the C terminus of GFP.
GST fusion proteins were constructed in a modified pGEX 4T2 (Amersham
Biosciences) in which the NotI site had been converted into
an XbaI site. Untagged Sch340-595 was inserted
as a XhoI-XbaI fragment. Sch535-595
and Sch544-595 were excised as a
BglII-BamHI fragments from the corresponding GFP
plasmids and inserted into the BamHI site of pGEX 4T2.
PCR-amplified fragments were fully sequenced to ensure that no unwanted
mutations were introduced. pCW7-expressing Myc-tagged ubiquitin was
previously described (15).
Constructions of ezrin/Sch chimeras
bx, where y is the
percent of the t = 0 signal, and x is time
in hours, calculated with Excel) giving a correlation coefficient R2 > 0.95 were taken into account to calculate
the half-life according to the formula t1/2 = (ln
50
ln a)/
b. This procedure gave less than 10%
variation between experiments.
80 °C or to a phospho-screen from 1 day to 1 week.
20 °C, and
rinsed twice with PBS and then once with PBS supplemented with 1 mg/ml
bovine serum albumin. Primary antibodies were incubated in PBS/bovine
serum albumin for 20 min. Coverslips were rinsed twice with PBS, once with PBS/bovine serum albumin, and then incubated for 20 min with appropriate Alexa488-conjugated or Cy3-conjugated anti-mouse or anti-rabbit Ab (1:200, Jackson ImmunoResearch) and Hoechst 33258 (10 µg/ml, Sigma). The GFP signals were also obtained after methanol fixation. After 4 washes in PBS, cells were mounted in Mowiol and
photographed using a Leica microscope equipped with a CCD camera
(Princeton Instruments) or a Leica confocal laser-scanning microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
F, but Not Ezrin
F, Forms Aggresomes upon Transient
Transfection--
To compare the effects of the
F deletion on the
tumor suppressor gene product and a related ERM protein, we transiently
transfected ezrin
F and Sch
F in LLC-PK1 cells. The two proteins
were tagged at their C terminus with a VSV G epitope so we examined
their localization by VSV G immunofluorescence. The localizations of Sch
F and ezrin
F were drastically different even though their level of expression was similar (see below). In the majority of transfected cells, Sch
F accumulated in a juxtanuclear area,
whereas ezrin
F was diffusely distributed in the cytosol (Fig.
1, a and b). The
juxtanuclear accumulation of mutant Sch has been already noticed with
other mutations of the FERM domain (7, 17). The two localizations of
Sch
F and ezrin
F were both clearly different from those of wild
type ezrin and Sch, which are found at the plasma membrane (data not
shown (18, 19)).
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Fig. 1.
Sch F, but not
ezrin
F, forms aggresomes upon transient
transfection in LLC-PK1 cells. Cells were transfected with
ezrin
F (a) or Sch
F (b-l) and examined by
immunofluorescence on a standard microscope (a and
b) or a confocal microscope (c-l). A single
confocal section is shown. VSV G immunofluorescence reveals a diffuse
localization of ezrin
F (a) but a juxtanuclear aggregation
of Sch
F (b). Sch
F aggregates are clustered around the
centrosome (c,
-tubulin mAb (green); VSV G pAb
(red)). Sch
F transfectants were left untreated
(d) or treated with nocodazole (e) to
depolymerize microtubules. Nocodazole treatment abrogates juxtanuclear
accumulation of Sch
F aggregates (d and e,
-tubulin (green); mAb, VSV G pAb (red)). Vimentin
filaments are reorganized around clustered Sch
F aggregates
(f, vimentin mAb (green), VSV G pAb
(red)). Sch
F aggresomes are composed of ubiquitinylated
material (cotransfection with Myc-tagged ubiquitin; g, Myc
mAb (green); h, VSV G pAb (red);
i, merge). Sch
F aggresomes recruit proteasomes
(j, proteasome pAb green; k, VSV G mAb
(red); l, merge). Bars, 5 µm.
F was reminiscent of a recently
described structure called the aggresome (20). The aggresome is a
structure composed of clustered aggregates of misfolded proteins, when
such proteins have been overexpressed or their degradation has been
inhibited. Aggregates of Sch
F fulfilled the criteria of aggresomes
as follows. (i) Aggregates of Sch
F accumulated at the centrosome, as
revealed by
-tubulin staining (Fig. 1c). Furthermore, in
some cells containing Sch
F aggregates,
-tubulin staining of the
centrosome was absent (data not shown), suggesting that the
accumulation of misfolded material can impede access of
-tubulin
antibodies to the centrosome. (ii) The accumulation of Sch
F at the
centrosome was dependent on the integrity of the microtubule
cytoskeleton (Fig. 1, d and e). Most
Sch
F-transfected cells formed aggresomes in control conditions, but
only a few did in the presence of nocodazole, which depolymerizes
microtubules. Dispersed aggregates of Sch
F were observed throughout
the cytosol of nocodazole-treated cells. (iii) A cage-like structure of
vimentin filaments surrounded the Sch
F aggresome (Fig.
1f).
F aggresome was composed of ubiquitinylated
material, since it was stained with Myc antibodies when Myc-tagged
ubiquitin was cotransfected with Sch
F (Fig. 1, g-i).
Moreover, proteasomes, which are normally diffusely distributed in the
cytoplasm and the nucleus, were recruited to the aggresome (Fig. 1,
j-l). Another pathogenetic mutant of Sch with a misfolded FERM domain, Sch
39-121, formed similar aggresomes (data not shown). Aggresome formation by Sch
F, but not by ezrin
F, was observed in
many cell lines (HeLa, Chinese hamster ovary, NIH3T3, Cos7, A431, A549,
CV1, baby hamster kidney cells, OK, and Madin-Darby canine kidney
cells; data not shown). These results emphasize that in transient
transfections aggresome formation is a robust behavior specific to
mutant Sch when compared with mutant ezrin.
F aggresomes, we performed
transmission electron microscopy. When Sch
F or ezrin
F transiently transfected LLC-PK1 cells were examined, amorphous electron dense structures were found only in Sch
F-expressing cells (Fig.
2). These structures, which were found in
the vicinity of the nucleus and Golgi vesicles, were not enclosed by
membranes, indicating that they indeed corresponded to aggregates.
These aggregates of several hundreds of nm in size were presumably very
dense since their periphery was stained more intensely with heavy metal
salts than their center. The concentration of intermediate filaments around the aggregates at the ultrastructural level supported the reorganization of vimentin seen by immunofluorescence.
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Fig. 2.
Ultrastructure of
Sch F aggresomes. LLC-PK1 cells were
transiently transfected with Sch
F and processed for transmission
electron microscopy. The aggregates (A) appeared as
amorphous electron dense material. The aggregates were embedded in a
dense meshwork of intermediate filaments (IF), in close
proximity to the Golgi apparatus (G) and the nucleus
(N). Bar, 200 nm.
F Mutation Impairs the Proper Folding of Both Sch and Ezrin
FERM Domains--
Because aggresome formation is thought to be a
general response to misfolded proteins, we reasoned that perhaps ezrin
was not misfolded by the
F mutation. This would be surprising given that the crystallographic structures of the FERM domain of ERM proteins
and Sch have revealed a very similar organization (9, 10). We assessed
experimentally the global folding of the FERM domain with chymotrypsin
because the FERM domain of both ezrin and Sch is known to resist
chymotryptic digestion (8, 21). For this purpose, we transfected
LLC-PK1 cells with cDNAs encoding wild type or
F schwannomin or
ezrin. Pools of stable transfectants were derived. In these stable
transfectants, both mutant proteins were diffusely localized and
soluble in RIPA buffer (data not shown). The 35S-labeled
exogenous proteins were immunoprecipitated through the VSV G epitope,
and the immunoprecipitated proteins were submitted to chymotrypsin.
With both wild type ezrin and Sch a series of proteolytically resistant
fragments were detected down to about 35 kDa, which is the molecular
mass of the FERM domain (Fig. 3). In
contrast, ezrin
F and Sch
F were no longer resistant to
chymotrypsin. Thus, the
F mutation impairs the proper folding of the
FERM domain in both ezrin and Sch.
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Fig. 3.
The F mutation
impairs the proper folding of the FERM domain of both ezrin and
Sch. Stable LLC-PK1 transfectants expressing wild type or mutant
ezrin (Ezr) or Sch were 35S metabolically
labeled. These exogenous proteins were immunoprecipitated through their
VSV G tag and submitted or not to chymotryptic digestion. Resistant
fragments were not observed with mutant proteins, indicating misfolding
of their FERM domain.
F and Sch
F, we assumed there was a sequence determining
aggresome formation. To identify this determinant, we constructed
chimeras between ezrin
F and Sch
F. We first swapped the misfolded
FERM domains of ezrin
F and Sch
F, giving rise to the chimeras SE
and ES1 (Fig. 4). The two chimeras were
able to form aggresomes, suggesting that there are at least two
determinants in the Sch sequence. However, aggresomes formed by SE were
smaller and looser than the ones formed by ES1. This indicated that the
main determinant of aggresome formation by Sch
F was not the
misfolded FERM domain but, rather, the C-terminal half of the molecule.
Therefore, we focused on the C-terminal aggresome determinant. The
chimera ES3 (Sch535-595) as well as the chimera ES2
(Sch444-595) formed aggresomes. Thus, the 61 amino acid
sequence Sch535-595 contains the major C-terminal
aggresome determinant. A further N-terminal deletion of this sequence
by 26 amino acids in the chimera ES4 (Sch561-595) or by
only 9 amino acids in the chimera ES5 (Sch544-595)
abrogated its ability to induce aggresomes. The C terminus of ERM
proteins and Sch contains a sequence that is able to bind to the FERM
domain of both types of proteins. A second isoform of Sch with an
alternatively spliced exon has a different C terminus that is unable to
bind to the FERM domain (11, 22, 23). We constructed a chimera ES3iso2
in which the Sch sequence starts at position 535 and ends with this
alternate C terminus (580-590). ES3iso2 also formed aggresomes,
suggesting that aggresome formation is independent from the ability of
Sch C-terminal sequence to bind to the FERM domain.
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Fig. 4.
Ezrin/Sch chimeras reveal an aggresome
determinant in the C terminus of Sch. A, schematic
representation of the various chimeras with which LLC-PK1 cells were
transiently transfected. B, aggresome formation was examined
by VSV G immunofluorescence. Bar, 5 µm. C, to
detect the various chimeras, transiently transfected cells were lysed
under denaturing conditions. The chimeras were immunoprecipitated with
VSV G pAb and detected by immunoblotting with VSV G mAb.
F, ES1, ES2, and to a smaller
extent of ES3, ES3iso2, and SE. This ladder is likely due to
ubiquitination, since Sch
F is efficiently degraded by the
ubiquitin-proteasome pathway (14). These conjugates were at the limit
of detection with ezrin
F, ES4, and ES5, which did not form
aggresomes. Thus, this correlation between aggresome formation and
ubiquitination confirms the observation that the aggregated material is
ubiquitinated (Fig. 1).
F and
ezrin
F, in which both proteins are soluble due to moderate
overexpression, we recently found that Sch
F is degraded 3 times
faster than ezrin
F (1.7 versus 5.4 h of half-life
(14)). We examined whether this correlation between aggresome formation
in transient transfections and fast turnover of the misfolded proteins
in stable transfectants holds true with our series of chimeras. We
selected stable transfectants for each of them and measured their
degradation rate by a pulse-chase analysis followed by VSV G
immunoprecipitations from RIPA extracts (Fig.
5). The chimeras had relatively short
half-lives, from 0.9 to 1.8 h, closer to the one Sch
F than
ezrin
F. Importantly the aggresome-forming chimeras were not degraded
faster than the others. For example, ES3 had a half-life of 1.7 h
compared with 1.4 or 0.9 h for ES4 or ES5, respectively, 2 chimeras that do not form aggresomes. Even though in stable
transfectants the most part of misfolded proteins was extracted by the
RIPA buffer, it was still possible that some residual aggregated
material was not analyzed by this method. So we also assayed the
stability of the chimeras by immunoblotting total cellular lysates
after different times of a cycloheximide treatment that prevents
protein synthesis. This experiment confirmed that the rate of
degradation of aggresome-forming chimeras was similar to the one of the
other chimeras (Fig. 5). Because fast degradation and aggresome
formation were uncoupled in this series of chimeras, these experiments
unambiguously dismiss the hypothesis that accumulation of aggregated
proteins at the centrosome relates to the efficiency of
degradation.
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Fig. 5.
Aggresome formation by ezrin/Sch chimeras is
independent from their rate of degradation. Stable LLC-PK1
transfectants expressing the various chimeras were pulse-labeled with
35S for 15 min and chased for 0, 2, 4, or 6 h as
indicated. VSV G immunoprecipitates were autoradiographed after
SDS-PAGE. Alternatively, the cells were treated with cycloheximide
(CHX) to prevent protein synthesis for 0, 2, 4, or 6 h,
and total cellular lysates were immunoblotted with VSV G
antibodies.
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Fig. 6.
Aggresome formation by GFP-Sch fusions.
LLC-PK1 cells were transiently transfected with indicated GFP-Sch
fusions. A, immunoblot analysis of total cellular lysates
using GFP antibodies. B, localization of GFP-Sch fusions.
Bar, 5 µm. C, pulse-chase analysis of stable
transfectants. Cells were pulse-labeled with 35S for 15 min
and chased for 0, 6, 24, or 48 h, as indicated.
View larger version (98K):
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Fig. 7.
In vitro aggregation of
GST-Sch535-595. Purified GST-Sch535-595
and GST-Sch544-595 were kept 3 days at 4 °C in a buffer
containing 50% glycerol. A, total, pellet, and supernatant
fractions were analyzed by Coomassie staining after SDS-PAGE. By
densitometry quantification of the supernatant compared with the total,
more than 50% of GST-Sch535-595 had precipitated. The
pellet was resuspended in one-fifth the original volume. B,
GST-Sch535-595 aggregates found in the pellet, as observed
by electron microscopy. Bar, 100 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
F, accumulates in juxtanuclear aggregates called
aggresomes. The
F mutation impairs the proper folding of the FERM
domain of Sch (8). The aggresome is a structure in which misfolded proteins accumulate when they are overexpressed or their degradation is
inhibited (20). These aggresomes were clustered around the centrosome
as a result of transport along microtubules. A large number of vimentin
intermediate filaments were observed to intersperse and encage the
Sch
F aggregates. In addition, these aggresomes were composed of
ubiquitinated Sch
F and recruited proteasomes that have been shown to
associate dynamically with the centrosome (25).
F, the related protein ezrin
F never
formed aggresomes upon overexpression by transient transfection even
though the folding of ezrin FERM domain was similarly impaired. Thus,
ezrin
F seems to be an exception to the rule that all misfolded proteins form aggresomes (20). However, upon inhibition of proteasomes in stable transfectants, ezrin
F formed very small aggresomes, whereas in the same conditions, Sch
F formed large ones (data not
shown). In consequence, there is a large quantitative difference in the
propensity of these two proteins to form aggresomes but not an absolute
qualitative difference. We took advantage of the fact that ezrin
F
never formed aggresomes upon transient transfection to map determinants
of aggresome formation in Sch
F using ezrin/Sch chimeras. We
delineated Sch535-595 as a major determinant. This
sequence induced a chimera composed at 90% of ezrin
F to form
aggresomes. Most importantly, this small sequence when fused to GFP was
sufficient to form aggresomes and when fused to GST to aggregate
in vitro. This is the smallest sequence scoring positive in
all three assays. To our knowledge, this is the first time that such a
small aggresome determinant was isolated.
-helices by several versions of software and, more
specifically, a coiled coil in its first 20 amino acids (26). The
predicted coiled coil is lost with the 9-amino acid deletion displayed
by Sch544-595, which does not form aggresomes. However, it
is unclear whether this coiled coil actually forms and is responsible
for the behavior of this sequence. No consensus sequences has emerged
from the studies of aggregating proteins so far, and aggresome
determinants have to be isolated experimentally.
F is compared with ezrin
F
(this study (14)) lend support to this hypothesis. However, when the
ezrin/Sch chimeras used to delineate the Sch535-595
aggresome determinant were analyzed for their degradation rates, this
correlation was lost. A similar result was obtained with GFP-Sch fusion
proteins. This formally establishes that degradation and aggresome
formation are distinct processes, since they can be separated in
experimental chimeras.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Bornens, L. Goutebroze, R. Kopito, and C. Maison for generous gifts of antibodies and plasmids. We thank Drs. N. Benaroudj and A.-M. Lennon for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from Ligue Nationale contre le Cancer and by the Association pour la Recherche contre le Cancer Grant ARC 5599.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.
§ Present address: Dept of Cell Biology, Harvard Medical School, Boston, MA 02115-5730.
¶ These authors contributed equally to this work.
** To whom correspondence should be addressed. Tel.: 33142346372; Fax: 33142346377; E-mail: monique.arpin@curie.fr.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M210639200
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ABBREVIATIONS |
---|
The abbreviations used are: Sch, schwannomin; ERM, ezrin-radixin-moesin; GFP, green fluorescent protein; GST, glutathione S-transferase; VSV, vesicular stomatitis virus; Ab, antibody; pAb, polyclonal Ab; mAb, monoclonal Ab; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation assay buffer; MES, 4-morpholinoethanesulfonic acid.
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