Isolation and Characterization of an Aggresome Determinant in the NF2 Tumor Suppressor*

Alexis GautreauDagger §, Bruno T. FievetDagger , Estelle Brault||, Claude AntonyDagger , Anne HoudusseDagger , Daniel LouvardDagger , and Monique ArpinDagger **

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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

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, SchDelta 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 Delta 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 Delta F102 deletion into ezrin. Strikingly, SchDelta F, but not ezrinDelta 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

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 beta -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 gamma -tubulin mAb (1:1000; Sigma), 7A3 vimentin mAb (1:100, gift of C. Maison, Institut Curie, Paris), N356 alpha -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.

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 EzrDelta F and SchDelta 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).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Constructions of ezrin/Sch chimeras

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-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.

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 -80 °C or to a phospho-screen from 1 day to 1 week.

Immunofluorescence-- Cells on glass coverslips were rinsed twice with PBS, fixed with 100% methanol for 5 min at -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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SchDelta F, but Not EzrinDelta F, Forms Aggresomes upon Transient Transfection-- To compare the effects of the Delta F deletion on the tumor suppressor gene product and a related ERM protein, we transiently transfected ezrinDelta F and SchDelta 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 SchDelta F and ezrinDelta F were drastically different even though their level of expression was similar (see below). In the majority of transfected cells, SchDelta F accumulated in a juxtanuclear area, whereas ezrinDelta 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 SchDelta F and ezrinDelta 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)).


View larger version (75K):
[in this window]
[in a new window]
 
Fig. 1.   SchDelta F, but not ezrinDelta F, forms aggresomes upon transient transfection in LLC-PK1 cells. Cells were transfected with ezrinDelta F (a) or SchDelta 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 ezrinDelta F (a) but a juxtanuclear aggregation of SchDelta F (b). SchDelta F aggregates are clustered around the centrosome (c, gamma -tubulin mAb (green); VSV G pAb (red)). SchDelta F transfectants were left untreated (d) or treated with nocodazole (e) to depolymerize microtubules. Nocodazole treatment abrogates juxtanuclear accumulation of SchDelta F aggregates (d and e, alpha -tubulin (green); mAb, VSV G pAb (red)). Vimentin filaments are reorganized around clustered SchDelta F aggregates (f, vimentin mAb (green), VSV G pAb (red)). SchDelta F aggresomes are composed of ubiquitinylated material (cotransfection with Myc-tagged ubiquitin; g, Myc mAb (green); h, VSV G pAb (red); i, merge). SchDelta F aggresomes recruit proteasomes (j, proteasome pAb green; k, VSV G mAb (red); l, merge). Bars, 5 µm.

The juxtanuclear accumulation of SchDelta 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 SchDelta F fulfilled the criteria of aggresomes as follows. (i) Aggregates of SchDelta F accumulated at the centrosome, as revealed by gamma -tubulin staining (Fig. 1c). Furthermore, in some cells containing SchDelta F aggregates, gamma -tubulin staining of the centrosome was absent (data not shown), suggesting that the accumulation of misfolded material can impede access of gamma -tubulin antibodies to the centrosome. (ii) The accumulation of SchDelta F at the centrosome was dependent on the integrity of the microtubule cytoskeleton (Fig. 1, d and e). Most SchDelta F-transfected cells formed aggresomes in control conditions, but only a few did in the presence of nocodazole, which depolymerizes microtubules. Dispersed aggregates of SchDelta F were observed throughout the cytosol of nocodazole-treated cells. (iii) A cage-like structure of vimentin filaments surrounded the SchDelta F aggresome (Fig. 1f).

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 SchDelta F aggresome was composed of ubiquitinylated material, since it was stained with Myc antibodies when Myc-tagged ubiquitin was cotransfected with SchDelta 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, SchDelta 39-121, formed similar aggresomes (data not shown). Aggresome formation by SchDelta F, but not by ezrinDelta 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.

To examine the ultrastructure of SchDelta F aggresomes, we performed transmission electron microscopy. When SchDelta F or ezrinDelta F transiently transfected LLC-PK1 cells were examined, amorphous electron dense structures were found only in SchDelta 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.


View larger version (136K):
[in this window]
[in a new window]
 
Fig. 2.   Ultrastructure of SchDelta F aggresomes. LLC-PK1 cells were transiently transfected with SchDelta 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.

The Delta 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 Delta 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 Delta 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, ezrinDelta F and SchDelta F were no longer resistant to chymotrypsin. Thus, the Delta F mutation impairs the proper folding of the FERM domain in both ezrin and Sch.


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 3.   The Delta 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.

Delineation of a Sch Determinant for Aggresome Formation Using Ezrin/Sch Chimeras-- To explain the different behaviors of ezrinDelta F and SchDelta F, we assumed there was a sequence determining aggresome formation. To identify this determinant, we constructed chimeras between ezrinDelta F and SchDelta F. We first swapped the misfolded FERM domains of ezrinDelta F and SchDelta 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 SchDelta 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.


View larger version (34K):
[in this window]
[in a new window]
 
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.

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 SchDelta F, ES1, ES2, and to a smaller extent of ES3, ES3iso2, and SE. This ladder is likely due to ubiquitination, since SchDelta F is efficiently degraded by the ubiquitin-proteasome pathway (14). These conjugates were at the limit of detection with ezrinDelta 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).

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 SchDelta F and ezrinDelta F, in which both proteins are soluble due to moderate overexpression, we recently found that SchDelta F is degraded 3 times faster than ezrinDelta 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 SchDelta F than ezrinDelta 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.


View larger version (41K):
[in this window]
[in a new window]
 
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.

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.


View larger version (62K):
[in this window]
[in a new window]
 
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.

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.


View larger version (98K):
[in this window]
[in a new window]
 
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

Here we showed that a mutant form of the NF2 tumor suppressor, SchDelta F, accumulates in juxtanuclear aggregates called aggresomes. The Delta 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 SchDelta F aggregates. In addition, these aggresomes were composed of ubiquitinated SchDelta F and recruited proteasomes that have been shown to associate dynamically with the centrosome (25).

Strikingly, in contrast to SchDelta F, the related protein ezrinDelta F never formed aggresomes upon overexpression by transient transfection even though the folding of ezrin FERM domain was similarly impaired. Thus, ezrinDelta F seems to be an exception to the rule that all misfolded proteins form aggresomes (20). However, upon inhibition of proteasomes in stable transfectants, ezrinDelta F formed very small aggresomes, whereas in the same conditions, SchDelta 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 ezrinDelta F never formed aggresomes upon transient transfection to map determinants of aggresome formation in SchDelta F using ezrin/Sch chimeras. We delineated Sch535-595 as a major determinant. This sequence induced a chimera composed at 90% of ezrinDelta 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.

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 alpha -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.

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 SchDelta F is compared with ezrinDelta 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.

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.

    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.

    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

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Gusella, J. F., Ramesh, V., MacCollin, M., and Jacoby, L. B. (1999) Biochim. Biophys. Acta 1423, 29-36
2. Bretscher, A., Chambers, D., Nguyen, R., and Reczek, D. (2000) Annu. Rev. Cell Dev. Biol. 16, 113-143[CrossRef][Medline] [Order article via Infotrieve]
3. Gautreau, A., Louvard, D., and Arpin, M. (2002) Curr. Opin. Cell Biol. 14, 104-109[CrossRef][Medline] [Order article via Infotrieve]
4. Morrison, H., Sherman, L. S., Legg, J., Banine, F., Isacke, C., Haipek, C. A., Gutmann, D. H., Ponta, H., and Herrlich, P. (2001) Genes Dev. 15, 968-980[Abstract/Free Full Text]
5. Shaw, R. J., Paez, J. G., Curto, M., Yaktine, A., Pruitt, W. M., Saotome, I., O'Bryan, J. P., Gupta, V., Ratner, N., Der, C. J., Jacks, T., and McClatchey, A. I. (2001) Dev. Cell 1, 63-72[Medline] [Order article via Infotrieve]
6. Algrain, M., Turunen, O., Vaheri, A., Louvard, D., and Arpin, M. (1993) J. Cell Biol. 120, 129-139[Abstract]
7. Deguen, B., Merel, P., Goutebroze, L., Giovannini, M., Reggio, H., Arpin, M., and Thomas, G. (1998) Hum. Mol. Genet. 7, 217-226[Abstract/Free Full Text]
8. Brault, E., Gautreau, A., Lamarine, M., Callebaut, I., Thomas, G., and Goutebroze, L. (2001) J. Cell Sci. 114, 1901-1912[Abstract/Free Full Text]
9. Pearson, M. A., Reczek, D., Bretscher, A., and Karplus, P. A. (2000) Cell 101, 259-270[Medline] [Order article via Infotrieve]
10. Shimizu, T., Seto, A., Maita, N., Hamada, K., Tsukita, S., and Hakoshima, T. (2002) J. Biol. Chem. 277, 10332-10336[Abstract/Free Full Text]
11. Nguyen, R., Reczek, D., and Bretscher, A. (2001) J. Biol. Chem. 276, 7621-7629[Abstract/Free Full Text]
12. Gronholm, M., Sainio, M., Zhao, F., Heiska, L., Vaheri, A., and Carpen, O. (1999) J. Cell Sci. 112, 895-904[Abstract/Free Full Text]
13. Deguen, B., Goutebroze, L., Giovannini, M., Boisson, C., van der Neut, R., Jaurand, M. C., and Thomas, G. (1998) Int. J. Cancer 77, 554-560[CrossRef][Medline] [Order article via Infotrieve]
14. Gautreau, A., Manent, J., Fiévet, B., Louvard, D., Giovannini, M., and Arpin, M. (2002) J. Biol. Chem. 277, 31279-31282[Abstract/Free Full Text]
15. Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121-127[Medline] [Order article via Infotrieve]
16. Gautreau, A., Louvard, D., and Arpin, M. (2000) J. Cell Biol. 150, 193-203[Abstract/Free Full Text]
17. Koga, H., Araki, N., Takeshima, H., Nishi, T., Hirota, T., Kimura, Y., Nakao, M., and Saya, H. (1998) Oncogene 17, 801-810[CrossRef][Medline] [Order article via Infotrieve]
18. Crepaldi, T., Gautreau, A., Comoglio, P. M., Louvard, D., and Arpin, M. (1997) J. Cell Biol. 138, 423-434[Abstract/Free Full Text]
19. Maeda, M., Matsui, T., Imamura, M., and Tsukita, S. (1999) Oncogene 18, 4788-4797[CrossRef][Medline] [Order article via Infotrieve]
20. Johnston, J. A., Ward, C. L., and Kopito, R. R. (1998) J. Cell Biol. 143, 1883-1898[Abstract/Free Full Text]
21. Franck, Z., Gary, R., and Bretscher, A. (1993) J. Cell Sci. 105, 219-231[Abstract/Free Full Text]
22. Gonzalez-Agosti, C., Wiederhold, T., Herndon, M. E., Gusella, J., and Ramesh, V. (1999) J. Biol. Chem. 274, 34438-34442[Abstract/Free Full Text]
23. Gutmann, D. H., Haipek, C. A., and Lu, K. H. (1999) J. Neurosci. Res 58, 706-716[CrossRef][Medline] [Order article via Infotrieve]
24. Fabunmi, R. P., Wigley, W. C., Thomas, P. J., and DeMartino, G. N. (2000) J. Biol. Chem. 275, 409-413[Abstract/Free Full Text]
25. Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R., Muallem, S., DeMartino, G. N., and Thomas, P. J. (1999) J. Cell Biol. 145, 481-490[Abstract/Free Full Text]
26. Lupas, A., Van Dyke, M., and Stock, J. (1991) Science 252, 1162-1164[Medline] [Order article via Infotrieve]
27. Kopito, R. R. (2000) Trends Cell Biol. 10, 524-530[CrossRef][Medline] [Order article via Infotrieve]
28. Anton, L. C., Schubert, U., Bacik, I., Princiotta, M. F., Wearsch, P. A., Gibbs, J., Day, P. M., Realini, C., Rechsteiner, M. C., Bennink, J. R., and Yewdell, J. W. (1999) J. Cell Biol. 146, 113-124[Abstract/Free Full Text]
29. Garcia-Mata, R., Bebok, Z., Sorscher, E. J., and Sztul, E. S. (1999) J. Cell Biol. 146, 1239-1254[Abstract/Free Full Text]
30. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Nature 416, 507-511[CrossRef][Medline] [Order article via Infotrieve]
31. Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.