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• W2024492847 abstract "Schwannomin (Sch) is the product of theNF2 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 ΔF118, 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 vivoand 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. Schwannomin (Sch) is the product of theNF2 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 ΔF118, 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 vivoand 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. 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 (1Gusella J.F. Ramesh V. MacCollin M. Jacoby L.B. Biochim. Biophys. Acta. 1999; 1423: 29-36Google Scholar). TheNF2 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), 1The 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 also known as merlin, is highly related to ERM (ezrin, radixin, moesin) proteins (2Bretscher A. Chambers D. Nguyen R. Reczek D. Annu. Rev. Cell Dev. Biol. 2000; 16: 113-143Google Scholar). 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 (3Gautreau A. Louvard D. Arpin M. Curr. Opin. Cell Biol. 2002; 14: 104-109Google Scholar, 4Morrison H. Sherman L.S. Legg J. Banine F. Isacke C. Haipek C.A. Gutmann D.H. Ponta H. Herrlich P. Genes Dev. 2001; 15: 968-980Google Scholar, 5Shaw 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. McClatchey A.I. Dev. Cell. 2001; 1: 63-72Google Scholar). For both Sch and ERM proteins, the FERM domain is responsible for their localization at the cytoplasmic side of the plasma membrane (6Algrain M. Turunen O. Vaheri A. Louvard D. Arpin M. J. Cell Biol. 1993; 120: 129-139Google Scholar, 7Deguen B. Merel P. Goutebroze L. Giovannini M. Reggio H. Arpin M. Thomas G. Hum. Mol. Genet. 1998; 7: 217-226Google Scholar). The FERM domain interacts with membrane proteins and with filamentous actin (3Gautreau A. Louvard D. Arpin M. Curr. Opin. Cell Biol. 2002; 14: 104-109Google Scholar, 8Brault E. Gautreau A. Lamarine M. Callebaut I. Thomas G. Goutebroze L. J. Cell Sci. 2001; 114: 1901-1912Google Scholar). The crystal structures of FERM domains from ERM proteins and Sch have confirmed that they display a similar overall structure (9Pearson M.A. Reczek D. Bretscher A. Karplus P.A. Cell. 2000; 101: 259-270Google Scholar,10Shimizu T. Seto A. Maita N. Hamada K. Tsukita S. Hakoshima T. J. Biol. Chem. 2002; 277: 10332-10336Google Scholar). 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 (11Nguyen R. Reczek D. Bretscher A. J. Biol. Chem. 2001; 276: 7621-7629Google Scholar). 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 (2Bretscher A. Chambers D. Nguyen R. Reczek D. Annu. Rev. Cell Dev. Biol. 2000; 16: 113-143Google Scholar). Oligomers also form between ERM proteins and Sch (12Gronholm M. Sainio M. Zhao F. Heiska L. Vaheri A. Carpen O. J. Cell Sci. 1999; 112: 895-904Google Scholar). 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, SchΔF118. 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. 13Deguen B. Goutebroze L. Giovannini M. Boisson C. van der Neut R. Jaurand M.C. Thomas G. Int. J. Cancer. 1998; 77: 554-560Google Scholar). We recently found that the ΔF mutation impairs the proper folding of Sch FERM domain (8Brault E. Gautreau A. Lamarine M. Callebaut I. Thomas G. Goutebroze L. J. Cell Sci. 2001; 114: 1901-1912Google Scholar). Because the phenylalanine affected by this deletion is conserved in ERM proteins, we introduced the equivalent ΔF102 deletion into ezrin. Strikingly, SchΔF, but not ezrinΔF, formed aggresomes upon transient transfection. We mapped the principal determinant of this behavior with ezrin/Sch chimeras. 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 β-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 γ-tubulin mAb (1:1000; Sigma), 7A3 vimentin mAb (1:100, gift of C. Maison, Institut Curie, Paris), N356 α-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. Ezrin/Sch chimeras were designed according to a multiple alignment of ERM members andNF2 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 EzrΔF and SchΔF constructs in pCB6 (7Deguen B. Merel P. Goutebroze L. Giovannini M. Reggio H. Arpin M. Thomas G. Hum. Mol. Genet. 1998; 7: 217-226Google Scholar, 14Gautreau A. Manent J. Fiévet B. Louvard D. Giovannini M. Arpin M. J. Biol. Chem. 2002; 277: 31279-31282Google Scholar) and contained at their C terminus the VSV G tag. Briefly, chimeras were obtained as described in Table Ieither 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 asXhoI-BamHI fragments at the C terminus of GFP. GST fusion proteins were constructed in a modified pGEX 4T2 (AmershamBiosciences) in which the NotI site had been converted into an XbaI site. Untagged Sch340–595 was inserted as a XhoI-XbaI fragment. Sch535–595and Sch544–595 were excised as aBglII-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 (15Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Google Scholar).Table IConstructions of ezrin/Sch chimerasChimeraN-terminalC-terminalJunction sequencecDNA fusionPlasmidsDigestSESch(1–341)Ezr(326–586)LAREKKXho1 of SchpCB6-SchΔFXho1-Xba1ES1Ezr(1–323)Sch(340–595)QQLAREXho1 of SchpCB6-EzrΔFKpn1-XholES2Ezr(1–434)Sch(444–595)EEASEREcoNI of EzrpCB6-EzrΔFEcoNI-XbalES3Ezr(1–526)Sch(535–595)NERLQEPCR-mediated recombinationpCB6-EzrΔFEcoNI-XbalES4Ezr(1–552)Sch(561–595)NDILIINPCR-mediated recombinationpCB6-EzrΔFEcoNI-XbalES5Ezr(1–535)Sch(544–595)TLSTEIBlp1 of EzrpCB6-EzrΔFBlp1-BlplES3iso2Ezr(1–526)Sch(535–590)NERLQESubcloningpCB6-ES3Stu1-Xbal Open table in a new tab Transfection of the different cell lines was performed by electroporation as described (16Gautreau A. Louvard D. Arpin M. J. Cell Biol. 2000; 150: 193-203Google Scholar). 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). 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 coefficientR 2 > 0.95 were taken into account to calculate the half-life according to the formula t 12 = (ln 50 − ln a)/−b. This procedure gave less than 10% variation between experiments. 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 mmEDTA, 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 (AmershamBiosciences) 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 mmdithiothreitol, 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 1m salicylate for 20 min. Dried gels were exposed to films at −80 °C or to a phospho-screen from 1 day to 1 week. 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. 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. To compare the effects of the ΔF deletion on the tumor suppressor gene product and a related ERM protein, we transiently transfected ezrinΔF and SchΔF 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 SchΔF and ezrinΔF were drastically different even though their level of expression was similar (see below). In the majority of transfected cells, SchΔF accumulated in a juxtanuclear area, whereas ezrinΔF 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 (7Deguen B. Merel P. Goutebroze L. Giovannini M. Reggio H. Arpin M. Thomas G. Hum. Mol. Genet. 1998; 7: 217-226Google Scholar, 17Koga H. Araki N. Takeshima H. Nishi T. Hirota T. Kimura Y. Nakao M. Saya H. Oncogene. 1998; 17: 801-810Google Scholar). The two localizations of SchΔF and ezrinΔF were both clearly different from those of wild type ezrin and Sch, which are found at the plasma membrane (data not shown (18Crepaldi T. Gautreau A. Comoglio P.M. Louvard D. Arpin M. J. Cell Biol. 1997; 138: 423-434Google Scholar, 19Maeda M. Matsui T. Imamura M. Tsukita S. Oncogene. 1999; 18: 4788-4797Google Scholar)). The juxtanuclear accumulation of SchΔF was reminiscent of a recently described structure called the aggresome (20Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Google Scholar). 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 SchΔF fulfilled the criteria of aggresomes as follows. (i) Aggregates of SchΔF accumulated at the centrosome, as revealed by γ-tubulin staining (Fig. 1 c). Furthermore, in some cells containing SchΔF aggregates, γ-tubulin staining of the centrosome was absent (data not shown), suggesting that the accumulation of misfolded material can impede access of γ-tubulin antibodies to the centrosome. (ii) The accumulation of SchΔF at the centrosome was dependent on the integrity of the microtubule cytoskeleton (Fig. 1, d and e). Most SchΔF-transfected cells formed aggresomes in control conditions, but only a few did in the presence of nocodazole, which depolymerizes microtubules. Dispersed aggregates of SchΔF were observed throughout the cytosol of nocodazole-treated cells. (iii) A cage-like structure of vimentin filaments surrounded the SchΔF aggresome (Fig.1 f). 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 (14Gautreau A. Manent J. Fiévet B. Louvard D. Giovannini M. Arpin M. J. Biol. Chem. 2002; 277: 31279-31282Google Scholar). Here we found that the SchΔF aggresome was composed of ubiquitinylated material, since it was stained with Myc antibodies when Myc-tagged ubiquitin was cotransfected with SchΔF (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, SchΔ39–121, formed similar aggresomes (data not shown). Aggresome formation by SchΔF, but not by ezrinΔF, 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 SchΔF aggresomes, we performed transmission electron microscopy. When SchΔF or ezrinΔF transiently transfected LLC-PK1 cells were examined, amorphous electron dense structures were found only in SchΔF-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. Because aggresome formation is thought to be a general response to misfolded proteins, we reasoned that perhaps ezrin was not misfolded by the ΔF 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 (9Pearson M.A. Reczek D. Bretscher A. Karplus P.A. Cell. 2000; 101: 259-270Google Scholar, 10Shimizu T. Seto A. Maita N. Hamada K. Tsukita S. Hakoshima T. J. Biol. Chem. 2002; 277: 10332-10336Google Scholar). 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 (8Brault E. Gautreau A. Lamarine M. Callebaut I. Thomas G. Goutebroze L. J. Cell Sci. 2001; 114: 1901-1912Google Scholar, 21Franck Z. Gary R. Bretscher A. J. Cell Sci. 1993; 105: 219-231Google Scholar). For this purpose, we transfected LLC-PK1 cells with cDNAs encoding wild type or ΔF 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, ezrinΔF and SchΔF were no longer resistant to chymotrypsin. Thus, the ΔF mutation impairs the proper folding of the FERM domain in both ezrin and Sch. To explain the different behaviors of ezrinΔF and SchΔF, we assumed there was a sequence determining aggresome formation. To identify this determinant, we constructed chimeras between ezrinΔF and SchΔF. We first swapped the misfolded FERM domains of ezrinΔF and SchΔF, 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 SchΔF 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 (11Nguyen R. Reczek D. Bretscher A. J. Biol. Chem. 2001; 276: 7621-7629Google Scholar, 22Gonzalez-Agosti C. Wiederhold T. Herndon M.E. Gusella J. Ramesh V. J. Biol. Chem. 1999; 274: 34438-34442Google Scholar, 23Gutmann D.H. Haipek C.A. Lu K.H. J. Neurosci. Res. 1999; 58: 706-716Google Scholar). 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. 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. 4 C). We noticed a ladder of conjugates of SchΔF, ES1, ES2, and to a smaller extent of ES3, ES3iso2, and SE. This ladder is likely due to ubiquitination, since SchΔF is efficiently degraded by the ubiquitin-proteasome pathway (14Gautreau A. Manent J. Fiévet B. Louvard D. Giovannini M. Arpin M. J. Biol. Chem. 2002; 277: 31279-31282Google Scholar). These conjugates were at the limit of detection with ezrinΔF, 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). The centrosome is associated with active proteasomes, suggesting that this location is a privileged site for degradation of ubiquitinated proteins (24Fabunmi R.P. Wigley W.C. Thomas P.J. DeMartino G.N. J. Biol. Chem. 2000; 275: 409-413Google Scholar, 25Wigley W.C. Fabunmi R.P. Lee M.G. Marino C.R. Muallem S. DeMartino G.N. Thomas P.J. J. Cell Biol. 1999; 145: 481-490Google Scholar). 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 SchΔF and ezrinΔF, in which both proteins are soluble due to moderate overexpression, we recently found that SchΔF is degraded 3 times faster than ezrinΔF (1.7 versus 5.4 h of half-life (14Gautreau A. Manent J. Fiévet B. Louvard D. Giovannini M. Arpin M. J. Biol. Chem. 2002; 277: 31279-31282Google Scholar)). 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 SchΔF than ezrinΔF. 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. 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. 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-Sch544–595were 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 Co" @default.
• W2024492847 created "2016-06-24" @default.
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• W2024492847 date "2003-02-01" @default.
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• W2024492847 title "Isolation and Characterization of an Aggresome Determinant in theNF2 Tumor Suppressor" @default.
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