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• W2099403408 abstract "Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90β-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90β-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90β gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90α gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90β-specific function. Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90β-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90β-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90β gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90α gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90β-specific function. Living cells continuously cope with protein folding and assembly. Because cell fate (e.g. proliferation, differentiation and even death) is determined and implemented by a plethora of varied proteins, the folding and assembly of these proteins are crucial to the life of a cell. However, many proteins do not achieve correct folding or proper assembly autonomously in the cell; instead, they require the assistance of molecular chaperones (1Bukau B. Deuerling E. Pfund C. Craig E.A. Cell. 2000; 101: 119-122Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 2Deuerling E. Bukau B. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 261-277Crossref PubMed Scopus (99) Google Scholar, 3Frydman J. Annu. Rev. Biochem. 2001; 70: 603-647Crossref PubMed Scopus (917) Google Scholar, 4Hartl F.U. Hayer-Hartl M. Science. 2002; 295: 1852-1858Crossref PubMed Scopus (2749) Google Scholar, 5Mosser D.D. Morimoto R.I. Oncogene. 2004; 23: 2907-2918Crossref PubMed Scopus (434) Google Scholar, 6Young J.C. Agashe V.R. Siegers K. Hartl F.U. Nat. Rev. Mol. Cell Biol. 2004; 5: 781-791Crossref PubMed Scopus (925) Google Scholar). The 90-kDa heat shock protein (Hsp90) 4The abbreviations used are: Hsp, heat shock protein; BCR, B cell antigen receptor; CHX, cycloheximide; EDEM, endoplasmic reticulum degradation-enhancing α-mannosidase-like protein; ER, endoplasmic reticulum; GA, geldanamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; HSF, heat shock factor; Hsp90, 90-kDa heat shock protein; Ig, immunoglobulin; μ, the IgM heavy chain; μm, a membrane-bound form of the μ chain; μs, a secreted form of the μ chains; RT, reverse transcription; XBP-1, X-box binding protein-1; YBAP1, a Y-box protein-associated acidic protein; YB-1, Y-box-binding protein 1; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; BLNK, B cell linker protein.4The abbreviations used are: Hsp, heat shock protein; BCR, B cell antigen receptor; CHX, cycloheximide; EDEM, endoplasmic reticulum degradation-enhancing α-mannosidase-like protein; ER, endoplasmic reticulum; GA, geldanamycin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; HSF, heat shock factor; Hsp90, 90-kDa heat shock protein; Ig, immunoglobulin; μ, the IgM heavy chain; μm, a membrane-bound form of the μ chain; μs, a secreted form of the μ chains; RT, reverse transcription; XBP-1, X-box binding protein-1; YBAP1, a Y-box protein-associated acidic protein; YB-1, Y-box-binding protein 1; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; BLNK, B cell linker protein. is one such molecular chaperone and is also one of the most abundant cytosolic proteins (7Pearl L.H. Prodromou C. Curr. Opin. Struct. Biol. 2000; 10: 46-51Crossref PubMed Scopus (276) Google Scholar, 8Picard D. Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar, 9Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 10Terasawa K. Minami M. Minami Y. J. Biochem. (Tokyo). 2005; 137: 443-447Crossref PubMed Scopus (128) Google Scholar, 11Wegele H. Müller L. Buchner J. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 1-44Crossref PubMed Scopus (508) Google Scholar, 12Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (702) Google Scholar). The Hsp90 family is highly conserved during evolution and is essential in yeast (13Borkovich K.A. Farrelly F.W. Finkelstein D.B. Taulien J. Lindquist S. Mol. Cell. Biol. 1989; 9: 3919-3930Crossref PubMed Scopus (523) Google Scholar), Caenorhabditis elegans (14Birnby D.A. Link E.M. Vowels J.J. Tian H. Colacurcio P.L. Thomas J.H. Genetics. 2000; 155: 85-104Crossref PubMed Google Scholar) and Drosophila melanogaster (15Cutforth T. Rubin G.M. Cell. 1994; 77: 1027-1036Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 16van der Straten A. Rommel C. Dickson B. Hafen E. EMBO J. 1997; 16: 1961-1969Crossref PubMed Scopus (119) Google Scholar, 17Yue L. Karr T.L. Nathan D.F. Swift H. Srinivasan S. Lindquist S. Genetics. 1999; 151: 1065-1079Crossref PubMed Google Scholar). Concordant with these facts, many Hsp90 client proteins (i.e. Hsp90 substrates) are key molecules in signal transduction, including protein kinases and transcription factors (7Pearl L.H. Prodromou C. Curr. Opin. Struct. Biol. 2000; 10: 46-51Crossref PubMed Scopus (276) Google Scholar, 8Picard D. Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar, 9Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 10Terasawa K. Minami M. Minami Y. J. Biochem. (Tokyo). 2005; 137: 443-447Crossref PubMed Scopus (128) Google Scholar, 11Wegele H. Müller L. Buchner J. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 1-44Crossref PubMed Scopus (508) Google Scholar, 12Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (702) Google Scholar). Although the proteome-wide function of Hsp90 as a capacitor for morphological evolution that buffers cryptic genetic variation is well known (18Queitsch C. Sangster T.A. Lindquist S. Nature. 2002; 417: 618-624Crossref PubMed Scopus (1030) Google Scholar, 19Rutherford S.L. Lindquist S. Nature. 1998; 396: 336-342Crossref PubMed Scopus (1689) Google Scholar, 20Sollars V. Lu X. Xiao L. Wang X. Garfinkel M.D. Ruden D.M. Nat. Genet. 2003; 33: 70-74Crossref PubMed Scopus (326) Google Scholar), elucidating the complete compendium of the Hsp90-interacting proteins remains a challenge (21Millson S.H. Truman A.W. King V. Prodromou C. Pearl L.H. Piper P.W. Eukaryot. Cell. 2005; 4: 849-860Crossref PubMed Scopus (128) Google Scholar, 22Zhao R. Davey M. Hsu Y.-C. Kaplanek P. Tong A. Parsons A.B. Krogan N. Cagney G. Mai D. Greenblatt J. Boone C. Emili A. Houry W.A. Cell. 2005; 120: 715-727Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar). Hsp90 is not required for the de novo folding of most proteins in yeast under normal conditions (23Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (311) Google Scholar), and reduction to 1/20 of its normal level can be tolerated (24Xu Y. Singer M.A. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 109-114Crossref PubMed Scopus (163) Google Scholar). By contrast, mouse mutant embryos lacking Hsp90β die because of defects in the development of the placental labyrinth (25Voss A.K. Thomas T. Gruss P. Development. 2000; 127: 1-11Crossref PubMed Google Scholar). Higher eukaryotes contain two Hsp90 isoforms that closely resemble each other (Hsp90α and Hsp90β). Although each forms a homodimer (26Minami Y. Kawasaki H. Miyata Y. Suzuki K. Yahara I. J. Biol. Chem. 1991; 266: 10099-10103Abstract Full Text PDF PubMed Google Scholar), no obvious difference in function between the two isoforms has been reported so far. The developmental deficiency observed in the Hsp90β mutant embryos may be attributable to a lack of specific functions that are fulfilled by only Hsp90β (25Voss A.K. Thomas T. Gruss P. Development. 2000; 127: 1-11Crossref PubMed Google Scholar); alternatively, the reduced expression level of Hsp90 may impinge on a specific aspect of the placental development. These possibilities raise two questions. First, do Hsp90α and Hsp90β have individual functions that cannot be accomplished by one another? Second, to what extent can the expression of Hsp90 be decreased without losing viability in higher eukaryotic cells? Anti-tumor agents such as geldanamycin (GA) compete with ATP for binding to the specific pocket of Hsp90 and ultimately block its ATPase-driven chaperone cycle (27Kamal A. Thao L. Sensintaffar J. Zhang L. Boehm M.F. Fritz L.C. Burrow F.J. Nature. 2003; 425: 407-410Crossref PubMed Scopus (1178) Google Scholar, 28Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. Cell. 1997; 90: 65-75Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar, 29Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1222) Google Scholar); therefore, they are efficient inhibitors of Hsp90 that can be used to dissect its functions (7Pearl L.H. Prodromou C. Curr. Opin. Struct. Biol. 2000; 10: 46-51Crossref PubMed Scopus (276) Google Scholar, 8Picard D. Cell. Mol. Life Sci. 2002; 59: 1640-1648Crossref PubMed Scopus (645) Google Scholar, 9Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar, 10Terasawa K. Minami M. Minami Y. J. Biochem. (Tokyo). 2005; 137: 443-447Crossref PubMed Scopus (128) Google Scholar, 11Wegele H. Müller L. Buchner J. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 1-44Crossref PubMed Scopus (508) Google Scholar, 12Young J.C. Moarefi I. Hartl F.U. J. Cell Biol. 2001; 154: 267-273Crossref PubMed Scopus (702) Google Scholar). Because GA binds to both Hsp90α and Hsp90β (30Lele Z. Hartson S.D. Martin C.C. Whitesell L. Matts R.L. Krone P.H. Dev. Biol. 1999; 210: 56-70Crossref PubMed Scopus (65) Google Scholar), this Hsp90-inhibitor is not suitable for discriminating between the functions of the two Hsp90 isoforms. In addition, our attempts to use small interfering RNA-directed gene knockdown of the specific isoforms of Hsp90 have not led to the successful depletion of either Hsp90α or Hsp90β. 5F. Shinozaki, M. Minami, K. Terasawa, and Y. Minami, unpublished data.5F. Shinozaki, M. Minami, K. Terasawa, and Y. Minami, unpublished data. Thus, to address the aforementioned issues, here we have generated cells from the chicken B lymphocyte line DT40 that are deficient in Hsp90β. We show that multiple defects are concurrently elicited in the mutant cells; in particular, components involved in B cell antigen receptor (BCR) signaling are impaired. Most strikingly, the expression level of the immunoglobulin (Ig) M heavy chain is profoundly reduced. These defects can be efficiently corrected not only by reintroducing the Hsp90β gene but also by introducing the Hsp90α gene; therefore, the defects observed in these cells are derived mainly from a reduction in the Hsp90 content and not from a loss of Hsp90β-specific function. Plasmid Constructs—Chicken Hsp90β genomic DNA was obtained from DT40-derived genomic DNA by PCR amplification based on previous studies (31Meng X. Baulieu E.-E. Catelli M.-G. Biochem. Biophys. Res. Commun. 1995; 206: 644-651Crossref PubMed Scopus (8) Google Scholar, 32Meng X. Jérôme V. Devin J. Baulieu E.-E. Catelli M.-G. Biochem. Biophys. Res. Commun. 1993; 190: 630-636Crossref PubMed Scopus (39) Google Scholar) and confirmed by DNA sequencing. Two targeting constructs, Hsp90β-neo and Hsp90β-hisD, were generated by replacing the DNA segment encompassing exons 4 to 6 with drug-resistant cassettes for neomycin (neo) and histidinol (hisD), respectively. Chicken Hsp90α and Hsp90β cDNAs were synthesized by reverse transcription (RT)-PCR using mRNA isolated from DT40 cells and were cloned either into the pApuro2 vector (made by introducing a multicloning site into the pApuro vector (33Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar)) for their (re)introduction into Hsp90β-deficient cells or into the Escherichia coli expression vector pET23a (Novagen) for the production of recombinant proteins. A cDNA fragment encoding a region of Hsp90β (residues 571–725) was introduced into a pGEX6P2 plasmid (Amersham Biosciences) to produce a glutathione S-transferase (GST) fusion protein. The cytoplasmic domain of chicken Igα was synthesized by RT-PCR using a previously described primer combination (34Pike K.A. Iacampo S. Fiedmann J.E. Ratcliffe M.J.H. J. Immunol. 2004; 172: 2210-2218Crossref PubMed Scopus (25) Google Scholar), and the cDNA fragment was introduced into a pGEX6P2 plasmid. Cell Culture, Transfection, and Screening—DT40 cells were cultured in RPMI1640 medium (Sigma) containing 10% (v/v) fetal bovine serum, 1% (v/v) chicken serum, 50 μm β-mercaptoethanol, and antibiotics (penicillin and streptomycin) at 39.5 °C under 5% CO2. Cells were electroporated at 25 microfarads and 550 V with a Gene Pulser II apparatus (Bio-Rad); ∼107 cells and 50 μg of linearized DNA were used for each transfection (35Buerstedde J. Takeda S. Cell. 1991; 67: 179-188Abstract Full Text PDF PubMed Scopus (476) Google Scholar). Stable transformants were selected with 2 mg/ml G-418, 1 mg/ml histidinol, and 0.5 μg/ml puromycin (all from Sigma). Genomic DNAs were isolated using a DNeasy Tissue Kit (Qiagen). The DNAs (5 μg) were digested with PvuII, separated in 0.8% (w/v) agarose gels, and transferred onto a Hybond N+ nylon membrane (Amersham Biosciences). Membranes were hybridized with an Hsp90β genomic DNA fragment, as shown in Fig. 1A, which was labeled using a Gene Images random prime labeling and detection system (Amersham Biosciences) as a probe. After incubating cells with tetrazolium salt WST-8 (Cell Counting Kit-8: Dojindo), the optical density was measured at 450 nm to assess cell viability. Northern Blot Analysis and RT-PCR—Total RNA was isolated by using an RNeasy Mini kit (Qiagen). Membranes (5 μg of RNA) were hybridized with the following fluorescently labeled cDNA fragments as probes: the EcoRI-PstI fragment of Hsp90α (0.63 kb); the BglII fragment of Hsp90β (0.8 kb); a PCR fragment of IgM (X01613) synthesized with the two primers 5′-CGGAACAACTGAACGGCAAC-3′ and 5′-GCCCTATCCACCGACTTCTG-3′ (0.3 kilobases (kb)); the PstI-HindIII fragment of Igα (0.16 kb); the BamHI-EcoNI fragment of Igβ (0.27 kb). Total RNAs (1μg) were used for the first-strand synthesis with oligo(dT) and Superscript II RNaseH– reverse transcriptase (Invitrogen). For semiquantitative RT-PCR (membrane-bound (μm) and secreted (μs) forms of the IgM heavy chain (μ) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), total cDNA was subjected to a 3-fold dilution. PCR amplification was performed with KOD-Plus DNA polymerase (Toyobo) using the following oligonucleotide primers: μm, 5′-ATCTCCTCCATTGGCCTCTGGAG-3′ and 5′-TTTCACCTTGATGAGGGTGACGG-3′; μs, 5′-CGGAACAACTGAACGGCAAC-3′ and 5′-CAATCGAGCGGCCGCTTAACG-3′; X-box binding protein-1 (XBP-1), 5′-CAGCCTCTCCCTGGAGAACCAGG-3′ and 5′-CAGAATCCATGTGGAGGTTGTCAGGAATGGTGAC-3′; endoplasmic reticulum (ER) degradation-enhancing α-mannosidase-like protein (EDEM), 5′-TCCTGGACTGCAGGTGTTGATAGGAG-3′ and 5′-ATTCAGATCAAGCCCACCATCCGATC-3′; GAPDH, 5′-TGGAGAGATGGCAGAGGTGCTG-3′ and 5′-GGATGCCATGTGGACCATCAAG-3′. Purification of Bacterially Expressed Recombinant Proteins—For GST fusion proteins (Hsp90β and Igα) or His-tagged proteins (Hsp90α and Hsp90β), the corresponding vectors were introduced into E. coli strain BL21(DE3) pLysS (Stratagene). Cultures were induced with 0.4 mm isopropyl-1-β-d-galactopyranoside after reaching an absorbance at 600 nm of 0.6 and were further incubated for 30 min or 1 h, respectively, at 37 °C. Cells were harvested and washed with phosphate-buffered saline (137 mm NaCl, 2.68 mm KCl, 8.1 mm Na2HPO4, 1.47 mm KH2PO4, pH 7.4) for GST fusion proteins or with TBS (50 mm Tris-HCl, pH 7.5, 300 mm NaCl) for His-tagged proteins. Cells were resuspended in the same buffer solutions containing 1 mm phenylmethylsulfonyl fluoride and disrupted by sonication. Recombinant proteins were purified using glutathione-Sepharose 4 (Amersham Biosciences) or nickel nitrilotriacetic acid-agarose (Qiagen). Antisera—Anti-heat shock factor 1–3 (HSF1-HSF3), anti-Hsp70, anti-Hsp90α (36Kawazoe Y. Tanabe M. Sasai N. Nagata K. Nakai A. Eur. J. Biochem. 1999; 265: 688-697Crossref PubMed Scopus (27) Google Scholar), anti-Syk (33Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar), and anti-BLNK (37Ishiai M. Kurosaki M. Pappu R. Okawa K. Ronko I. Fu C. Shibata M. Iwamatsu A. Chan A.C. Kurosaki T. Immunity. 1999; 10: 117-125Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) antisera have been described. Anti-mouse Hsp90 antisera were provided by Y. Miyata (Kyoto University). Antibodies specific for Y-box-binding protein 1 (YB-1) (directed against the N-terminal peptide of chicken YB-1) and a Y-box protein-associated acidic protein (YBAP1) (directed against recombinant proteins of chicken YBAP1) will be described in more detail elsewhere. 6K. Matsumoto, manuscript in preparation. Anti-chicken Hsp90β and anti-Igα antibodies were raised in rabbits using the GST fusion proteins. Anti-Hsp90α and anti-Hsp90β antisera (10 μl) were mixed with the His-tagged Hsp90β or Hsp90α proteins (100 μg), respectively, and incubated for 2 h at room temperature to eliminate mutual cross-reactivities between them; for anti-Hsp90β antisera, incubation was further continued overnight after the re-addition of Hsp90α protein (100 μg). Anti-chicken IgM monoclonal antibody M4 (Southern Biotechnology) and goat anti-chicken IgM antibody (Betyl Laboratories) were used for BCR stimulation and for immunoprecipitation/immunoblotting, respectively. The following antibodies were purchased: anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology); anti-Raf-1 (C-12 and E-10) and horseradish peroxidase-conjugated anti-goat IgG antibodies (Santa Cruz Biotechnology); anti-ERK, anti-phosphorylated ERK, anti-MEK, anti-phosphorylated MEK, anti-Akt and phospho-(Ser/Thr) Akt substrate antibodies (Cell Signaling Technology); anti-actin antibody (Sigma); horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (Amersham Biosciences). Stimulation of DT40 Cells—Cells were washed twice with phosphate-buffered saline and resuspended in serum-free RPMI1640 medium. After a 10-min incubation at 39.5 °C, cells were activated by the addition of either 4 μg/ml of anti-IgM antibodies (M4) or 20% (v/v) fetal bovine serum. Western Blot Analysis and Immunoprecipitation—Where indicated, cells were treated with 2 μm GA (Sigma), 50 μg/ml of cycloheximide (CHX) (Sigma), or 50 nm calyculin A (Cell Signaling Technology) before cell lysis. Cells were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mm NaCl, 20 mm Tris-HCl, pH 7.5, 1 mm EDTA) containing 50 mm NaF, 10 μm sodium molybdate, and 0.2 mm sodium vanadate (33Takata M. Sabe H. Hata A. Inazu T. Homma Y. Nukada T. Yamamura H. Kurosaki T. EMBO J. 1994; 13: 1341-1349Crossref PubMed Scopus (584) Google Scholar) supplemented with protease inhibitor mixture (Complete mini: Roche Applied Science). Cell lysates were cleared by centrifugation at 16,000 × g for 15 min and subjected to SDS-PAGE followed by transfer onto Immobilon-P membranes (Millipore). Blots were incubated with antibodies and subjected to chemiluminescence detection with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). The intensity of immunodetected protein bands was quantified using NIH image software after scanning of the blot with a densitometer. For immunoprecipitation, cell lysates were incubated with antibodies for 10 min on ice, either protein A- or protein G-Sepharose (Amersham Biosciences) was then added, and incubation was continued for a further 3 h. The immunoprecipitates were washed four times with Nonidet P-40 lysis buffer. To prepare μs proteins for immunoblotting, cells were cultured for 12 h in RPMI1640 with 10% fetal bovine serum, and the secreted μs proteins were immunoprecipitated and immunoblotted with anti-IgM antibodies (Betyl Laboratories). Phosphatase Treatment—Cell lysates (30 μl) were incubated with or without 60 units of λ protein phosphatase (New England Biolabs) for 30 min at 30 °C. Flow Cytometric Analysis—Cells were washed with a buffer solution (phosphate-buffered saline, 0.3% (w/v) bovine serum albumin, 0.02% (w/v) sodium azide) and incubated with the same solution containing goat anti-chicken IgM antibody for 30 min on ice. After washing twice, cells were incubated with fluorescein isothiocyanate-labeled rabbit anti-goat antibody (Sigma) for 30 min on ice. The stained cells were then resuspended in 1 ml of the above solution, and their fluorescent intensity was analyzed by a Beckman Epics XL flow cytometer (Beckman Coulter). In Vitro Kinase Assay—For the in vitro Raf-1 kinase assay, immunoprecipitates obtained with anti-Raf-1 antibody (E-10) were washed four times with Nonidet P-40 lysis buffer and then with ice-cold reaction buffer (20 mm Tris-HCl, pH 7.5, and 10 mm MgCl2). The kinase reaction was performed in 30 μl of the reaction buffer containing 250 μm ATP and 1 μg of recombinant MEK-1 substrate (Santa Cruz Biotechnology) at 30 °C for 30 min. Targeted Gene Disruption of Hsp90β in Chicken DT40 Cells—Two different targeting constructs were designed to disrupt exons encoding the ATP-binding domain of Hsp90β that is essential to its chaperone function (38Obermann W.M.J. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (479) Google Scholar, 39Panaretou B. Prodromou C. Roe S.M. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. EMBO J. 1998; 17: 4829-4836Crossref PubMed Scopus (610) Google Scholar) (Fig. 1A). We generated Hsp90β-deficient cells by the successive transfection of these two constructs (Fig. 1B). Southern blot analysis indicated that the constructs were properly targeted (Fig. 1B), and this result was corroborated by Northern blotting (Fig. 1C). Expression of Hsp90β protein was not detected in Hsp90β-deficient cells by Western blot analysis (Fig. 1D), albeit a faint band was seen probably due to cross-reactivity of anti-Hsp90β antibody with Hsp90α proteins. Notably, expression of Hsp90α protein did not increase in the Hsp90β-deficient cells (Fig. 1D); consistent with this, the level of Hsp90α mRNA was comparable with that in wild-type cells (Fig. 1C). The protein levels of Hsp70 and Hsp40 were also unchanged in the Hsp90β-deficient cells (data not shown). Taken together, we conclude that Hsp90β can be successfully depleted by gene disruption and that expression of Hsp90α does not increase to compensate for the lack of Hsp90β protein. Although the Hsp90β-deficient cells did not lose viability and their saturation densities did not differ from those of wild-type cells (data not shown), they grew more slowly; by contrast, heterozygous mutant cells seemed to grow normally (Fig. 1E). Thus, the gene knock-out of Hsp90β apparently affected an aspect of cell proliferation. Initially, we suspected that the heat shock response might be compromised in Hsp90β-deficient cells, because Hsp90 has been suggested to have a role in modulating HSF1 (40Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1509) Google Scholar). Western blotting showed, however, that the expression levels of the HSFs (avian cells express HSF1-HSF3) were unchanged (Fig. 2A), although HSF2 is not activated by heat stress (36Kawazoe Y. Tanabe M. Sasai N. Nagata K. Nakai A. Eur. J. Biochem. 1999; 265: 688-697Crossref PubMed Scopus (27) Google Scholar). Within 15 min of sustained exposure to 43 °C, an electrophoretic mobility shift of HSF3 was observed in both the wild-type and Hsp90β-deficient cells (Fig. 2B); by contrast, HSF1 did not show any detectable mobility shift (data not shown). This upshift pattern of HSF3 in the two cell clones was eliminated by phosphatase treatment (Fig. 2C), which reconfirms a previous study showing that the characteristic mobility shift of mammalian HSF1 results from stress-induced phosphorylation (indicating the transient activation) (41Satyal S.H. Chen D. Fox S.G. Kramer J.M. Morimoto R.I. Genes Dev. 1998; 12: 1962-1974Crossref PubMed Scopus (183) Google Scholar); HSF3 is dominant among the three HSFs in regulation of the heat shock response in DT40 cells (42Tanabe M. Kawazoe Y. Takeda S. Morimoto R.I. Nagata K. Nakai A. EMBO J. 1998; 17: 1750-1758Crossref PubMed Scopus (85) Google Scholar). Most importantly, an equal accumulation of Hsp70 after the upshift of HSF3 bands was detected in the two cell clones (Fig. 2B). We, therefore, conclude that the heat shock response is intact in Hsp90β-deficient cells. Expression of IgM Is Markedly Reduced in Hsp90β-deficient Cells—Signaling through the BCR is essential for B cell function (43Hendriks R.W. Middendorp S. Trends Immunol. 2004; 25: 249-256Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 44Kurosaki T. Nat. Rev. Immunol. 2002; 2: 354-363Crossref PubMed Scopus (170) Google Scholar). BCR cross-linking by anti-IgM antibody induced an immediate increase in tyrosine phosphorylation on several proteins (indicating successful stimulation), and ERK was phosphorylated concurrently in wild-type cells (Fig. 3A). Whereas tyrosine phosphorylation was reduced to some extent, ERK phosphorylation appeared to be almost unaltered in Hsp90β+/– cells (Fig. 3A). By contrast, the amounts of tyrosine-phosphorylated proteins and phosphorylated ERK were markedly decreased in Hsp90β-deficient cells (Fig. 3A). These observations strongly suggest that loss of Hsp90β impairs a factor or factors involved in early phases of BCR signaling. The BCR consists of the antigen binding membrane Ig (IgM in DT40 cells) (45Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inaba K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (136) Google Scholar) and the signaling heterodimers of Igα and Igβ (43Hendriks R.W. Middendorp S. Trends Immunol. 2004; 25: 249-256Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 44Kurosaki T. Nat. Rev. Immunol. 2002; 2: 354-363Crossref PubMed Scopus (170) Google Scholar). Western blotting revealed that the amount of the μ chain (i.e. the total amount of both μm and μs in cell lysates) was greatly reduced in Hsp90β-deficient cells, whereas Hsp90β+/– cells showed somewhat decreased but yet significant amounts of μ protein (Fig. 3B). These observations were corroborated by flow cytometric analysis of the expression of μ chains at the cell surface (i.e. μm) in Hsp90β+/– and Hsp90β–/– cells (Fig. 3C). Unexpectedly, μs protein was also drastically decreased in Hsp90β-deficient cells (Fig. 3B). Despite these findings, we observed only a slight, if any, reduction in μ transcripts in these mutant cells, as measured by Northern blotting (Fig. 3D) or by semiquantitative RT-PCR (Fig. 3E). These data have a strong resemblance to those of a recent study in which a profound depression in synthesis of IgM protein was detected in mouse B cells deficient in the transcription factor XBP-1, notwithstanding μ transcript levels similar to those of wild-type cells (46Tirosh B. Iwakoshi N.N. Glimcher L.H. Ploegh H.L. J. Exp. Med. 2005; 202: 505-516Crossref PubMed Scopus (62) Google Scholar). It has been shown that mRNA of XBP-1 must be spliced to promote secretion of IgM, and this splicing in turn depends on the production of μm (46Tirosh B. Iwakoshi N.N. Glimcher L.H. Ploegh H.L. J. Exp. Med. 2005; 202: 505-516Crossref PubMed Scopus (62) Google Scholar, 47Iwakoshi N.N. Lee A.-H. Vallabhajosyula P. Otipoby K.L. Rajewsky K. Glimcher L.H. Nat. Immunol. 2003; 4: 321-329Crossref PubMed Scopus (694) Google Scholar). We found, however, that splicing of XBP-1 mRNA in Hsp90β-deficient cells was normal (Fig. 3F). We, therefore, considered that the function of XBP-1 might be compromised. Because EDEM is an XBP-1-dependent target gene (48Lee A.-H. Iwakoshi N.N." @default.
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• W2099403408 title "Depletion of Hsp90β Induces Multiple Defects in B Cell Receptor Signaling" @default.
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