Fbs2 Is a New Member of the E3 Ubiquitin Ligase Family That Recognizes Sugar Chains
2003; Elsevier BV; Volume: 278; Issue: 44 Linguagem: Inglês
10.1074/jbc.m304157200
ISSN1083-351X
AutoresYasuko Yoshida, Fuminori Tokunaga, Tomoki Chiba, Kazuhiro Iwaï, Keiji Tanaka, Tadashi Tai,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoF-box proteins are substrate recognition components of Skp1-Cullin1-F-box protein-Roc1 (SCF) E3 ubiquitin-protein ligases. We reported previously that Fbs1 (F-box protein that recognizes sugar chains; equivalent to Fbx2 or NFB42) binds specifically to proteins attached with high mannose oligosaccharides and subsequently contributes to elimination of N-glycoproteins in cytosol (Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and Tai, T. (2002) Nature 418, 438–442). Here we report the identification of another F-box protein that recognizes N-glycan, Fbs2 (called Fbx6b or FBG2 previously). Although the expression of Fbs1 was restricted to the adult brain and testis, the Fbs2 transcript was widely expressed. The Fbs2 protein forms an SCFFbs2 ubiquitinligase complex that targets sugar chains in N-glycoproteins for ubiquitylation. Only glycoproteins bound to concanavalin A lectin and not to wheat germ agglutinin or Ricinus communis agglutinin interacted with Fbs2 in various tissues and cell lines. Pull-down analysis using various oligosaccharides revealed that Man3–9GlcNAc2 glycans were required for efficient Fbs2 binding, whereas modifications of mannose residues by other sugars or deletion of inner GlcNAc reduced Fbs2 binding. Fbs2 interacted with N-glycans of T-cell receptor α-subunit (TCRα), a typical substrate of the endoplasmic reticulum-associated degradation (ERAD) pathway, and the forced expression of mutant Fbs2ΔF, which lacks the F-box domain essential for forming the SCF complex, and decrease of endogenous Fbs2 by small interfering RNA led to inhibition of TCRα degradation in cells. Thus, Fbs2 is a novel member of F-box protein family that recognizes N-glycans and plays a role in ERAD. F-box proteins are substrate recognition components of Skp1-Cullin1-F-box protein-Roc1 (SCF) E3 ubiquitin-protein ligases. We reported previously that Fbs1 (F-box protein that recognizes sugar chains; equivalent to Fbx2 or NFB42) binds specifically to proteins attached with high mannose oligosaccharides and subsequently contributes to elimination of N-glycoproteins in cytosol (Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and Tai, T. (2002) Nature 418, 438–442). Here we report the identification of another F-box protein that recognizes N-glycan, Fbs2 (called Fbx6b or FBG2 previously). Although the expression of Fbs1 was restricted to the adult brain and testis, the Fbs2 transcript was widely expressed. The Fbs2 protein forms an SCFFbs2 ubiquitinligase complex that targets sugar chains in N-glycoproteins for ubiquitylation. Only glycoproteins bound to concanavalin A lectin and not to wheat germ agglutinin or Ricinus communis agglutinin interacted with Fbs2 in various tissues and cell lines. Pull-down analysis using various oligosaccharides revealed that Man3–9GlcNAc2 glycans were required for efficient Fbs2 binding, whereas modifications of mannose residues by other sugars or deletion of inner GlcNAc reduced Fbs2 binding. Fbs2 interacted with N-glycans of T-cell receptor α-subunit (TCRα), a typical substrate of the endoplasmic reticulum-associated degradation (ERAD) pathway, and the forced expression of mutant Fbs2ΔF, which lacks the F-box domain essential for forming the SCF complex, and decrease of endogenous Fbs2 by small interfering RNA led to inhibition of TCRα degradation in cells. Thus, Fbs2 is a novel member of F-box protein family that recognizes N-glycans and plays a role in ERAD. Selective protein degradation by the ubiquitin-proteasome pathway serves as a powerful regulatory mechanism in a wide variety of cellular processes. Ubiquitin conjugation requires the sequential activities of three enzymes or protein complexes called the ubiquitin-activating enzyme (E1), 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; chitobiose, di-N-acetylchitobiose; ConA, concanavalin A; WGA, wheat germ agglutinin; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GST, glutathione S-transferase; GTF, GlcNAc-terminated fetuin; HA, hemagglutinin A; RCA, Ricinus communis; SCF, Skp1-Cullin1-F-box protein-Roc1; siRNA, small interfering RNA; TBS, Tris-buffered saline; TCRα; T-cell receptor α-subunit; UGGT, UDP-glucose:glycoprotein glucosyltransferase.1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; chitobiose, di-N-acetylchitobiose; ConA, concanavalin A; WGA, wheat germ agglutinin; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GST, glutathione S-transferase; GTF, GlcNAc-terminated fetuin; HA, hemagglutinin A; RCA, Ricinus communis; SCF, Skp1-Cullin1-F-box protein-Roc1; siRNA, small interfering RNA; TBS, Tris-buffered saline; TCRα; T-cell receptor α-subunit; UGGT, UDP-glucose:glycoprotein glucosyltransferase. the ubiquitin-conjugating enzyme (E2), and the ubiquitin-protein ligase (E3) (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6792) Google Scholar). In the ubiquitin pathway, E3 plays an important role in the selection of target proteins for degradation, because each distinct E3 usually binds a protein substrate with a degree of selectivity for ubiquitylation. E3s are believed to exist as molecules with a large diversity, presumably in more than hundreds of species, which are classified into many subfamilies. One of the best characterized E3 families is the Skp1-Cullin1-F-box protein-Roc1 (SCF) complex (2Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar). The SCF is composed of a Cullin1/Cdc53, Skp1, Roc1/Rbx1/Hrt1, and one member of a large family of proteins called F-box proteins. F-box proteins typically have a bipartite structure with an N-terminal F box motif consisting of ∼40 amino acid residues and a C-terminal region that interacts with the substrate and, thereby, the function of the F-box protein is to trap target proteins (3Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 4Kipreos E.T. Pagano M. Genome. Biol. 2000; 1: 3002.1-3002.7Crossref Google Scholar). However, it remains elusive how E3s accurately recognize target proteins. Accumulating evidence suggests that phosphorylation of target proteins is a prerequisite for their recognition by SCF complexes (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6792) Google Scholar, 2Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar, 4Kipreos E.T. Pagano M. Genome. Biol. 2000; 1: 3002.1-3002.7Crossref Google Scholar). In addition, it has been shown that proline hydroxylation of the transcription factor hypoxia-induced factor 1α (HIF1α) serves as a signal for ubiquitylation by the SCF-like Cullin2-based VBC ubiquitin-ligase (5Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3828) Google Scholar, 6Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4367) Google Scholar). On the other hand, we have reported recently that Fbx2 forms an SCFFbx2 ubiquitin ligase complex that targets sugar chains in N-linked glycoproteins for ubiquitylation (7Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Crossref PubMed Scopus (297) Google Scholar). Thus, Fbx2 is a novel example of F-box proteins that have evolved to recognize protein modifications other than phosphorylation and hydroxylation. N-glycosylation acts as a targeting signal to eliminate intracellular glycoproteins by Fbx2-dependent ubiquitylation and subsequent proteasomal degradation.N-glycosylation of the proteins occurs when newly synthesized proteins enter the endoplasmic reticulum (ER) through the translocation channel “translocon.” N-glycans play an important role in glycoprotein transport and sorting (8Fiedler K. Simons K. Cell. 1995; 81: 309-312Abstract Full Text PDF PubMed Scopus (275) Google Scholar), in particular at the initial step of secretion that occurs in the ER compartment (9Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1061) Google Scholar, 10Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1955) Google Scholar). N-linked glycoproteins are subjected to “quality control” in which aberrant proteins are distinguished from properly folded proteins and retained in the ER (10Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1955) Google Scholar). The quality control system includes the calnexin-calreticulin cycle, a unique chaperone system that recognizes Glc1Man9–6GlcNAc2 and assists refolding of misfolded or unfolded proteins. When the improperly folded or incompletely assembled proteins fail to restore their functional states, they are degraded by the ER-associated degradation (ERAD) system, which involves a retrograde transfer of proteins from the ER to the cytosol followed by degradation by the proteasome (11Plemper R.K. Wolf D.H. Trends Biochem. Sci. 1999; 24: 266-270Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 12Brodsky J.L. McCracken A.A. Semin. Cell Dev. Biol. 1999; 10: 507-513Crossref PubMed Scopus (298) Google Scholar, 13Tsai B. Ye Y. Rapoport T.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 246-255Crossref PubMed Scopus (548) Google Scholar). Precisely how they are selected for ERAD remains unclear, but what is clear is that the trimming of N-glycans plays a key role in the selection process. Recent studies have demonstrated that Man8GlcNAc2 structures serve as part of the signal needed for ERAD and that a lectin for Man8GlcNAc2 in ER accelerates the turnover rate of the misfolded glycoprotein (14Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar, 15Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar, 16Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). It has been reported that many E3s are involved in the ERAD pathway, such as ER-embedded Hrd1 (17Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Crossref PubMed Scopus (378) Google Scholar) and Doa10 (18Swanson R. Locher M. Hochstrasser M. Genes Dev. 2001; 15: 2660-2674Crossref PubMed Scopus (367) Google Scholar), which have overlapping functions in yeast, and gp78 (19Fang D. Haraguchi Y. Jinno A. Soda Y. Shimizu N. Hoshino H. Biochem. Biophys. Res. Commun. 1999; 261: 357-363Crossref PubMed Scopus (22) Google Scholar), CHIP (20Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (699) Google Scholar) and Parkin (21Imai Y. Soda M. Inoue H. Hattori N. Mizuno Y. Takahashi R. Cell. 2001; 105: 891-902Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar), which ubiquitylate ER membrane proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and the Pael receptor in mammals. In addition, we have recently identified a novel member of the ERAD-linked E3 family, SCFFbx2, which participates in ERAD for selective elimination of glycoproteins (7Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Crossref PubMed Scopus (297) Google Scholar). Misfolding or misassembly might be the general feature of all substrates; however, Fbx2 is expressed mainly in neuronal cells in the adult brain (22Erhardt J.A. Hynicka W. DiBenedetto A. Shen N. Stone N. Paulson H. Pittman R.N. J. Biol. Chem. 1998; 273: 35222-35227Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Winston et al. (3Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar) and Ilyin et al. (23Ilyin G.P. Serandour A.L. Pigeon C. Rialland M. Glaise D. Guguen-Guillouzo C. Gene. 2002; 296: 11-20Crossref PubMed Scopus (32) Google Scholar) reported previously that several F-box proteins, including Fbx2, contain a conserved motif F-box-associated (FBA) domain or G-domain (sharing similarity with bacterial protein ApaG) in their C termini.The present study is an extension of the above mentioned research work and was designed to determine whether these F-box proteins also recognize N-glycans. Our results showed that Fbx6b/FBG2 bound several glycoproteins, but other F-box proteins failed to bind any of the glycoproteins tested so far. In considering the results of these functional studies, we renamed Fbx2/FBG1 and Fbx6b/FBG2 Fbs1 (F-box protein that recognizes sugar chains 1) and Fbs2, respectively. We found that Fbs2 is widely distributed in a variety of mouse tissues, differing from the restricted expression of Fbs1. Furthermore, a dominant negative Fbs2 mutant suppressed degradation of a typical ERAD substrate, the T cell receptor α subunit (TCRα). Taken together, we concluded that Fbs2 is a new member of the E3-Fbs subfamily for ubiquitylation of N-linked glycoproteins and plays a role in the ERAD pathway.EXPERIMENTAL PROCEDURESMaterials—Ribonuclease B, fetuin, asialofetuin type II, and thyroglobulin were purchased from Sigma. β-galactosidase (Streptococcus 6646K) and β-N-acetylhexosaminidase (Jack Bean) were purchased from Seikagaku-Kogyo (Tokyo, Japan), and N-glycosidase F was from Roche Applied Science. Affi-Gel 10 and 15 were from Bio-Rad and were used according to the instructions provided by the manufacturer. Di-N-acetylchitobiose (chitobiose) was purchased from Seikagaku-Kogyo, Man8GlcNAc2 was from Glyko (Upper Heyford, UK), Man5GlcNAc1 was from IsoSep AB, GlcNAc3Man3GlcNAc2 (asialo-, agalacto-, tri-antennary complex) and GlcNAc2Man3GlcNAc2(Fuc1) (asialo-, agalacto-, corefucosylated bi-antennary complex) were from Ludger (Oxford, UK), and Man3GlcNAc2, Man5GlcNAc2, and Man5GlcNAc2-Asn were purchased from Sigma. Man9GlcNAc2 was a kind gift from Y. Ito. ConA, RCA, and WGA lectin-agaroses were purchased from Seikagaku-Kogyo.Isolation of cDNAs Coding for F-box Proteins and Plasmid Construction—The sequences of mouse Fbs2/Fbx6b/FBG2 (accession number AF176526), Fbx17/FBG4 (accession number AF176532), and FBG3 (accession number XM_204068) cDNAs were obtained from the GenBank™ data base. The cDNAs for mouse Fbs2, Fbx17, and FBG3 were amplified by PCR with Taq polymerase (Sigma) from mouse kidney cDNA. The PCR primers were as follows: 5′-TCT CTG GGA TCC CCA TGG TCC ACA TCA AGG AG-3′ and 5′-GAG CCT GAG CGG CCG CTA ACG CCT TAG CCT TTG CCA-3′ for Fbs2; 5′-CTG ACC GGA TCC TCA TGG GAG CGC GGC CCT CG-3′ and 5′-ACC TGA AGG CGG CCG CAT CAC ATC ATG GTA GTC C-3′ for Fbx17; and 5′-ACG CCA GGA TCC CAG TAG GCA ACA TCA ACG-3′ and 5′-TGA GGA CTG CGG CCG CTC AGG AGG CAT CAG GGC AG-3′ for FBG3. PCR products were digested with BamHI/NotI, subcloned into pBluescriptII SK+, and sequenced. The cDNAs of wild-type or deletion mutants were amplified by PCR with appropriate primers and ligated into BamHI/NotI-cut pcDNA3-FLAG or pVL1393-His expression vector. The coding residues of the Fbs2 deletion mutant ΔF were from amino acids 47 to 295.Overlay Assay—Cultured cells and mouse tissues were homogenized in 10 volumes of TBS (20 mm Tris-HCl, pH 7.5, and 150 mm NaCl) containing 0.5% Nonidet P-40 and protease inhibitor mixture (complete EDTA-free; Roche Applied Science). After centrifugation of the homogenate at 15,000 × g for 30 min, each supernatant (15-mg proteins) was incubated with 50 μl of various lectin-agaroses under gentle rotation at 4 °C for 2 h. The agaroses were washed with ice-cold TBS containing 0.5% Nonidet P-40, and bound proteins were boiled with SDS sample buffer. Proteins were separated with SDS-PAGE and blotted onto a membrane (Immobilon, Millipore, Bedford, MA). To prepare the [35S]methionine-Fbs1 and Fbs2 probes, we constructed Fbs1-Metx3 and Fbs2-Metx3 plasmids for Fbs1 (ΔN-2) (7Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Crossref PubMed Scopus (297) Google Scholar) and Fbs2 (ΔF), respectively, with three additional methionines at the C termini by subcloning them into pcDNA3-FLAG. The TnT Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used for generating [35S]methionine-Fbs1, Fbs2, and βTrCP1. Membranes were blocked by 5% skim milk in TBS, washed, and then incubated with probes in 1% skim milk at 4 °C for 18 h with gentle shaking. Membranes were washed with TBS containing 0.05% Tween 20, air-dried, and analyzed by autoradiography.Northern Blot Analyses—Tissues from 8-week-old ICR mice were employed for RNA extraction using the guanidine thiocyanate method. 15 μg of total RNA was fractionated on a denaturing formaldehyde-agarose gel (1%) and then transferred onto a nylon membrane (Nytran, Schleicher & Schuell). Northern blots were probed with the full-length Fbs1 or Fbs2 cDNA that had been labeled with [α32P]dCTP. The blots were washed at 65 °C in 0.2× standard saline citrate containing 0.1% SDS and then exposed and analyzed with a Molecular Imager FX (Bio-Rad).In Vitro Ubiquitylation Assays—Recombinant His-Ubc4 and His-Ubc12, His-NEDD8, and GST-ubiquitin were produced in Escherichia coli. Recombinant His-E1 (Uba1), His-APP-BP1/T7-Uba3, and SCFFbs2 (FLAG-Skp1/Cul1-HA/His-Fbs2/T7-Roc1) were produced by baculovirus-infected HiFive or Sf9 insect cells. The SCFFbs2 complex was obtained by simultaneously infecting four baculoviruses. These proteins were affinity-purified by a HiTrap HP column (Amersham Biosciences) as described previously (24Kawakami T. Chiba T. Suzuki T. Iwai K. Yamanaka K. Minato N. Suzuki H. Shimbara N. Hidaka Y. Osaka F. Omata M. Tanaka K. EMBO J. 2001; 20: 4003-4012Crossref PubMed Scopus (271) Google Scholar).The methods for preparation of GlcNAc-terminated fetuin (GTF) and deglycosylated fetuin (DGF) were described previously (7Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Crossref PubMed Scopus (297) Google Scholar). One microgram each of GTF or DGF was incubated in 50 μl of the reaction mixture containing the ATP-regenerating system, 0.5 μg E1, 1 μg of Ubc4 (E2), 2 μg of SCFFbs2, and 6.5 μg of recombinant GST-ubiquitin in the presence or absence of the NEDD8 system (24Kawakami T. Chiba T. Suzuki T. Iwai K. Yamanaka K. Minato N. Suzuki H. Shimbara N. Hidaka Y. Osaka F. Omata M. Tanaka K. EMBO J. 2001; 20: 4003-4012Crossref PubMed Scopus (271) Google Scholar) consisting of NEDD8 (10 μg), APP-BP1/T7-Uba3 (0.5 μg), and Ubc12 (0.5 μg) at 30 °C. After terminating the reaction by the addition of 25 μl of 3× SDS-PAGE sample buffer, the proteins in 8 μl of the boiled supernatants were separated with 4–20% SDS-PAGE, and the high molecular mass ubiquitylated proteins were detected by immunoblotting with an anti-fetuin (Chemicon) or anti-GST (Ab-1; Calbiochem, La Jolla, CA) antibody.Pull-down Assay—Each 2.0 mg of glycoprotein was immobilized to 0.5 ml of Affi-Gel 10 or 15. Each cell extract prepared with TBS containing 0.5% Nonidet P-40 from FLAG-tagged Fbs2 (ΔF) or Fbs1 (ΔN-2)-expressing cells (25 μg) was incubated with 15 μl of various glycoprotein-immobilized beads, and bound proteins were eluted by boiling with SDS sample buffer or incubation with 15 μl of various concentrations of oligosaccharides at room temperature for 10 min. The eluates were separated by spin filtration.RNA Interference Experiment—Twenty-one nucleotide dsRNAs were prepared by Dharmacon (Lafayette, CO). The siRNA sequences targeting Fbs2 mRNA (GenBank™ accession number NM 018438) corresponded to the coding regions 285–304 (GAGGAUAUGUUUGCAUGGC) and 754–773 (ACAGCAGCAUUGUCGUCAG) relative to the first nucleotide of the start codon. A nonspecific control duplex was purchased from Dharmacon. 293T cells were transfected with siRNA and/or plasmid DNA by the use of LipofectAMINE Plus (Invitrogen). Total RNA was isolated with TRIZOL reagent (Invitrogen). Reverse transcription PCR was performed using total RNA of 293T cells as a template. The 5′ and 3′ primers were 5′-CCTCCTGGCGGGACCTCATCG-3′ and 5′-ACCAGCTGGGACTTGAGGCAC-3′ for Fbs2 and 5′-GAGCTGAACGGGAAGCTCAC-3′ and 5′-ACCACCCTGTTGCTGTAGC-3′ for the glyceroaldehyde-3-phosphate dehydrogenase, respectively.Metabolic Labeling and Immunoprecipitation—293T cells were transiently transfected with1 μg of the TCRα-HA expression plasmid and 1 μg of the Fbs2 plasmid or pcDNA3. Twenty-four hours after transfection, 293T cells were starved for 30 min in methionine- and cysteine-free Dulbecco's modified Eagle's medium containing 10% dialyzed fetal calf serum. Cells were then labeled for 30 min with 150 μCi of Pro-Mix l-35S in vitro cell-labeling mix (Amersham Biosciences) per milliliter. For pulse-chase experiments, cells were washed after labeling and chased with complete Dulbecco's modified Eagle's medium containing 10% fetal calf serum for different lengths of time at 37 °C. In experiments with tunicamycin, cells were treated with 5 μm tunicamycin (Wako Pure Chemical Industries, Osaka, Japan) 2 h prior to and throughout the labeling period. In the experiments of Fbs2 knock-down by siRNA, cells that had been co-transfected with 4.2 μg (300 pmol) of siRNA duplex and 1 μg of the TCRα-HA expression plasmid and cultured for 48 h were metabolically labeled with the cell-labeling mix. After labeling or chase, cells were washed with ice-cold phosphate-buffered saline and lysed with TBS buffer containing 0.1% SDS and 1% Nonidet P-40. In co-immunoprecipitation experiments, TBS buffer containing 1% Triton X-100 was used for lysing. After one cycle of freezethaw, cell lysates were cleared by centrifugation, and the supernatants were used for immunoprecipitation. Briefly, the supernatants were precleared with protein A-Sepharose (Amersham Biosciences), mouse monoclonal antibodies, anti-FLAG (M2; Sigma), and anti-HA (HA11, BABCO) were then added, and incubation was performed at 4 °C with rotation. Immune complexes were then incubated with protein A-Sepharose, collected by centrifugation, and washed four times with the lysis buffer. For protein analysis, immune complexes were dissociated by heating in SDS-PAGE sample buffer and loaded onto SDS-PAGE. After drying, gels were exposed, and radioactive band intensity was measured using Molecular Imager FX. All experiments described in this study were approved by the institutional ethics review committee for animal experimentation.RESULTSFbs2 Interacts with High Mannose Oligosaccharide-containing Glycoproteins—We reported recently that Fbs1/Fbx2/NFB42 forms a SCFFbs1 ubiquitin ligase complex that targets N-linked high mannose oligosaccharides in glycoproteins for ubiquitylation (7Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Crossref PubMed Scopus (297) Google Scholar). Fbs1 interacts with substrate glycoproteins through its C-terminal domain, which shows high homology with other F-box proteins (3Winston J.T. Koepp D.M. Zhu C. Elledge S.J. Harper J.W. Curr. Biol. 1999; 9: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 23Ilyin G.P. Serandour A.L. Pigeon C. Rialland M. Glaise D. Guguen-Guillouzo C. Gene. 2002; 296: 11-20Crossref PubMed Scopus (32) Google Scholar). To explore the presence of other F-box proteins that recognize N-glycans, we isolated mouse cDNA clones that encode F-box proteins homologous to Fbs1 and examined the properties of the proteins with regard to interaction with N-linked glycoproteins. The mouse Fbs2/Fbx6b/FBG2, Fbx17/FBG4, and FBG3 cDNA clones obtained by reverse transcription PCR were expressed in 293T cells as FLAG-tagged, full-length F-box proteins, and these cell extracts were incubated with various N-linked glycoproteins. Only Fbs2 bound several glycoproteins, whereas the other F-box proteins failed to bind any of the glycoproteins tested (see Fig. 3, and data not shown).Next, we investigated the interaction of these F-box proteins with oligosaccharides on endogenous proteins by overlay assay. To enrich glycoproteins that were fractionated by their oligosaccharide structures, we prepared proteins bound to lectins from the extracts of several cell lines and adult mouse tissues. These glycoproteins were then separated by SDS-PAGE and detected by each 35S-labeled F-box protein. In glycoproteins bound to ConA, a lectin that binds to high mannose oligosaccharides, many protein bands were detected by Fbs1 and Fbs2 probes (Fig. 1). These glycoproteins did not bind to βTrCP1, which is a F-box protein with WD repeats for substrate recognition (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6792) Google Scholar, 2Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar) (Fig. 1). On the other hand, a few proteins reacted with Fbs1 and Fbs2 in the glycoproteins bound to RCA120, a lectin that binds to terminal Galβ1–4GlcNAc or WGA and is specific for terminal GlcNAc or sialic acids (Fig. 1). The broad 75–80-kDa protein band(s) and the 60-kDa protein in the WGA-bound fraction prepared from brain extract seemed to be identical to that bound to ConA. Because many proteins contain several and/or structurally diversified oligosaccharides, these 75–80-kDa and 60-kDa proteins may be modified by high mannose oligosaccharides and complex type glycans. The whole pattern of the protein bands detected by the Fbs2 probe was almost the same as those detected by Fbs1, but the strength of binding activity with Fbs2 seemed to be somewhat weak compared with Fbs1. Any glycoproteins concentrated with ConA, RCA120, and WGA lectins failed to be detected with Fbx17 and FBG3 probes (data not shown). These results suggest that high mannose oligosaccharides are important for the substrate recognition by Fbs2 as well as Fbs1.Fig. 1Interaction of Fbs2 and Fbs1 with endogenous glycoproteins containing N-linked high mannose type oligosaccharides. Each extract of Neuro2a (N2a), P19, PC12 cells, and tissues from adult mice (brain and testis) was incubated with ConA, RCA, or WGA lectin-agarose. The lectin-bound proteins were separated with 4–20% SDS-PAGE and then transferred to membranes that were used for the overlay assay. 35S-labeled Fbs2/Fbx6b ΔF, Fbs1/Fbx2 ΔN-2, and βTrCP1 ΔF were used for the probes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of Fbs Genes in Mouse Tissues—Fbs1 is reported to be expressed in the adult rat brain but not in non-neural tissues (22Erhardt J.A. Hynicka W. DiBenedetto A. Shen N. Stone N. Paulson H. Pittman R.N. J. Biol. Chem. 1998; 273: 35222-35227Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We compared the expression patterns of Fbs1 and Fbs2 by Northern blot analysis in 12 different mouse tissues. This analysis demonstrated that the Fbs1 transcript of ∼1.5 kb mRNA was expressed in the brain and, to a lesser extent, in the testis (Fig. 2). Western blot analysis using Fbs1 polyclonal antiserums confirmed that Fbs1 protein is present only in the brain and testis among the 12 different tissues examined (data not shown). The transcription of Fbs1 appeared in 1-week-old mice, reaching the highest level in the adult brain. On the other hand, an ∼1.8-kb transcript of Fbs2 was detected in various tissues, mainly in the heart, intestine, kidney, liver, lung, muscle, stomach, and testis as well as other tissues (brain, skin, spleen, and thymus) with low abundance. Fbs2 expression was not observed in the fetus, but became detectable after birth and increased in the adult brain. Thus, although the expression of Fbs1 is restricted to the brain and testis, the Fbs2 transcript is ubiquitously expressed in all tissues examined.Fig. 2Expression of Fbs genes in mouse tissues. Total RNA was extracted from adult mouse tissues or mice brains at different stages of development. A membrane preloaded with 15 μg of total RNA per lane was hybridized with radiolabeled mouse Fbs1 and Fbs2 cDNAs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In Vitro Ubiquitylation Assay of Fbs2—To directly demonstrate the E3 ubiquitin ligase activity in SCFFbs2, we devised a fully reconstituted system for ubiquitylation of GTF in the presence of the NEDD8 system. To this end, we produced all components required for these systems as recombinant or purified proteins (see “Experimental Procedures”). Ubiquitylation of GTF by GST-tagged ubiquitin was detected by immunoblotting using an anti-fetuin or anti-GST antibody. No ubiquitylation activity was detected in the absence of E1, E2, ATP, SCFFbs2, or GTF (Fig. 3A). The amount of ubiquitylation increased in a time-dependent manner (Fig. 3B). In addition, N-glycanase F-treated fetuin (DGF) wa
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