Ubiquitination-mediated Regulation of Biosynthesis of the Adhesion Receptor SHPS-1 in Response to Endoplasmic Reticulum Stress
2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês
10.1074/jbc.m311463200
ISSN1083-351X
AutoresReiko Murai-Takebe, Tetsuya Noguchi, Takeshi Ogura, Toshiyuki Mikami, Kazunori Yanagi, Kenjiro Inagaki, Hiroshi Ohnishi, Takashi Matozaki, Masato Kasuga,
Tópico(s)Autophagy in Disease and Therapy
ResumoMisfolding of proteins during endoplasmic reticulum (ER) stress results in the formation of cytotoxic aggregates. The ER-associated degradation pathway counteracts such aggregation through the elimination of misfolded proteins by the ubiquitin-proteasome system. We now show that SHP substrate-1 (SHPS-1), a transmembrane glycoprotein that regulates cytoskeletal reorganization and cell-cell communication, is a physiological substrate for the Skp1-Cullin1-NFB42-Rbx1 (SCFNFB42) E3 ubiquitin ligase, a proposed mediator of ER-associated degradation. SCFNFB42 mediated the polyubiquitination of immature SHPS-1 and its degradation by the proteasome. Ectopic expression of NFB42 both suppressed the formation of aggresome-like structures and the phosphorylation of the translational regulator eIF2α induced by overproduction of SHPS-1 as well as increased the amount of mature SHPS-1 at the cell surface. An NFB42 mutant lacking the F box domain had no such effects. Our results suggest that SCFNFB42 regulates SHPS-1 biosynthesis in response to ER stress. Misfolding of proteins during endoplasmic reticulum (ER) stress results in the formation of cytotoxic aggregates. The ER-associated degradation pathway counteracts such aggregation through the elimination of misfolded proteins by the ubiquitin-proteasome system. We now show that SHP substrate-1 (SHPS-1), a transmembrane glycoprotein that regulates cytoskeletal reorganization and cell-cell communication, is a physiological substrate for the Skp1-Cullin1-NFB42-Rbx1 (SCFNFB42) E3 ubiquitin ligase, a proposed mediator of ER-associated degradation. SCFNFB42 mediated the polyubiquitination of immature SHPS-1 and its degradation by the proteasome. Ectopic expression of NFB42 both suppressed the formation of aggresome-like structures and the phosphorylation of the translational regulator eIF2α induced by overproduction of SHPS-1 as well as increased the amount of mature SHPS-1 at the cell surface. An NFB42 mutant lacking the F box domain had no such effects. Our results suggest that SCFNFB42 regulates SHPS-1 biosynthesis in response to ER stress. Adhesion molecules on the surface of cells play pivotal roles in many physiological processes, including cell growth, differentiation, and migration. Dysregulation of specific adhesion molecules has thus been shown to result in pathological conditions such as inflammation, neurodegeneration, and cancer metastasis. We previously identified SHP substrate-1 (SHPS-1) 1The abbreviations used are: SHPS-1, SHP substrate-1; NFB42, neural F box protein of 42 kDa; Skp1, S phase kinase-associated protein 1; ER, endoplasmic reticulum; GFP, green fluorescent protein; HA, hemagglutinin epitope; FBS, fetal bovine serum; CHO, Chinese hamster ovary; mAb, monoclonal antibody; eIF2α, eukaryotic initiation factor 2α; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ERAD, ER-associated degradation; UPR, unfolded protein response; E3, ubiquitin-protein isopeptide ligase; SCF, Skp1-Cullin1-F box. (1Fujioka Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasuga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (394) Google Scholar, 2Yamao T. Matozaki T. Amano K. Matsuda Y. Takahashi N. Ochi F. Fujioka Y. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 231: 61-67Crossref PubMed Scopus (56) Google Scholar), an adhesion receptor of the immunoglobulin superfamily also known as SIRPα1 (3Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (565) Google Scholar), BIT (4Sano S. Ohnishi H. Omori A. Hasegawa J. Kubota M. FEBS Lett. 1997; 411: 327-334Crossref PubMed Scopus (76) Google Scholar), MFR (5Saginario C. Sterling H. Beckers C. Kobayashi R. Solimena M. Ullu E. Vignery A. Mol. Cell. Biol. 1998; 18: 6213-6223Crossref PubMed Scopus (125) Google Scholar), and p84 neural adhesion molecule (6Comu S. Weng W. Olinsky S. Ishwad P. Mi Z. Hempel J. Watkins S. Lagenaur C.F. Narayanan V. J. Neurosci. 1997; 17: 8702-8710Crossref PubMed Google Scholar). The extracellular region of SHPS-1 comprises three immunoglobulin-like domains, the most amino-terminal of which associates with the ligand CD47 (7Jiang P. Lagenaur C.F. Narayanan V. J. Biol. Chem. 1999; 274: 559-562Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Through its interaction with CD47, SHPS-1 contributes to the phagocytosis of red blood cells by macrophages (8Oldenborg P.A. Zheleznyak A. Fang Y.F. Lagenaur C.F. Gresham H.D. Lindberg F.P. Science. 2000; 288: 2051-2054Crossref PubMed Scopus (1371) Google Scholar), to macrophage multinucleation (9Han X. Sterling H. Chen Y. Saginario C. Brown E.J. Frazier W.A. Lindberg F.P. Vignery A. J. Biol. Chem. 2000; 275: 37984-37992Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), to T cell activation (10Seiffert M. Brossart P. Cant C. Cella M. Colonna M. Brugger W. Kanz L. Ullrich A. Bühring H.J. Blood. 2001; 97: 2741-2749Crossref PubMed Scopus (158) Google Scholar), and to neutrophil transmigration (11Liu Y. Bühring H.J. Zen K. Burst S.L. Schnell F.J. Williams I.R. Parkos C.A. J. Biol. Chem. 2002; 277: 10028-10036Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The cytoplasmic region of SHPS-1 acts as a scaffold for the assembly of multiprotein complexes (1Fujioka Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasuga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (394) Google Scholar, 3Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (565) Google Scholar, 12Timms J.F. Swanson K.D. Marie-Cardine A. Raab M. Rudd C.E. Schraven B. Neel B.G. Curr. Biol. 1999; 9: 927-930Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). By recruiting the protein-tyrosine phosphatase SHP-2 to the cell membrane, SHPS-1 negatively or positively regulates intracellular signaling initiated either by tyrosine kinase-coupled receptors for growth factors or by cell adhesion to extracellular matrix proteins (13Cant C.A. Ullrich A. Cell. Mol. Life Sci. 2001; 58: 117-124Crossref PubMed Scopus (52) Google Scholar, 14Oshima K. Ruhul Amin A.R. Suzuki A. Hamaguchi M. Matsuda S. FEBS Lett. 2002; 519: 1-7Crossref PubMed Scopus (94) Google Scholar). Our studies with mice lacking most of the cytoplasmic region of SHPS-1 have also revealed that this protein is an upstream signaling component important for SHP-2-mediated regulation of cell migration and cytoskeletal reorganization (15Inagaki K. Yamao T. Noguchi T. Matozaki T. Fukunaga K. Takada T. Hosooka T. Akira S. Kasuga M. EMBO J. 2000; 19: 6721-6731Crossref PubMed Scopus (126) Google Scholar, 16Yamao T. Noguchi T. Takeuchi O. Nishiyama U. Morita H. Hagiwara T. Akahori H. Kato T. Inagaki K. Okazawa H. Hayashi Y. Matozaki T. Takeda K. Akira S. Kasuga M. J. Biol. Chem. 2002; 277: 39833-39839Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Expression of SHPS-1 in the central nervous system is regulated both during embryonic development and postnatally, and this regulation has been implicated in synapse formation or maintenance (6Comu S. Weng W. Olinsky S. Ishwad P. Mi Z. Hempel J. Watkins S. Lagenaur C.F. Narayanan V. J. Neurosci. 1997; 17: 8702-8710Crossref PubMed Google Scholar, 17Mi Z.P. Jiang P. Weng W.L. Lindberg F.P. Narayanan V. Lagenaur C.F. J. Comp. Neurol. 2000; 416: 335-344Crossref PubMed Scopus (61) Google Scholar). SHPS-1 is also down-regulated during cell transformation and thus might play a role in carcinogenesis (14Oshima K. Ruhul Amin A.R. Suzuki A. Hamaguchi M. Matsuda S. FEBS Lett. 2002; 519: 1-7Crossref PubMed Scopus (94) Google Scholar). Little is known, however, of the mechanisms by which the cellular abundance of SHPS-1 is determined, although several oncogene products down-regulate SHPS-1 mRNA (18Machida K. Matsuda S. Yamaki K. Senga T. Thant A.A. Kurata H. Miyazaki K. Hayashi K. Okuda T. Kitamura T. Hayakawa T. Hamaguchi M. Oncogene. 2000; 19: 1710-1718Crossref PubMed Scopus (24) Google Scholar). In the present study, we sought to provide insight into SHPS-1 function by identifying proteins that regulate SHPS-1 or transduce signals emanating from its cytoplasmic region. We now demonstrate a physical and functional interaction between SHPS-1 and both neural F box protein of 42 kDa (NFB42) and S phase kinase-associated protein 1 (Skp1) in mouse brain and melanocytes. NFB42 (recently renamed Fbx2) was first identified as a gene product that is highly enriched in the nervous system (19Erhardt 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 (55) Google Scholar). F box proteins contain an F box domain and mediate substrate recognition by Skp1-Cullin1-F box protein-Rbx1 (SCF)-type E3 ubiquitin-protein ligase complexes (20Craig K.L. Tyers M. Prog. Biophys. Mol. Biol. 1999; 72: 299-328Crossref PubMed Scopus (246) Google Scholar). Ectopic expression of NFB42 was shown to inhibit the proliferation of neuroblastoma cells (19Erhardt 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 (55) Google Scholar). Skp1 is an invariant component of SCF-type E3 enzymes and is thought to control the cell cycle (21Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (1007) Google Scholar). NFB42 and Skp1 are thus implicated in the control of neuronal proliferation by protein ubiquitination, although the biological roles of these proteins remain to be established. Skp1 also participates in various signal transduction pathways, including that responsible for glucose-dependent reassembly of V-ATPase complexes (22Seol J.H. Shevchenko A. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 4: 384-391Crossref Scopus (216) Google Scholar). We now show that NFB42 and Skp1, together with Cullin1, constitute an SCF-type E3 ubiquitin ligase (SCFNFB42) and that this complex catalyzes the polyubiquitination of immature forms of SHPS-1. Forced expression of NFB42 resulted in elimination of misfolded SHPS-1 molecules from the endoplasmic reticulum (ER) by the ubiquitin-proteasome proteolytic pathway, an effect that was associated with marked inhibition of the formation of cellular aggregates containing SHPS-1. Furthermore, this elimination of misfolded SHPS-1 from the ER led to substantial up-regulation of SHPS-1 expression at the cell surface. We also provide evidence that SCFNFB42 functions to maintain ER homeostasis during cellular stress by supporting the biosynthesis of SHPS-1. Expression Vectors—The cDNAs encoding the SHPS-1 mutants SHPS-1–4F, in which all four tyrosine residues in the cytoplasmic region (Tyr408, Tyr432, Tyr449, and Tyr473) are replaced by phenylalanine, and SHPS-1-JM, in which two juxtamembrane lysine residues (Lys401 and Lys402) are replaced by asparagine and glutamine, respectively, were generated by site-directed mutagenesis with the full-length mouse SHPS-1 cDNA (2Yamao T. Matozaki T. Amano K. Matsuda Y. Takahashi N. Ochi F. Fujioka Y. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 231: 61-67Crossref PubMed Scopus (56) Google Scholar) as a template and a Transformer Site-directed Mutagenesis kit (Clontech). The wild-type and mutant SHPS-1 cDNAs were then inserted separately into the EcoRI and NotI site of pTracer-CMV (Invitrogen). To generate a cDNA encoding SHPS-1 fused to green fluorescent protein (GFP), we performed the polymerase chain reaction (PCR) with a full-length human SHPS-1 cDNA (2Yamao T. Matozaki T. Amano K. Matsuda Y. Takahashi N. Ochi F. Fujioka Y. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 231: 61-67Crossref PubMed Scopus (56) Google Scholar) as a template, a T7 primer, and the antisense primer 5′-AAAGTCGACTTCTTCTACAAGG-3′. The PCR product was digested with EcoRI and SalI and then inserted into pEGFP N2 (Clontech). The pSRα vector encoding wild-type rat SHPS-1 was described previously (1Fujioka Y. Matozaki T. Noguchi T. Iwamatsu A. Yamao T. Takahashi N. Tsuda M. Takada T. Kasuga M. Mol. Cell. Biol. 1996; 16: 6887-6899Crossref PubMed Scopus (394) Google Scholar). The pcDNA3 vectors encoding hemagglutinin epitope (HA)-tagged versions of either wild-type rat NFB42 (amino acids 2–296) or the deletion mutants NFBΔP (residues 53–296), NFBΔPΔF (residues 95–296), or NFBPF (residues 2–94) (19Erhardt 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 (55) Google Scholar) were kindly provided by R. Pittman (University of Pennsylvania). To generate cDNAs encoding Myc epitope-tagged versions of wild-type NFB42 and NFBΔPΔF, we amplified the corresponding coding regions by PCR with the full-length NFB42 cDNA as template. The PCR products were digested with EcoRI and SalI and then inserted in-frame into pCl-neo (Invitrogen) that had been modified to add the coding sequence for the Myc epitope to the 5′ end of the inserted cDNA. The pcDNA3 vectors encoding Myc epitope-tagged forms of mouse Skp1 and mouse Cullin1 were kindly provided by S. Hatakeyama (Kyushu University, Japan). Cells, Transfection, and Antibodies—Melan-a cells were maintained in modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 12-O-tetradecanoylphorbol 13-acetate (200 ng/ml) (Sigma). Chinese hamster ovary (CHO) cells stably expressing wild-type mouse SHPS-1 (CHO-mSHPS-1 cells) were established as described previously (23Takada T. Matozaki T. Takeda H. Fukunaga K. Noguchi T. Fujioka Y. Okazaki I. Tsuda M. Yamao T. Ochi F. Kasuga M. J. Biol. Chem. 1998; 273: 9234-9242Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and were maintained in Ham's F-12 medium (Sigma) supplemented with 10% FBS. Transient transfection of CHO or Melan-a cells (∼4 × 105 cells/60-mm dish) was performed with the use of a LipofectAMINE Transfection Kit (Invitrogen). In some experiments, cells were cultured in the presence of 10 μm MG132 (Calbiochem) or 2 μm thapsigargin (Sigma), or with the corresponding vehicle (0.025% dimethyl sulfoxide and 0.2% ethanol, respectively). A rat monoclonal antibody (mAb) to mouse SHPS-1 (24Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (89) Google Scholar) and rabbit polyclonal antibodies to NFB42 (19Erhardt 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 (55) Google Scholar) were described previously. Mouse mAbs to human SIRPα1 and to Skp1 were obtained from Transduction Laboratories; rabbit polyclonal antibodies to SHPS-1 and to Ser51-phosphorylated eukaryotic initiation factor 2α (eIF2α) were from Upstate Biotechnology, Inc.; rabbit polyclonal antibodies to eIF2α were from Cell Signaling Technology; rabbit polyclonal antibodies to Cullin1 were from Zymed Laboratories Inc.; a mouse mAb to ubiquitin (P4D1) and normal rat or mouse IgG were from Santa Cruz Biotechnology; Texas Red-conjugated sheep polyclonal antibodies to mouse IgG were from Amersham Biosciences; and Alexa Fluor 488-conjugated goat polyclonal antibodies to rat IgG were from Molecular Probes. The mAb 9E10 to the Myc tag and mAb 12CA5 to the HA tag were purified from the culture supernatants of mouse hybridoma cells. Purification of SHPS-1-binding Proteins—All procedures were performed at 4 °C. The entire brain from one male mouse (12 weeks of age) was disrupted in 10 ml of ice-cold buffer A (10 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, aprotinin (100 units/ml), 1 mm benzamidine, 10 μm leupeptin, 1 mm sodium vanadate) with a Polytron homogenizer. The crude extract was centrifuged at 100,000 × g for 60 min, and the resulting supernatant (protein, 10 mg/ml) was incubated for 3 h in a 15-ml tube on a gently rotating platform with protein G-Sepharose beads (500 μl of beads) (Amersham Biosciences) that had been covalently coupled with 500 μg of the mAb to mouse SHPS-1. The beads were then washed four times with buffer A and once with 10 mm Tris-HCl (pH 6.8), and bound proteins were eluted with 5 ml of acidic buffer containing 100 mm glycine (pH 2.5). Fractions (1 ml each) were collected, neutralized by the addition of 0.1 volume of 1 m Tris-HCl (pH 8.8), and subjected to dot-immunoblot analysis with polyclonal antibodies to SHPS-1. Two peak fractions containing SHPS-1 were combined and concentrated with a Centricon 30 (Amicon) filtration device to a final volume of 50 μl. Mass Spectrometry—Proteins were resolved by SDS-PAGE on a 12.5% gel and stained with silver. Bands of interest were excised from the gel, washed in a solution comprising acetonitrile and 100 mm NH4HCO3 (1:1, v/v), and subjected to in-gel digestion with trypsin overnight at 37 °C. Peptides were then extracted with a solution of 50% acetonitrile and 5% formic acid, lyophilized, resuspended in 0.1% acetic acid, and analyzed with an LCQ ion-trap mass spectrometer equipped with a nanospray ion source and an HP1100 liquid chromatography system (ThermoQuest). Peptide sequences were identified by searching the NCBI nonredundant sequence data base with the obtained tandem mass spectra with the use of Mascot software (Matrix Science). Immunoprecipitation, Lectin Binding, and Immunoblot Analysis— Cells were lysed on ice in lysis buffer (10 mm Tris-HCl (pH 7.6), 150 mm NaCl, 1% Triton X-100) containing 1 mm phenylmethylsulfonyl fluoride, aprotinin (100 units/ml), and 1 mm sodium vanadate. The lysates were centrifuged at 10,000 × g for 15 min at 4 °C. For immunoprecipitation, the supernatants (protein, ∼5 mg/ml) were incubated for 3 h at 4 °C with antibody-coupled protein G-Sepharose beads. Alternatively, the supernatants were incubated for 1 h at 4 °C in the presence of 1 mm MnCl2 and 1 mm MgCl2 with agarose beads conjugated with Galanthus nivalis agglutinin (EY Laboratories). Both types of beads were then washed four times with lysis buffer, suspended in Laemmli sample buffer, and subjected to SDS-PAGE and immunoblot analysis with various antibodies and the ECL detection system (Amersham Biosciences). Immunofluorescence Analysis—All procedures were performed at room temperature. Cells seeded on glass coverslips were washed with phosphate-buffered saline (PBS), fixed with 3% paraformaldehyde in PBS for 20 min, and incubated with 50 mm NH4Cl for 10 min. Cells were then permeabilized for 2 min in PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) before incubation first for 1 h in PBS containing 0.1% Triton X-100, 10% FBS, and 0.5% BSA and then for 1 h with mAb 9E10 to the Myc tag. After washing twice with PBS containing 0.5% BSA, the cells were incubated with Texas Red-conjugated secondary antibodies for 30 min, washed three times with PBS containing 0.5% BSA, and examined with a laser-scanning confocal microscope (Bio-Rad model MRC-1024). Flow Cytometry—Detached cells (0.5 × 106 to 1 × 106) were washed with buffer B (PBS containing 1 mm EDTA and 2% FBS) and then incubated with the mAb to mouse SHPS-1 (20 μg/ml) or with control rat IgG (20 μg/ml) for 1 h on ice. They were washed with buffer B and incubated for 1 h on ice with Alexa Fluor 488-conjugated secondary antibodies (10 μg/ml). The stained cells were washed twice, suspended in 1 ml of buffer B, and then analyzed with a FACSCalibur flow cytometer (BD Biosciences). Data were processed with CellQuest software (BD Biosciences). SHPS-1 Forms a Complex with NFB42 and Skp1 in Vivo— SHPS-1 is particularly abundant in the central nervous system (4Sano S. Ohnishi H. Omori A. Hasegawa J. Kubota M. FEBS Lett. 1997; 411: 327-334Crossref PubMed Scopus (76) Google Scholar, 6Comu S. Weng W. Olinsky S. Ishwad P. Mi Z. Hempel J. Watkins S. Lagenaur C.F. Narayanan V. J. Neurosci. 1997; 17: 8702-8710Crossref PubMed Google Scholar, 24Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (89) Google Scholar). We therefore adopted an immunoaffinity approach to isolate proteins that interact with SHPS-1 in the brain. A detergent-solubilized extract of mouse brain was applied to protein G-Sepharose beads that had been coupled with a mAb that reacts with the extracellular portion of mouse SHPS-1 (24Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (89) Google Scholar). After extensive washing of the beads, bound material was eluted, resolved by SDS-PAGE, and visualized by silver staining (Fig. 1A). Bands corresponding to polypeptides that copurified with SHPS-1 were excised from the gel and subjected to in-gel digestion with trypsin, and the resulting peptides were analyzed by tandem mass spectrometry. The peptide sequences so obtained were used to search the NCBI sequence data base with Mascot software. Data base searches revealed that peptide sequences derived from proteins of ∼40 and ∼20 kDa were identical to sequences of rat NFB42 (AF098301) (19Erhardt 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 (55) Google Scholar) and mouse Skp1 (AF083214) (25Hatakeyama S. Kitagawa M. Nakayama K. Shirane M. Matsumoto M. Hattori K. Higashi H. Nakano H. Okumura K. Onoe K. Good R.A. Nakayama K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3859-3863Crossref PubMed Scopus (182) Google Scholar), respectively (Fig. 1A). NFB42 and Skp1 were shown previously to interact directly through the F box domain of the former (19Erhardt 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 (55) Google Scholar), suggesting that they associate with SHPS-1 as a complex. Immunoblot analysis confirmed that NFB42 and Skp1 associate with SHPS-1 in mouse brain extract, although Skp1 was barely detectable in immunoprecipitates prepared with a mAb that reacts with the cytoplasmic region of human SIRPα1 (Fig. 1B). Endogenous SHPS-1 was also coprecipitated with antibodies to NFB42 (Fig. 1C). Immunoblot analysis also revealed that an immortal line of mouse pigmented melanocytes, Melan-a (26Bennett D.C. Cooper P.J. Hart I.R. Int. J. Cancer. 1987; 39: 414-418Crossref PubMed Scopus (407) Google Scholar), expresses SHPS-1, NFB42, and Skp1 at levels similar to those observed in mouse brain extract (Fig. 1B). A substantial proportion of endogenous NFB42 was associated with SHPS-1 in Melan-a cells, although this proportion was markedly less than that apparent in mouse brain. Skp1 interaction with SHPS-1 was not detected in these cells. The interaction between SHPS-1 and NFB42 was also demonstrated in CHO cells transiently expressing recombinant SHPS-1 and HA-tagged NFB42 (Fig. 1D); NFB42 associated preferentially with immature forms of SHPS-1 in these cells, hardly interacting at all with the mature forms (Fig. 1D). Structural Requirements for SHPS-1-NFB42 Interaction— Immunoprecipitation and immunoblot analysis with a brain extract prepared from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region (15Inagaki K. Yamao T. Noguchi T. Matozaki T. Fukunaga K. Takada T. Hosooka T. Akira S. Kasuga M. EMBO J. 2000; 19: 6721-6731Crossref PubMed Scopus (126) Google Scholar) revealed that NFB42 and Skp1 each associated with the mutant protein, albeit to a lesser extent than with wild-type SHPS-1 (Fig. 2A), suggesting that the extracellular or membrane-proximal cytoplasmic region of SHPS-1 mediates the interaction with NFB42. We next examined the ability to interact with NFB42 of SHPS-1 mutants in which two juxtamembrane lysine residues in the cytoplasmic region were replaced by asparagine and glutamine, respectively, or in which all four cytoplasmic tyrosine residues were changed to phenylalanine. Each of these mutant proteins associated with HA-tagged NFB42 in CHO cells to an extent similar to that observed with wild-type SHPS-1 (Fig. 2B). Thus, neither the positively charged juxtamembrane region nor cytoplasmic tyrosine residues of SHPS-1 appeared to play a major role in the interaction of this protein with NFB42. To map the SHPS-1-binding site in NFB42, we employed the following three NFB42 mutant proteins: NFBΔP, which lacks the NH2-terminal PEST domain; NFBΔPΔF, which lacks both the PEST and F box domains; and NFBPF, which comprises only the PEST and F box domains (Fig. 2C). The HA-tagged versions of these mutant proteins were transiently expressed in CHO cells that stably express wild-type mouse SHPS-1 (CHO-mSHPS-1 cells) and were then tested for their ability to interact with SHPS-1. NFBΔP and NFBΔPΔF, but not NFBPF, bound to SHPS-1 (Fig. 2D), suggesting that the COOH-terminal domain of NFB42 mediates this interaction. Consistent with previous observations (19Erhardt 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 (55) Google Scholar), NFBΔP, but not NFBΔPΔF, also bound Skp1. SCFNFB42 Ubiquitin Ligase Mediates Polyubiquitination and Proteasomal Degradation of Immature SHPS-1—SCF-type ubiquitin ligase (E3) complexes include the invariant component Skp1, an F box protein that is responsible for substrate recognition, and Cullin1, which serves as a scaffold for organization of the other subunits (27Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1089) Google Scholar). Both NFB42 (Fig. 3A) and Skp1 (Fig. 3B) interact with Cullin1, suggesting that these three proteins constitute an SCFNFB42 ubiquitin ligase. Ectopic expression of NFB42 in CHO-mSHPS-1 cells revealed polyubiquitination of SHPS-1 in the presence of the specific proteasome inhibitor MG132 (Fig. 3C). Treatment of cells with MG132 also substantially increased the amount of immature SHPS-1 associated with NFB42 (Fig. 3D). Together, these results suggested that SCFNFB42 acts as an E3 that catalyzes the polyubiquitination of immature SHPS-1 and that the ubiquitinated protein is subsequently eliminated by proteasomal degradation. SHPS-1 Is a Substrate for ER-associated Degradation—The possibility that SCFNFB42-mediated degradation down-regulates the biological function of SHPS-1 appeared unlikely given the lack of a positive effect of MG132 on the abundance of mature SHPS-1 (Fig. 3D). Rather, the selective association of immature forms of SHPS-1 with NFB42 suggested a role for SCFNFB42 in the elimination of SHPS-1 molecules misfolded during biosynthesis. To test this possibility, we determined whether SHPS-1 serves as a substrate for ER-associated degradation (ERAD), a pathway by which misfolded proteins in the ER are translocated to the cytosol, polyubiquitinated, and degraded by the proteasome (28Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (539) Google Scholar, 29Ellgaard L. Helenius A. Curr. Opin. Cell Biol. 2001; 13: 431-437Crossref PubMed Scopus (337) Google Scholar, 30Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (407) Google Scholar). Fluorescence microscopy of Melan-a cells expressing a fusion construct of SHPS-1 and GFP (SHPS-1-GFP) revealed that the recombinant protein was localized predominantly to both the cell surface and the perinuclear region (Fig. 4A). A small proportion of SHPS-1-GFP was also detected as small aggregates throughout the cytoplasm. Similar analysis of cells exposed to MG132 showed that most SHPS-1-GFP was present either in large aggregates adjacent to the nucleus (Fig. 4B) or in small aggregates in the cytoplasm (data not shown). The large perinuclear aggregates exhibited the characteristic features of aggresomes, cellular inclusion bodies that consist of polyubiquitinated proteins and which are formed by ERAD substrates in response to inhibition of proteasome activity (31Johnston J.A. Ward C.L. Kopito R.R. J. Cell Biol. 1998; 143: 1883-1898Crossref PubMed Scopus (1805) Google Scholar, 32Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1636) Google Scholar). Our results thus suggested that SHPS-1 is a substrate for the ERAD pathway. SCFNFB42 Promotes Elimination of Misfolded SHPS-1 by the ERAD Pathway—Wild-type NFB42 was distributed diffusely in the cytosol of Melan-a cells incubated in the absence (data not shown) or presence (Fig. 4G) of MG132. Overexpression of wild-type NFB42 induced redistribution of a substantial proportion of SHPS-1-GFP to the cell surface and markedly suppressed the formation of aggresome-like bodies and cytoplasmic aggregates in cells treated with MG132 (Fig. 4C; data not shown). In contrast, expression of NFBΔPΔF, which does not form a functional ubiquitin ligase, promoted the formation of these structures (Fig. 4D; data not shown), indicative of a dominant negative effect of this mutant. The staining of wild-type NFB42 overlapped partially with SHPS-1-GFP fluorescence in a reticular pattern throughout the cytoplasm (Fig. 4I), consistent with colocalization of these proteins at or in the ER. However, extensive colocalization of the two proteins was not detected at the cell periphery, where mature SHPS-1 is expected to reside, even in the presence of MG132. A substantial proportion of NFBΔPΔF colocalized with SHPS-1-GFP in the aggresome-like bodies of MG132-treated cells (Fig. 4J). Binding experiments in vitro with G. nivalis agglutinin lectin, which specifically recognizes terminal mannose residues of glycoproteins (33Shibuya N. Goldstein I.J. Van Damme E.J. Peumans W.J. J. Biol. Chem. 1988; 263: 728-734Abstract Full Text PDF PubMed Google Scholar), revealed that recombinant SHPS
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