Ectodomain Shedding of SHPS-1 and Its Role in Regulation of Cell Migration
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m313085200
ISSN1083-351X
AutoresHiroshi Ohnishi, Hisae Kobayashi, Hideki Okazawa, Yoshihide Ohe, Kyoko Tomizawa, Ryuji Sato, Takashi Matozaki,
Tópico(s)Galectins and Cancer Biology
ResumoSHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein. SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein. The extracellular region of various transmembrane proteins is cleaved by proteases, such as metalloproteinases, and released as a soluble protein fragment (1Massague J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (598) Google Scholar, 2Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (682) Google Scholar). This process, known as "ectodomain shedding," often influences the mode of action or biological activity of the affected protein. For instance, ectodomain shedding of heparin-binding epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; ADAM, a disintegrin and metalloproteinase; ConA, concanavalin A; FAK, focal adhesion kinase; FBS, fetal bovine serum; HB-EGF, heparin-binding EGF; LPA, lysophosphatidic acid; mAb, monoclonal antibody; MMP, matrix metalloproteinase; OPT, 1,10-phenanthroline; pAbs, polyclonal antibodies; PBS, phosphate-buffered saline; PKC, protein kinase C; SH2, Src homology 2; SHPS-1, SH2 domain-containing protein-tyrosine phosphatase substrate-1; TPA, 12-O-tetradecanoylphorbol 13-acetate. 1The abbreviations used are: EGF, epidermal growth factor; ADAM, a disintegrin and metalloproteinase; ConA, concanavalin A; FAK, focal adhesion kinase; FBS, fetal bovine serum; HB-EGF, heparin-binding EGF; LPA, lysophosphatidic acid; mAb, monoclonal antibody; MMP, matrix metalloproteinase; OPT, 1,10-phenanthroline; pAbs, polyclonal antibodies; PBS, phosphate-buffered saline; PKC, protein kinase C; SH2, Src homology 2; SHPS-1, SH2 domain-containing protein-tyrosine phosphatase substrate-1; TPA, 12-O-tetradecanoylphorbol 13-acetate.-like growth factor (HB-EGF), a membrane-anchored EGF-related protein (3Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1038) Google Scholar), results in its activation, in that the released fragment binds to EGF receptors and thereby stimulates cell proliferation (3Higashiyama S. Abraham J.A. Miller J. Fiddes J.C. Klagsbrun M. Science. 1991; 251: 936-939Crossref PubMed Scopus (1038) Google Scholar, 4Hashimoto K. Higashiyama S. Asada H. Hashimura E. Kobayashi T. Sudo K. Nakagawa T. Damm D. Yoshikawa K. Taniguchi N. J. Biol. Chem. 1994; 269: 20060-20066Abstract Full Text PDF PubMed Google Scholar). In contrast, the membrane-anchored forms of c-kit ligand (5Flanagan J.G. Chan D.C. Leder P. Cell. 1991; 64: 1025-1035Abstract Full Text PDF PubMed Scopus (612) Google Scholar) and of ephrins, the latter of which are ligands of Eph receptor tyrosine kinases (6Davis S. Gale N.W. Aldrich T.H. Maisonpierre P.C. Lhotak V. Pawson T. Goldfarb M. Yancopoulos G.D. Science. 1994; 266: 816-819Crossref PubMed Scopus (630) Google Scholar), are fully functional, whereas their soluble forms generated by ectodomain shedding exhibit little or no biological activity. Ectodomain shedding is thus an important regulator of the function of certain membrane proteins that contribute to cell-cell communication.SHPS-1 (SHP substrate-1) (7Fujioka 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 (382) Google Scholar), also known as SIRPα (8Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (538) Google Scholar), BIT (9Ohnishi H. Kubota M. Ohtake A. Sato K. Sano S. J. Biol. Chem. 1996; 271: 25569-25574Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and p84 neural adhesion molecule (10Comu 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), is a receptor-like transmembrane protein that is particularly abundant in neurons and macrophages (10Comu 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, 11Seiffert M. Cant C. Chen Z. Rappold I. Brugger W. Kanz L. Brown E.J. Ullrich A. Buhring H.J. Blood. 1999; 94: 3633-3643Crossref PubMed Google Scholar), although other cell types, such as fibroblasts, also express this protein (7Fujioka 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 (382) Google Scholar). The putative extracellular region of SHPS-1 comprises three immunoglobulin (Ig)–like domains with multiple N-linked glycosylation sites, whereas the cytoplasmic region of the protein contains four YXX(L/V/I) motifs, which are putative tyrosine phosphorylation sites and binding sites for the Src homology 2 (SH2) domains of the protein-tyrosine phosphatases SHP-2 and SHP-1 (7Fujioka 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 (382) Google Scholar, 8Kharitonenkov A. Chen Z. Sures I. Wang H. Schilling J. Ullrich A. Nature. 1997; 386: 181-186Crossref PubMed Scopus (538) Google Scholar). Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors, including insulin and EGF, as well as by cell adhesion to extracellular matrix proteins (12Tsuda M. Matozaki T. Fukunaga K. Fujioka Y. Imamoto A. Noguchi T. Takada T. Yamao T. Takeda H. Ochi F. Yamamoto T. Kasuga M. J. Biol. Chem. 1998; 273: 13223-13229Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). SHP-2 participates in the positive regulation of cell migration (13Yu D.H. Qu C.K. Henegariu O. Lu X. Feng G.S. J. Biol. Chem. 1998; 273: 21125-21131Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 14Manes S. Mira E. Gomez-Mouton C. Zhao Z.J. Lacalle R.A. Martinez A.C. Mol. Cell. Biol. 1999; 19: 3125-3135Crossref PubMed Scopus (220) Google Scholar, 15Kodama A. Matozaki T. Fukuhara A. Kikyo M. Ichihashi M. Takai Y. Mol. Biol. Cell. 2000; 11: 2565-2575Crossref PubMed Scopus (106) Google Scholar, 16Lacalle R.A. Mira E. Gomez-Mouton C. Jimenez-Baranda S. Martinez A.-C. Manes S. J. Cell Biol. 2002; 157: 277-289Crossref PubMed Scopus (71) Google Scholar), and SHPS-1 recruits and activates SHP-2 at the cell membrane in response to growth factors or to integrin-mediated cell adhesion (9Ohnishi H. Kubota M. Ohtake A. Sato K. Sano S. J. Biol. Chem. 1996; 271: 25569-25574Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 17Takada 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). Characterization of immortalized fibroblasts from mice that lack most of the cytoplasmic region of SHPS-1 revealed a marked impairment of cell migration associated with an increased formation of actin stress fibers and focal adhesions (18Inagaki 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 (120) Google Scholar). These observations suggest that the tyrosine phosphorylation of SHPS-1 and the consequent association of SHPS-1 with SHP-2 promote cell migration through regulation of cytoskeletal reorganization.CD47, also named IAP, is implicated as a ligand for SHPS-1 (11Seiffert M. Cant C. Chen Z. Rappold I. Brugger W. Kanz L. Brown E.J. Ullrich A. Buhring H.J. Blood. 1999; 94: 3633-3643Crossref PubMed Google Scholar, 19Jiang P. Lagenaur C.F. Narayanan V. J. Biol. Chem. 1999; 274: 559-562Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). This protein, which was originally identified in association with αvβ3 integrin (20Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (308) Google Scholar), is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, five putative membrane-spanning segments, and a short cytoplasmic tail (21Brown E.J. Frazier W.A. Trends Cell Biol. 2001; 11: 130-135Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). CD47 and SHPS-1 appear to constitute a cell-cell communication system (the CD47-SHPS-1 system) that plays an important role in a variety of cell functions. We have recently shown that the CD47-SHPS-1 system and SHP-2 contribute to the inhibition of cell migration by cell-cell contact (22Motegi S. Okazawa H. Ohnishi H. Sato R. Kaneko Y. Kobayashi H. Tomizawa K. Ito T. Honma N. Bühring H.J. Ishikawa O. Matozaki T. EMBO J. 2003; 22: 2634-2644Crossref PubMed Scopus (74) Google Scholar). Neutrophil migration to sites of inflammation is markedly impaired in CD47 knockout mice (23Lindberg F.P. Bullard D.C. Caver T.E. Gresham H.D. Beaudet A.L. Brown E.J. Science. 1996; 274: 795-798Crossref PubMed Scopus (292) Google Scholar) and monoclonal antibodies (mAbs) to CD47 inhibit neutrophil transmigration (24Liu Y. Merlin D. Burst S.L. Pochet M. Madara J.L. Parkos C.A. J. Biol. Chem. 2001; 276: 40156-40166Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), suggesting that the CD47-SHPS-1 system might mediate bidirectional inhibitory regulation of cell migration. The binding of CD47 on red blood cells to SHPS-1 on macrophages also inhibits phagocytosis of the red blood cells by the macrophages (25Oldenborg P.A. Zheleznyak A. Fang Y.F. Lagenaur C.F. Gresham H.D. Lindberg F.P. Science. 2000; 288: 2051-2054Crossref PubMed Scopus (1226) Google Scholar). Thus, SHPS-1 appears to play multiple roles in cellular activities in a manner dependent on or independent of its interaction with CD47. It has not been known, however, whether SHPS-1 undergoes ectodomain shedding, a process that might potentially regulate its function.We now show that SHPS-1 indeed undergoes ectodomain shedding. Furthermore, we have characterized the molecular mechanism of this process as well as examined its physiological role in the regulation of cell migration.EXPERIMENTAL PROCEDURESPrimary Antibodies and Reagents—A rat mAb (αp84) to mouse SHPS-1 was purified from culture supernatants of hybridoma cells kindly provided by C. F. Lagenaur (University of Pittsburgh). Rabbit polyclonal antibodies (pAbs) to the cytoplasmic region of SHPS-1 were obtained from ProSci. A mouse mAb to SHP-2 was from Transduction Laboratories. A mouse mAb to paxillin and rabbit pAbs to focal adhesion kinase (FAK) (for immunoprecipitation) were from Upstate Biotechnology. Rabbit pAbs to FAK (for immunoblot analysis) and peroxidase-conjugated mAb PY20 to phosphotyrosine were from Santa Cruz Biotechnology. A mouse mAb to vinculin as well as 12-O-tetradecanoylphorbol 13-acetate (TPA), lysophosphatidic acid (LPA), and 1,10-phenanthroline (OPT) were from Sigma. IC-3 was from Biomol Research Laboratories. KB-R7785 was kindly provided by S. Higashiyama (Ehime University).Cells and Cell Culture—All cells were maintained at 37 °C under a humidified atmosphere of 5% CO2 in air. The mouse melanoma cell line B16F10 was kindly provided by T. Horikawa (Kobe University). The mouse macrophage cell line RAW264.7 was kindly provided by Y. Kaneko (Gunma University). Primary mouse hippocampal neurons were prepared and cultured as described previously (26Ohnishi H. Yamada M. Kubota M. Hatanaka H. Sano S. J. Neurochem. 1999; 72: 1402-1408Crossref PubMed Scopus (38) Google Scholar). CHO cells stably expressing an active form of H-Ras (CHO-Ras cells) were kindly provided by S. Shirahata (Kyushu University). CHO-Ras cells stably expressing mouse SHPS-1 (CHO-Ras-SHPS-1-WT cells) as well as those expressing mouse CD47 (CHO-Ras-CD47 cells) were kindly provided by N. Honma (Kirin Brewery Co. Ltd.). CHO cells stably expressing mouse wild-type SHPS-1 (CHO-SHPS-1-WT cells) were kindly provided by T. Noguchi (Kobe University). CHO-Ras, CHO-Ras-SHPS-1-WT, and CHO-Ras-CD47 cells were cultured in αMEM (Sigma) supplemented with 2 mm l-glutamine, 10 mm Hepes-NaOH (pH 7.4), 10% fetal bovine serum (FBS), and Geneticin (500 μg/ml) (Invitrogen). CHO and CHO-SHPS-1-WT cells were cultured in F-12 medium (Sigma) supplemented with 10% FBS.Determination of Ectodomain Shedding of SHPS-1 by Concanavalin A (ConA)-mediated Precipitation, Immunoprecipitation, and Immunoblot Analysis—The supernatants of cell cultures (∼4 × 106 cells in a 60-mm dish) were collected and centrifuged at 21,000 × g for 15 min at 4 °C, and the resulting supernatants were then incubated for 2 h at 4 °C with ConA-coupled agarose beads (Amersham Biosciences). Alternatively, the cell-free conditioned medium was incubated for 4 h at 4 °C with the mAb αp84 bound to protein G-Sepharose beads (Amersham Biosciences). Both types of beads were then washed twice with 1 ml of WG buffer (50 mm Hepes-NaOH (pH 7.6), 150 mm NaCl, 0.1% Triton X-100), resuspended in SDS sample buffer, and subjected to SDS-PAGE followed by immunoblot analysis either with the αp84 mAb or with pAbs to SHPS-1 as described previously (22Motegi S. Okazawa H. Ohnishi H. Sato R. Kaneko Y. Kobayashi H. Tomizawa K. Ito T. Honma N. Bühring H.J. Ishikawa O. Matozaki T. EMBO J. 2003; 22: 2634-2644Crossref PubMed Scopus (74) Google Scholar). Immune complexes were detected with an ECL detection kit (Amersham Biosciences). Lysates of cultured cells were also prepared by incubation on ice with 1 ml of lysis buffer (20 mm Tris-HCl (pH 7.6), 140 mm NaCl, 1 mm EDTA, 1% Nonidet P-40) containing 1 mm phenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), and 1 mm sodium vanadate. The lysates were centrifuged at 21,000 × g for 15 min at 4 °C, and the resulting supernatants were subjected to immunoblot analysis. Immunoprecipitation and immunoblot analysis of paxillin and FAK were also performed by similar procedures. For preparation of membrane and cytosolic fractions, CHO-Ras-SHPS-1-WT cells were lysed on ice in 1 ml of hypotonic buffer (20 mm Tris-HCl (pH 7.6), 1 mm EDTA) containing 1 mm phenylmethylsulfonyl fluoride, aprotinin (10 μg/ml), and 1 mm sodium vanadate; the lysate was subjected to centrifugation at 436,000 × g for 60 min at 4 °C; and the resulting supernatant and pellet were designated the cytosolic and membrane fractions, respectively.Preparation of SHPS-1-Fc and CD47-Fc Fusion Proteins—An SHPS-1-Fc fusion protein, which contained the extracellular region of mouse SHPS-1 (amino acids 1–371) fused to the Fc portion of human IgG, and a CD47-Fc fusion protein, which contained the extracellular region of mouse CD47 (amino acids 1–161) fused to the Fc portion of human IgG, were produced and purified as described previously (27Sato R. Ohnishi H. Kobayashi H. Kiuchi D. Hayashi A. Kaneko Y. Honma N. Okazawa H. Hirata Y. Matozaki T. Biochem. Biophys. Res. Commun. 2003; 309: 584-590Crossref PubMed Scopus (19) Google Scholar).Analysis for the Binding of Fc Fusion Proteins to CHO-Ras Cells— The binding of SHPS-1-Fc to CHO-Ras cells stably expressing CD47 was determined essentially as described previously (27Sato R. Ohnishi H. Kobayashi H. Kiuchi D. Hayashi A. Kaneko Y. Honma N. Okazawa H. Hirata Y. Matozaki T. Biochem. Biophys. Res. Commun. 2003; 309: 584-590Crossref PubMed Scopus (19) Google Scholar). Briefly, confluent CHO-Ras cells stably expressing CD47 in 96-well plates were incubated for 30 min at 37 °C with various concentrations of SHPS-1-Fc in the culture medium described above, after which the cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated for 30 min at 4 °C with horseradish peroxidase-conjugated goat pAbs to the Fc fragment of human IgG (Jackson ImmunoResearch). The cells were again washed with PBS, and Fc fusion protein binding was determined by measurement of peroxidase activity with o-phenylenediamine dihydrochloride (Sigma) as substrate. The absorbance of each well at 492 nm was monitored with a microplate reader. The binding of CD47-Fc to CHO-Ras cells stably expressing wild-type or a mutant SHPS-1 was also performed as described above.Identification of the SHPS-1 Peptide Bond Cleaved by Matrix Metalloproteinases—Incubation of SHPS-1-Fc with MMP-1 or MMP-9 was performed according to the method described by Franzke et al. (28Franzke C.-W. Tasanen K. Schäcke H. Zhou Z. Tryggvason K. Mauch C. Zigrino P. Sunnarborg S. Lee D.C. Fahrenholz F. Bruckner-Tuderman L. EMBO J. 2002; 21: 5026-5035Crossref PubMed Scopus (186) Google Scholar), with minor modifications. The purified fusion protein (15 μg) was incubated for 24 h at 37 °C with 5 μg of MMP-9 or MMP-1 (Calbiochem) in 120 μl of a solution containing 50 mm Tris-HCl (pH 7.4), 0.2 m NaCl, 5 mm CaCl2, 0.1 mm ZnCl2, and 2 mmp-aminophenyl mercuric acetate, after which the reaction mixture was evaporated. The samples were then subjected to SDS-PAGE, the separated proteins were transferred to a polyvinylidene difluoride membrane, and the membrane was stained with Coomassie Brilliant Blue R-250. Stained bands corresponding to proteins of 35 and 30 kDa were excised, and their NH2-terminal amino acid sequences were determined with a gas-phase amino acid sequence analyzer (Procise 492; Applied Biosystems) as described previously (29Ohe Y. Ohnishi H. Okazawa H. Tomizawa K. Kobayashi H. Okawa K. Matozaki T. Biochem. Biophys. Res. Commun. 2003; 308: 719-725Crossref PubMed Scopus (23) Google Scholar).Generation of CHO-Ras and CHO Cells Expressing a Mutant SHPS-1—To generate the plasmid for expression of the cleavage-resistant SHPS-1 mutant (SHPS-1-FLAG-JM), we performed the polymerase chain reaction with an expression plasmid for wild-type mouse SHPS-1 (full-length cDNA for mouse SHPS-1 (30Yamao 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) subcloned into the EcoRI and NotI sites of pTracer-CMV (Invitrogen)) as the template and the primers 5′-CCGATATCGACTACAAGGACGACGATGACAAGACCCACAACTGGAATGTCTTC-3′ (sense) and 5′-CCGATATCCCCTTGATCACTCGAGTG-3′ (antisense). The sense primer encodes the artificial amino acid sequence DIDYKDDDDK, the last eight residues of which correspond to the FLAG epitope tag. The amplification product thus encoded a mutant SHPS-1 protein (SHPS-1-FLAG-JM) with this 10-amino acid sequence instead of the juxtamembrane sequence SMQTFPGNNA368 (Fig. 5A); it was digested with EcoRV and then self-ligated, and its entire nucleotide sequence was verified by DNA sequence analysis with an ABI PRISM310 Genetic Analyzer (Applied Biosystems). CHO-Ras cells were then transfected with the plasmid containing the mutant SHPS-1 cDNA with the use of LipofectAMINE 2000 (Invitrogen). The cells were cultured in αMEM supplemented with 2 mm l-glutamine, 10 mm Hepes-NaOH (pH 7.6), 10% FBS, and Zeocin (200 μg/ml) (Invitrogen), and colonies were isolated 14–21 days after transfection. CHO cells were also transfected with the plasmid containing the mutant SHPS-1 cDNA and selected as described above. The cells were cultured in F-12 medium (Sigma) supplemented with 10% FBS and Zeocin (200 μg/ml). Colonies were isolated 14–21 days after transfection, and several cell lines expressing the mutant SHPS-1 protein were identified by immunoblot analysis of cell lysates with the mAb αp84.Cell Migration Assay—Cell migration was assayed with a Transwell apparatus (Corning) as described previously (22Motegi S. Okazawa H. Ohnishi H. Sato R. Kaneko Y. Kobayashi H. Tomizawa K. Ito T. Honma N. Bühring H.J. Ishikawa O. Matozaki T. EMBO J. 2003; 22: 2634-2644Crossref PubMed Scopus (74) Google Scholar). In brief, CHO, CHO-Ras, and derived cell lines were detached from culture dishes and resuspended in αMEM containing 10% FBS. A portion of the cell suspension (1 × 105 cells in 100 μl) was then transferred to a polycarbonate filter (pore size, 8 μm; Corning) in the upper compartment of a Transwell apparatus, and 600 μl of fresh culture medium was placed in the lower compartment. The number of cells that had migrated into the lower compartment after incubation for 16 h at 37 °C was then counted in triplicate with a hemocytometer and was expressed as a percentage of the total number of cells added to the upper compartment.Immunofluorescence Analysis—CHO-Ras and derived cell lines were plated on cover glasses 12–24 h before analysis. They were fixed for 20 min either with 4% paraformaldehyde in PBS for vinculin staining or with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS for actin and paxillin staining. The cells were then permeabilized for 1 h at room temperature in buffer G (PBS containing 5% goat serum and 0.1% Triton X-100) before incubation for 1 h at room temperature with a mAb to vinculin (1/400 dilution), a mAb to paxillin (5 μg/ml), or rhodamine-conjugated phalloidin (2 units/ml) (Molecular Probes) diluted in buffer G. They were then washed three times with PBS. For staining of vinculin or paxillin, the cells were incubated for 30 min with Alexa488-conjugated sheep pAbs to mouse IgG (Molecular Probes) and then washed another three times with PBS. All cells were examined with a confocal laser-scanning microscope (LSM 5 Pascal, Carl Zeiss).Cell Spreading and Adhesion Assay—Detached cells were suspended in fresh culture medium and transferred at a density of 1 × 105 cells/ml to 35-mm dishes coated with fibronectin (Asahi Technoglass). After incubation for 1 or 4 h, the cells were examined with a light microscope equipped with phase-contrast optics (Leica DM IRBE), and digital images of random fields were captured with a charge-coupled device camera (Penguin 600CL, Pixera).RESULTSEctodomain of SHPS-1 in Conditioned Medium of SHPS-1-expressing Cells—We first examined whether the ectodomain of SHPS-1 was shed from CHO-Ras-SHPS-1-WT cells, CHO cells that were transformed as a result of expression of an active form of H-Ras and that also express mouse SHPS-1. Given that SHPS-1 is extensively glycosylated and therefore binds to ConA (7Fujioka 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 (382) Google Scholar), we incubated the conditioned medium obtained by overnight culture of these cells with ConA-coupled agarose beads and then examined bead-bound proteins by immunoblot analysis with a mAb (αp84) that specifically recognizes the extracellular region of mouse SHPS-1 (31Chuang W. Lagenaur C.F. Dev. Biol. 1990; 137: 219-232Crossref PubMed Scopus (85) Google Scholar). An immunoreactive protein of ∼100 kDa was detected in the conditioned medium from CHO-Ras-SHPS-1-WT cells (Fig. 1A) but not in that from parental CHO-Ras cells (data not shown). The same protein was also observed in immunoprecipitates prepared with the αp84 mAb from the medium conditioned by CHO-Ras-SHPS-1-WT cells (Fig. 1A). In contrast, immunoblot analysis with rabbit pAbs that react with the cytoplasmic region of SHPS-1 failed to detect the ∼100-kDa protein in precipitates prepared from this conditioned medium with either ConA or the αp84 mAb (Fig. 1A). Immunoblot analysis with these pAbs to SHPS-1 did detect an ∼20-kDa protein in lysates of CHO-Ras-SHPS-1-WT cells but not in those of parental CHO-Ras cells (Fig. 1B). This ∼20-kDa protein appeared to be localized predominantly in a membrane fraction, rather than a cytosolic fraction, of CHO-Ras-SHPS-1-WT cells (Fig. 1B). The molecular size of intact SHPS-1 in lysates of CHO-Ras-SHPS-1-WT cells was ∼120 kDa (Fig. 1A), whereas that of the transmembrane plus cytoplasmic regions of SHPS-1 was estimated to be ∼20 kDa on the basis of the predicted amino acid sequence of SHPS-1 (30Yamao 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). Together, these results thus suggested that the ∼100-kDa protein detected in the conditioned medium of CHO-Ras-SHPS-1-WT cells was the extracellular domain of SHPS-1, whereas the ∼20-kDa protein present in the membrane fraction of these cells was the transmembrane-cytoplasmic region of SHPS-1 remaining after cleavage (see also Fig. 5B).Fig. 1Presence of the ectodomain of SHPS-1 in the conditioned medium of cultured cells.A, conditioned medium of CHO-Ras cells stably expressing SHPS-1 (CHO-Ras-SHPS-1-WT cells) was incubated with ConA-coupled agarose beads or with protein G-Sepharose beads containing bound mAb αp84 to SHPS-1. The proteins precipitated by each type of beads were subjected to immunoblot analysis with αp84 (left panel) or with pAbs to SHPS-1 (αSHPS-1) (right panel). Whole cell lysates were also similarly subjected to immunoblot analysis. The positions of full-length SHPS-1 and of the ectodomain of SHPS-1 (SHPS-1-EX) are indicated by lines and an arrow, respectively. B, cell lysates prepared from parental CHO-Ras cells or CHO-Ras-SHPS-1-WT cells (left panel) as well as membrane and cytosolic fractions prepared from CHO-Ras-SHPS-1-WT cells (right panel) were subjected to immunoblot analysis with pAbs to SHPS-1. The position of an immunoreactive protein of ∼20-kDa is indicated by arrows. C, conditioned medium of primary cultured mouse hippocampal neurons, B16F10 mouse melanoma cells, or RAW264.7 mouse macrophages was incubated with ConA-coupled agarose beads, after which the bead-bound proteins (ConA ppt.), as well as total cell lysates, were subjected to immunoblot analysis with the αp84 mAb to SHPS-1. All data are representative of three separate experiments.View Large Image Figure ViewerDownload (PPT)Endogenous SHPS-1 was detected in lysates prepared from primary cultured mouse hippocampal neurons, B16F10 mouse melanoma cells, or RAW264.7 mouse macrophages (Fig. 1C). We also detected ∼80- to 100-kDa proteins that were reactive with the αp84 mAb (Fig. 1C), but not with the pAbs to SHPS-1 (data not shown), in the conditioned media obtained from these cells. The molecular sizes of the SHPS-1 fragments in these conditioned media were also ∼20 kDa smaller than that of the intact SHPS-1 protein in the corresponding cell lysates. These results indicated that the ectodomain of SHPS-1 is shed into the medium of cultured cells that express endogenous SHPS-1 as well as into that of CHO-Ras-SHPS-1-WT cells. We also observed that the ectodomain of human SHPS-1 was present in the culture medium of CHO-Ras cells expressing the intact human protein (data not shown).Regulation of Ectodomain Shedding of SHPS-1—We next examined whether the shedding of the ectodomain of SHPS-1 occurs in response to extracellular or intracellular stimulation. Minimal ectodomain shedding of SHPS-1 was apparent after incubation for 4 h in serum-free medium of CHO cells that stably express mouse SHPS-1 (CHO-SHPS-1-WT cells) (Fig. 2A). In contrast, incubation of the cells for 4 h in the presence of either 10% FBS or 1 μm LPA resulted in a marked increase in the amount of the SHPS-1 ectodomain in the medium (Fig. 2A). LPA induces activation of protein kinase C (PKC) (32Moolenaar W.H. Curr. Opin. Cell Biol. 1995; 7: 203-210Crossref PubMed Scopus (222) Google Scholar), and PKC activation in turn triggers ectodomain shedding of various membrane-associated proteins (33Pandiella A. Massague J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1726-1730Crossref PubMed Scopus (181) Google Scholar, 34Arribas J. Coodly L. Vollmer P. Kishimoto T.K. Rose-John S. Massague J. J. Biol. Chem. 1996; 271: 11376-11382Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar
Referência(s)