FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2130170015> ?p ?o ?g. }

• W2130170015 endingPage "660" @default.
• W2130170015 startingPage "647" @default.
• W2130170015 abstract "In transmembrane (TM) domains, tetraspanin KAI1/CD82 contains an Asn, a Gln, and a Glu polar residue. A mutation of all three polar residues largely disrupts the migration-, invasion-, and metastasis-suppressive activities of KAI1/CD82. Notably, KAI1/CD82 inhibits the formation of microprotrusions and the release of microvesicles, while the mutation disrupts these inhibitions, revealing the connections of microprotrusion and microvesicle to KAI1/CD82 function. The TM polar residues are needed for proper interactions between KAI1/CD82 and tetraspanins CD9 and CD151, which also regulate cell movement, but not for the association between KAI1/CD82 and α3β1 integrin. However, KAI1/CD82 still efficiently inhibits cell migration when either CD9 or CD151 is absent. Hence, KAI1/CD82 interacts with tetraspanin and integrin by different mechanisms and is unlikely to inhibit cell migration through its associated proteins. Moreover, without significantly affecting the glycosylation, homodimerization, and global folding of KAI1/CD82, the TM interactions maintain the conformational stability of KAI1/CD82, evidenced by the facts that the mutant is more sensitive to denaturation and less associable with tetraspanins and supported by the modeling analysis. Thus, the TM interactions mediated by these polar residues determine a conformation either in or near the tightly packed TM region and this conformation and/or its change are needed for the intrinsic activity of KAI1/CD82. In contrast to immense efforts to block the signaling of cancer progression, the perturbation of TM interactions may open a new avenue to prevent cancer invasion and metastasis. In transmembrane (TM) domains, tetraspanin KAI1/CD82 contains an Asn, a Gln, and a Glu polar residue. A mutation of all three polar residues largely disrupts the migration-, invasion-, and metastasis-suppressive activities of KAI1/CD82. Notably, KAI1/CD82 inhibits the formation of microprotrusions and the release of microvesicles, while the mutation disrupts these inhibitions, revealing the connections of microprotrusion and microvesicle to KAI1/CD82 function. The TM polar residues are needed for proper interactions between KAI1/CD82 and tetraspanins CD9 and CD151, which also regulate cell movement, but not for the association between KAI1/CD82 and α3β1 integrin. However, KAI1/CD82 still efficiently inhibits cell migration when either CD9 or CD151 is absent. Hence, KAI1/CD82 interacts with tetraspanin and integrin by different mechanisms and is unlikely to inhibit cell migration through its associated proteins. Moreover, without significantly affecting the glycosylation, homodimerization, and global folding of KAI1/CD82, the TM interactions maintain the conformational stability of KAI1/CD82, evidenced by the facts that the mutant is more sensitive to denaturation and less associable with tetraspanins and supported by the modeling analysis. Thus, the TM interactions mediated by these polar residues determine a conformation either in or near the tightly packed TM region and this conformation and/or its change are needed for the intrinsic activity of KAI1/CD82. In contrast to immense efforts to block the signaling of cancer progression, the perturbation of TM interactions may open a new avenue to prevent cancer invasion and metastasis. Functionally, KAI1/CD82 suppresses tumor metastasis and cell migration.1Jackson P Marreiros A Russell PJ KAI1 tetraspanin and metastasis suppressor.Int J Biochem Cell Biol. 2005; 37: 530-534Crossref PubMed Scopus (49) Google Scholar, 2Tonoli H Barrett JC CD82 metastasis suppressor gene: a potential target for new therapeutics?.Trends Mol Med. 2005; 11: 563-570Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 3Liu WM Zhang XA KAI1/CD82, a tumor metastasis suppressor.Cancer Letter. 2006; 240: 183-194Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar The role of KAI1/CD82 in metastasis suppression was originally discovered in metastatic prostate cancer,4Dong J-T Lamb PW Rinker-Schaeffer CW Vukanovic J Ichikawa T Issacs JT Barrett JC KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2.Science. 1995; 268: 884-886Crossref PubMed Scopus (762) Google Scholar and then it was found that KAI1/CD82 expression also suppresses the invasion and/or metastasis of other epithelial malignancies.1Jackson P Marreiros A Russell PJ KAI1 tetraspanin and metastasis suppressor.Int J Biochem Cell Biol. 2005; 37: 530-534Crossref PubMed Scopus (49) Google Scholar, 2Tonoli H Barrett JC CD82 metastasis suppressor gene: a potential target for new therapeutics?.Trends Mol Med. 2005; 11: 563-570Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 3Liu WM Zhang XA KAI1/CD82, a tumor metastasis suppressor.Cancer Letter. 2006; 240: 183-194Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Although the mechanism remains unclear, recent studies have shown that KAI1/CD82 may inhibit cell motility by regulating the biological activities of its associated proteins and/or reorganizing plasma membrane microdomains.1Jackson P Marreiros A Russell PJ KAI1 tetraspanin and metastasis suppressor.Int J Biochem Cell Biol. 2005; 37: 530-534Crossref PubMed Scopus (49) Google Scholar, 2Tonoli H Barrett JC CD82 metastasis suppressor gene: a potential target for new therapeutics?.Trends Mol Med. 2005; 11: 563-570Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 3Liu WM Zhang XA KAI1/CD82, a tumor metastasis suppressor.Cancer Letter. 2006; 240: 183-194Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar In other words, KAI1/CD82 may suppress cancer metastasis by directly inhibiting cancer cell movement. In addition, KAI1/CD82 overexpression was reported to induce apoptosis of cancer cells5Ono M Handa K Withers DA Hakomori S Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation.Cancer Res. 1999; 59: 2335-2339PubMed Google Scholar, 6Schoenfeld N Bauer MKA Grimm S The metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates.FASEB J. 2003; 18: 158-160PubMed Google Scholar by releasing intracellular glutathione and by accumulating intracellular reactive oxygen intermediates.6Schoenfeld N Bauer MKA Grimm S The metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates.FASEB J. 2003; 18: 158-160PubMed Google Scholar Recently, KAI1/CD82 was found to bind Duffy antigen receptor for chemokine proteins expressing on endothelium, which results in the senescence of KAI1/CD82-positive cancer cells in primary lesions and the metastasis of KAI1/CD82-negative cancer cells to distant organs.7Bandyopadhyay S Zhan R Chaudhuri A Watabe M Pai SK Hirota S Hosobe S Tsukada T Miura K Takano Y Saito K Pauza ME Hayashi S Wang Y Mohinta S Mashimo T Iiizumi M Furuta E Watabe K Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression.Nat Med. 2006; 12: 933-938Crossref PubMed Scopus (188) Google Scholar Structurally, KAI1/CD82 protein is a member of the tetraspanin superfamily, members of which can be found in all eukaryotic organisms and engage in a wide spectrum of biological functions.8Boucheix C Rubinstein E Tetraspanins.Cell Mol Life Sci. 2001; 58: 1189-1205Crossref PubMed Scopus (535) Google Scholar, 9Hemler ME Tetraspanin functions and associated microdomains.Nat Rev Mol Cell Biol. 2005; 6: 801-811Crossref PubMed Scopus (994) Google Scholar Consistent with the role that a fungus tetraspanin plays in cellular invasion, many tetraspanins in humans regulate cell movement.8Boucheix C Rubinstein E Tetraspanins.Cell Mol Life Sci. 2001; 58: 1189-1205Crossref PubMed Scopus (535) Google Scholar A prominent feature of tetraspanins is that they associate with each other and with other transmembrane (TM) and intracellular signaling molecules to form a transmembrane multimolecular complex called tetraspanin web or tetraspanin-enriched microdomain (TEM).8Boucheix C Rubinstein E Tetraspanins.Cell Mol Life Sci. 2001; 58: 1189-1205Crossref PubMed Scopus (535) Google Scholar, 9Hemler ME Tetraspanin functions and associated microdomains.Nat Rev Mol Cell Biol. 2005; 6: 801-811Crossref PubMed Scopus (994) Google Scholar KAI1/CD82 associates with other tetraspanins such as CD9 and CD81 in the plasma membrane.3Liu WM Zhang XA KAI1/CD82, a tumor metastasis suppressor.Cancer Letter. 2006; 240: 183-194Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar In addition, KAI1/CD82 associates with a list of other TM proteins such as integrins, Ig superfamily proteins, and growth factor receptors, which are also the components of the tetraspanin web or TEM.3Liu WM Zhang XA KAI1/CD82, a tumor metastasis suppressor.Cancer Letter. 2006; 240: 183-194Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Approximately one-third of amino acid residues of KAI1/CD82 are embedded in the lipid bilayer and form four TM domains.10Imai T Fukudome K Takagi S Nagira M Furuse M Fukuhara N Nishimura M Hinuma Y Yoshie O C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.J Immunol. 1992; 149: 2879-2886PubMed Google Scholar, 11Gil ML Vita N Lebel-Binay S Miloux B Chalon P Kaghad M Marchiol-Fournigault C Conjeaud H Caput D Ferrara P A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line. cloning and preliminary functional studies.J Immunol. 1992; 148: 2826-2833PubMed Google Scholar, 12Nojima Y Hirose T Tachibana K Tanaka T Shi L Doshen J Freeman GJ Schlossman SF Morimoto C The 4F9 antigen is a member of the tetra spans transmembrane protein family and functions as an accessory molecule in T cell activation and adhesion.Cell Immunol. 1993; 152: 249-260Crossref PubMed Scopus (49) Google Scholar Interestingly, the TM domains of most, if not all, tetraspanins contain several conserved polar residues. For example, the first, third, and fourth TM domains of KAI1/CD82 contain respectively, a highly hydrophilic amino acid residue that is fully buried in the membrane lipid bilayer. Although the precise role of these polar residues in tetraspanin function remains unknown, recent studies suggest that the strong polar residues in CD9, CD81, and UPIb are involved in molecular packing, ie, the interactions between TM segments.13Kovalenko OV Metcalf DG DeGrado WF Hemler ME Structural organization and interactions of transmembrane domains in tetraspanin proteins.BMC Struct Biol. 2005; 5: 11Crossref PubMed Scopus (82) Google Scholar, 14Seigneuret M Complete predicted three-dimensional structure of the facilitator transmembrane proteins and hepatitis C virus receptor CD81: conserved and variable structural domains in the tetraspanin family.Biophysical J. 2006; 90: 212-227Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 15Tu L Kong XP Sun TT Kreibich G Integrity of all four transmembrane domains of the tetraspanin uroplakin Ib is required for its exit from the ER.J Cell Sci. 2006; 119: 5077-5086Crossref PubMed Scopus (26) Google Scholar This notion agrees with earlier observations made from other TM proteins that TM polar residues can mediate peptide-peptide interactions within the lipid bilayer.16Gratkowski H Lear JD DeGrado WF Polar side chains drive the association of model transmembrane peptides.Proc Natl Acad Sci USA. 2001; 98: 880-885Crossref PubMed Scopus (286) Google Scholar, 17Zhou FX Merianos HJ Brunger AT Engelman DM Polar residues drive association of polyleucine transmembrane helices.Proc Natl Acad Sci USA. 2001; 98: 2250-2255Crossref PubMed Scopus (316) Google Scholar, 18Call ME Pyrdol J Wiedmann M Wucherpfennig KW The organizing principle in the formation of the T cell receptor-CD3 complex.Cell. 2002; 111: 967-979Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 19Call ME Schnell JR Xu C Lutz RA Chou JJ Wucherpfennig KW The structure of the zetazeta transmembrane dimer reveals features essential for its assembly with the T cell receptor.Cell. 2006; 127: 355-368Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 20Cosson P Lankford SP Bonifacino JS Klausner RD Membrane protein association by potential intramembrane charge pairs.Nature. 1991; 351: 414-416Crossref PubMed Scopus (208) Google Scholar Furthermore, studies from T cell receptor demonstrated that the TM polar residues contribute to the assembly, cell surface expression, and signaling of T cell receptor,21Blumberg RS Alarcon J Sancho J McDermott FV Lopez P Breitmeyer J Terhorst C Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the alpha chain.J Biol Chem. 1990; 265: 14036-14043Abstract Full Text PDF PubMed Google Scholar, 22Alocover A Mariuzza RA Ermonval M Acuto O Lysine 271 in the transmembrane domain of the T-cell antigen receptor beta chain is necessary for its assembly with the CD3 complex but not for alpha/beta dimerization.J Biol Chem. 1990; 265: 4131-4135PubMed Google Scholar, 23Fuller-Espie S Hoffman Towler P Wiest DL Tietjen I Spain LM Transmembrane polar residues of TCR beta chain are required for signal transduction.Int Immunol. 1998; 10: 923-933Crossref PubMed Scopus (20) Google Scholar underscoring the biological significance of TM polar residues. Because the associations between KAI1/CD82 and other TM proteins in tetraspanin web or TEM may not result from the direct protein-protein interaction of either extracellular domains or intracellular domains, we hypothesized that these polar residues in KAI1/CD82's TM domains play important roles in the interactions between KAI1/CD82 and some of its associated proteins or the formation of KAI1/CD82-containing TEM. Because the associations of KAI/CD82 with the cell adhesion proteins and growth factor receptors in TEM are possibly needed for KAI1/CD82 motility-inhibitory activity, we also hypothesized that the TM polar residues of KAI1/CD82 are also functionally important. The monoclonal antibodies (mAbs) used in this study were CD82 mAbs M104,24Fukudome K Furuse M Imai T Nishimura M Takagi S Hinuma Y Yoshie O Identification of membrane antigen C33 recognized monoclonal antibodies inhibitory to HTLV-1 induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1–positive cells.J Virol. 1992; 66: 1394-1401Crossref PubMed Google Scholar TS82b25Charrin S Le Naour F Oualid M Billard M Faure G Hanash SM Boucheix C Rubinstein E The major CD9 and CD81 molecular partner. Identification and characterization of the complexes.J Biol Chem. 2001; 276: 14329-14337Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar (Diaclone SAS, Besancon, France), 4F9,12Nojima Y Hirose T Tachibana K Tanaka T Shi L Doshen J Freeman GJ Schlossman SF Morimoto C The 4F9 antigen is a member of the tetra spans transmembrane protein family and functions as an accessory molecule in T cell activation and adhesion.Cell Immunol. 1993; 152: 249-260Crossref PubMed Scopus (49) Google Scholar 6D7,12Nojima Y Hirose T Tachibana K Tanaka T Shi L Doshen J Freeman GJ Schlossman SF Morimoto C The 4F9 antigen is a member of the tetra spans transmembrane protein family and functions as an accessory molecule in T cell activation and adhesion.Cell Immunol. 1993; 152: 249-260Crossref PubMed Scopus (49) Google Scholar and 8E412Nojima Y Hirose T Tachibana K Tanaka T Shi L Doshen J Freeman GJ Schlossman SF Morimoto C The 4F9 antigen is a member of the tetra spans transmembrane protein family and functions as an accessory molecule in T cell activation and adhesion.Cell Immunol. 1993; 152: 249-260Crossref PubMed Scopus (49) Google Scholar; CD9 mAb MAB726White MM Foust JT Mauer AM Robertson JT Jennings LK Assessment of lumiaggregometry for research and clinical laboratories.Thromb Haemost. 1992; 67: 572-577PubMed Google Scholar and C9BB27Yauch RL Berditchevski F Harler MB Reichner J Hemler ME Highly stoichiometric, stable and specific association of integrin α3β1 with CD151 provides a major link to PI 4-kinase and may regulate cell migration.Mol Biol Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (266) Google Scholar; CD81 mAb M3824Fukudome K Furuse M Imai T Nishimura M Takagi S Hinuma Y Yoshie O Identification of membrane antigen C33 recognized monoclonal antibodies inhibitory to HTLV-1 induced syncytium formation: altered glycosylation of C33 antigen in HTLV-1–positive cells.J Virol. 1992; 66: 1394-1401Crossref PubMed Google Scholar; CD151 mAbs 5C1127Yauch RL Berditchevski F Harler MB Reichner J Hemler ME Highly stoichiometric, stable and specific association of integrin α3β1 with CD151 provides a major link to PI 4-kinase and may regulate cell migration.Mol Biol Cell. 1998; 9: 2751-2765Crossref PubMed Scopus (266) Google Scholar and 8C328Nishiuchi R Sanzen N Nada S Sumida Y Wada Y Okada M Takagi J Hasegawa H Sekiguchi K Potentiation of the ligand-binding activity of integrin α3β1 via association with CD151.Proc Natl Acad Sci USA. 2005; 102: 1939-1944Crossref PubMed Scopus (131) Google Scholar; integrin β1 mAb TS2/16 mAb29Hemler ME Sanchez-Madrid F Flotte TJ Krensky AM Burakoff SJ Bhan AK Springer TA Strominger JL J Immunol. 1984; 132: 3011-3018PubMed Google Scholar; and β-tubulin mAb (Sigma, St. Louis, MO). The tetraspanin mAbs were kindly provided directly or indirectly by Drs. O. Yoshie, E. Rubinstein, C. Morimoto, L. Jennings, M. Hemler, and K. Sekiguchi. A mouse IgG2b (clone MOPC 141; Sigma) was used as a negative control antibody in flow cytometry. The polyclonal antibody used in this study was integrin α3 antibody.30Zhang XA Bontrager AL Stipp CS Kraeft SK Bazzoni G Chen LB Hemler ME Phosphorylation of a conserved integrin alpha 3 QPSXXE motif regulates signaling, motility, and cytoskeletal engagement.Mol Biol Cell. 2001; 12: 351-365Crossref PubMed Scopus (53) Google Scholar The secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (Biosource International, Camarillo, CA). The extracellular matrix (ECM) proteins used in this study were human plasma fibronectin (FN; Invitrogen, San Diego, CA), mouse laminin 1 (LN; Invitrogen), and Matrigel (BD Bioscience, San Jose, CA). The prostate cancer cell line Du145 and the fibroblastoma cell line HT1080 were obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, and 100 μg/ml streptomycin. As previously described,31Zhou B Liu L Reddivari M Zhang XA The palmitoylation of metastasis suppressor KAI1/CD82 is important for its motility-inhibitory activity.Cancer Res. 2004; 64: 7455-7463Crossref PubMed Scopus (82) Google Scholar the asparagine (N), glutamine (Q), and glutamic acid (E) mutant (NQE) of KAI1/CD82, in which the N17, Q99, and E242 residues are simultaneously replaced by A residues, was generated by PCR-based site-directed mutagenesis using KAI1/CD82 wild-type cDNA as the template and constructed into pcDNA3.1(+) plasmid expression vector (Invitrogen). The introduced mutations were confirmed by DNA sequencing. The Du145 transfectant of Mock, KAI1/CD82 wild-type, and KAI1/CD82 NQE mutant were established as described previously.31Zhou B Liu L Reddivari M Zhang XA The palmitoylation of metastasis suppressor KAI1/CD82 is important for its motility-inhibitory activity.Cancer Res. 2004; 64: 7455-7463Crossref PubMed Scopus (82) Google Scholar Briefly, plasmid DNAs were transfected into Du145 cells via Lipofectamine 2000 (Invitrogen) and selected under G418 (Invitrogen) at a concentration of 1 mg/ml. Hundreds of G418-resistant clones were pooled, and the KAI1/CD82-positive cells were collected using flow cytometric cell sorting. The pooled Mock or KAI1/CD82-positive clones were the stable transfectants used in all subsequent experiments. HT1080-Mock and KAI1/CD82 stable transfectants were established in the same way as described above. Cells were harvested with 2 mmol/L EDTA/PBS, washed once with PBS, treated with DMEM supplemented with 5% goat serum at 4°C for 15 minutes, and then incubated with a primary mAb at 4°C for 1 hour. After removing unbound primary mAbs with two washes, cells were additionally incubated with FITC-conjugated goat anti-mouse IgG at 4°C for 30 minutes. The cells were analyzed on a FACSCalibur flow cytometer (BD Bioscience). As described in earlier studies,30Zhang XA Bontrager AL Stipp CS Kraeft SK Bazzoni G Chen LB Hemler ME Phosphorylation of a conserved integrin alpha 3 QPSXXE motif regulates signaling, motility, and cytoskeletal engagement.Mol Biol Cell. 2001; 12: 351-365Crossref PubMed Scopus (53) Google Scholar, 31Zhou B Liu L Reddivari M Zhang XA The palmitoylation of metastasis suppressor KAI1/CD82 is important for its motility-inhibitory activity.Cancer Res. 2004; 64: 7455-7463Crossref PubMed Scopus (82) Google Scholar circular glass coverslips (Fisher, Pittsburgh, PA) were coated with the ECM proteins fibronectin (10 μg/ml) or laminin 1 (10 μg/ml) at 4°C overnight and then blocked with 0.1% heat-inactivated bovine serum albumin at 37°C for 45 minutes. Cells were harvested in 2 mmol/L EDTA/PBS, washed once in PBS, and plated on the ECM-coated coverslips in serum-free DMEM at 37°C overnight. Cells were then fixed with 3% paraformaldehyde at room temperature for 15 minutes. If necessary, cells were permeabilized with 0.1% Brij 98 in PBS at room temperature for 2 minutes. Nonspecific binding sites were blocked with 20% goat serum in PBS at room temperature for 1 hour. Primary mAbs were diluted at a final concentration of ∼1 μg/ml in 20% goat serum/PBS and incubated with cells at room temperature for 1 hour followed by three washes with PBS. Cells were then labeled with FITC-conjugated goat anti-mouse IgG at room temperature for 1 hour, followed by four washes with PBS. Finally, the coverslips were mounted on glass slides in FluroSave reagent (Calbiochem, Carlsbad, CA). Digital images were captured using a Zeiss Axiophot fluorescent microscope at a magnification of ×63. For metabolic labeling with S35, Du145 transfectants were incubated with 40 μCi/ml S35Schweinitz A Steinmetzer T Banke IJ Arlt MJ Stürzebecher A Schuster O Geissler A Giersiefen H Zeslawska E Jacob U Krüger A Stürzebecher J Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents.J Biol Chem. 2004; 279: 33613-33622Crossref PubMed Scopus (128) Google Scholarl-methionine (Perkin-Elmer, Boston, MA) in methionine- and cysteine-free DMEM containing 10% dialyzed FCS at 37°C overnight. Cells were lysed in lysis buffer containing 1% Brij 97 (Sigma), 25 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L MgCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mmol/L sodium orthovanadate, and 2 mmol/L sodium fluoride. Insoluble material was removed by centrifugation at 14, 000 × g for 15 minutes, and the lysates were precleared by incubating with protein A- and G-Sepharose beads (Amersham, Uppsala, Sweden) at 4°C for 6 hours. Then the mAb-preabsorbed protein A- and G-Sepharose beads were incubated with cell lysate at 4°C overnight. Immune complexes were collected by centrifugation followed by four washes in lysis buffer. Immune complexes were eluted from the beads with Laemmli sample buffer and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) under a nonreducing condition. The gels were treated with autoradiography enhancement reagent (Perkin-Elmer), dried, and exposed to film (Kodak, Rochester, NY) at −80°C for 5 to 7 days. For cell surface biotinylation, Du145 transfectants were labeled with 100 μg/ml EZlink sulfo-NHS-LC biotin (Pierce, Rockford, IL) in PBS at room temperature for 1 hour followed by three washes with PBS. The biotinylated cells were lysed in 1% Brij 97 lysis buffer, and cell lysates were immunoprecipitated as described above. Immunoprecipitated proteins resolved by SDS-PAGE were transferred to nitrocellulose membrane (Schleicher & Schuell, Keene, NH) and incubated with horseradish peroxidase-conjugated extravidin (Sigma). Blots were visualized by chemiluminescence (Perkin-Elmer). Immunoprecipitation and immunoblotting were performed as described.32Zhang XA He B Zhou B Liu L Requirement of p130CAS-Crk coupling in KAI1/CD82-mediated suppression of cell migration.J Biol Chem. 2003; 278: 27319-27328Crossref PubMed Scopus (79) Google Scholar Briefly, cells were lysed with 1% NP40 or 1% Brij97 lysis buffer. Lysates were immunoprecipitated as described above, and the precipitates were then separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes and sequentially blotted with a primary antibody and a horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Sigma) secondary antibody followed by chemiluminescence detection. For Western blotting, lysates were directly separated by SDS-PAGE followed by the blotting procedures as indicated above. CD82 dimerization was assessed as described.33Kovalenko OV Yang X Kolesnikova TV Hemler ME Evidence for specific tetraspanin homodimers: inhibition of palmitoylation makes cysteine residues available for cross-linking.Biochem J. 2004; 377: 407-417Crossref PubMed Scopus (109) Google Scholar Briefly, the amino-specific cross-linker dithio-bis-succinimidylproprionate (DSP; Pierce, Rockford, IL) was dissolved in DMSO at 10 mg/ml immediately before use. DSP was added to the cell lysate prepared from 1% Brij97 lysis buffer (25 mmol/L Hepes, pH 7.4, 150 mmol/L NaCl, 2 mmol/L MgCl2, and aforementioned protease inhibitors) to a final concentration of 0.25 mg/ml. DSP was incubated with the cell lysate at 4°C for 1 hour, and 1% NP-40 in 20 mmol/L Tris-HCl was added to cell lysate for further incubation for 15 minutes. CD82 was immunoprecipitated and then immunoblotted with its mAb. Migration assays were performed in Transwell filter inserts in 24-well tissue culture plates (BD Bioscience) as described.31Zhou B Liu L Reddivari M Zhang XA The palmitoylation of metastasis suppressor KAI1/CD82 is important for its motility-inhibitory activity.Cancer Res. 2004; 64: 7455-7463Crossref PubMed Scopus (82) Google Scholar The polycarbonate membrane filters of Transwell contain pores 8 μm in diameter. Filters were spotted with FN (10 μg/ml) or LN-1 (10 μg/ml) on the lower surface of the Transwell inserts at 4°C overnight and then blocked with 0.1% heat-inactivated bovine serum albumin at 37°C for 30 minutes. Cells were detached at 90% confluence, washed once in PBS, and resuspended in serum-free DMEM containing 0.1% heat-inactivated bovine serum albumin. A 300-μl of cell suspension was added to inserts at a density of 3 × 104 cells/insert. DMEM containing 1% FCS was added to the lower wells. Migration was allowed to proceed at 37°C for 3 hours. Cells that did not migrate through the filters were removed using cotton swabs, and cells that migrated through the inserts were fixed and stained with Diff-Quick (Baxter, McGraw Park, IL). The number of migrated cells per microscopic field (at original magnification ×40) was counted visually under a light microscope. Data from five independent experiments were pooled and analyzed using a two-tailed, Student's t-test. Invasion was measured using Biocoat Matrigel invasion chambers (BD Biosciences) by following the manufacturer's protocol. Briefly, cells at the 80% to 90% confluent stage were detached. DMEM containing 10% FCS and 10 nmol/L stromal cell-derived factor 1 (R&D, Minneapolis, MN) was placed in the lower well, and 2.5 × 103 cells in 500 μl of serum-free medium were loaded to the upper chamber of the Matrigel-coated insert and incubated at 37°C for 22 hours. Cells that invaded to the lower surface of the filter were stained with Diff-Quick (American Scientific Products, McGraw Park, IL) and quantified with light microscope at original magnification ×100. The data were expressed as the average number of cells from five randomly selected fields and analyzed statistically using a two-tailed, Student's t-test. The experiments were repeated three times. The metastatic potentials of HT1080 transfectants were evaluated in mice using experimental lung metastasis assay as described.34Fuse C Ishida Y Hikita T Asai T Oku N Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma.J Biol Chem. 2007; 282: 8276-8283Crossref PubMed Scopus (39) Google Scholar, 35Schweinitz A Steinmetzer T Banke IJ Arlt MJ Stürzebecher A Schuster O Geissler A Giersiefen H Zeslawska E Jacob U Krüger A Stürzebecher J Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents.J Biol Chem. 2004; 279: 33613-33622Crossref PubMed Scopus (128) Google Scholar A total of 30 BALB/c nu/nu female mice (8-week old, Harlan, Indianapolis, IN) were included in the assay with 10 mice for each group/transfectant. HT1080 transfectant cells (1 × 106 cells in 0.1 ml PBS) were injected into each nude mouse through tail vein. The mice were sacrificed 3 weeks after the injection. Lungs were dissected, fixed in formalin solution, and photographed. The macrometastatic foci (>0.3 mm) on the lung surface were counted under the dissect microscope. Statistical analysis (Student's t-test) was performed with StatView software (SAS Institute Inc., Cary, NC), and the results were expressed as mean ± SD. The pSIREN-RetroQ retrovira" @default.
• W2130170015 created "2016-06-24" @default.
• W2130170015 creator A5017081696 @default.
• W2130170015 creator A5026830772 @default.
• W2130170015 creator A5052274720 @default.
• W2130170015 creator A5057141905 @default.
• W2130170015 creator A5067010771 @default.
• W2130170015 creator A5071096317 @default.
• W2130170015 creator A5083419129 @default.
• W2130170015 date "2009-02-01" @default.
• W2130170015 modified "2023-09-27" @default.
• W2130170015 title "Transmembrane Interactions Are Needed for KAI1/CD82-Mediated Suppression of Cancer Invasion and Metastasis" @default.
• W2130170015 cites W1491590651 @default.
• W2130170015 cites W1515952044 @default.
• W2130170015 cites W1547463001 @default.
• W2130170015 cites W1552764442 @default.
• W2130170015 cites W1561145742 @default.
• W2130170015 cites W1586743471 @default.
• W2130170015 cites W1970858060 @default.
• W2130170015 cites W1973927289 @default.
• W2130170015 cites W1979738242 @default.
• W2130170015 cites W198154431 @default.
• W2130170015 cites W1985831769 @default.
• W2130170015 cites W1987490683 @default.
• W2130170015 cites W1988791617 @default.
• W2130170015 cites W1991803711 @default.
• W2130170015 cites W1992837561 @default.
• W2130170015 cites W2003549561 @default.
• W2130170015 cites W2003783113 @default.
• W2130170015 cites W2007610219 @default.
• W2130170015 cites W2008442497 @default.
• W2130170015 cites W2011189175 @default.
• W2130170015 cites W2017767253 @default.
• W2130170015 cites W2019574809 @default.
• W2130170015 cites W2027483071 @default.
• W2130170015 cites W2028652903 @default.
• W2130170015 cites W2031644342 @default.
• W2130170015 cites W2048481017 @default.
• W2130170015 cites W2055584330 @default.
• W2130170015 cites W2059210858 @default.
• W2130170015 cites W2060572354 @default.
• W2130170015 cites W2060809301 @default.
• W2130170015 cites W2063186096 @default.
• W2130170015 cites W2066036926 @default.
• W2130170015 cites W2072796784 @default.
• W2130170015 cites W2078162414 @default.
• W2130170015 cites W2093137068 @default.
• W2130170015 cites W2116331760 @default.
• W2130170015 cites W2116349394 @default.
• W2130170015 cites W2118278603 @default.
• W2130170015 cites W2122199548 @default.
• W2130170015 cites W2125168459 @default.
• W2130170015 cites W2131553403 @default.
• W2130170015 cites W2145345239 @default.
• W2130170015 cites W2152313975 @default.
• W2130170015 cites W2153783648 @default.
• W2130170015 cites W2154859242 @default.
• W2130170015 cites W2161799300 @default.
• W2130170015 cites W2164497783 @default.
• W2130170015 cites W2165176916 @default.
• W2130170015 cites W2166518329 @default.
• W2130170015 cites W2171893470 @default.
• W2130170015 cites W4231644493 @default.
• W2130170015 doi "https://doi.org/10.2353/ajpath.2009.080685" @default.
• W2130170015 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2630572" @default.
• W2130170015 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19116362" @default.
• W2130170015 hasPublicationYear "2009" @default.
• W2130170015 type Work @default.
• W2130170015 sameAs 2130170015 @default.
• W2130170015 citedByCount "43" @default.
• W2130170015 countsByYear W21301700152012 @default.
• W2130170015 countsByYear W21301700152013 @default.
• W2130170015 countsByYear W21301700152014 @default.
• W2130170015 countsByYear W21301700152015 @default.
• W2130170015 countsByYear W21301700152016 @default.
• W2130170015 countsByYear W21301700152017 @default.
• W2130170015 countsByYear W21301700152018 @default.
• W2130170015 countsByYear W21301700152019 @default.
• W2130170015 countsByYear W21301700152020 @default.
• W2130170015 countsByYear W21301700152021 @default.
• W2130170015 countsByYear W21301700152022 @default.
• W2130170015 crossrefType "journal-article" @default.
• W2130170015 hasAuthorship W2130170015A5017081696 @default.
• W2130170015 hasAuthorship W2130170015A5026830772 @default.
• W2130170015 hasAuthorship W2130170015A5052274720 @default.
• W2130170015 hasAuthorship W2130170015A5057141905 @default.
• W2130170015 hasAuthorship W2130170015A5067010771 @default.
• W2130170015 hasAuthorship W2130170015A5071096317 @default.
• W2130170015 hasAuthorship W2130170015A5083419129 @default.
• W2130170015 hasBestOaLocation W21301700151 @default.
• W2130170015 hasConcept C121608353 @default.
• W2130170015 hasConcept C142724271 @default.
• W2130170015 hasConcept C170493617 @default.
• W2130170015 hasConcept C24530287 @default.
• W2130170015 hasConcept C2779013556 @default.
• W2130170015 hasConcept C2992691968 @default.
• W2130170015 hasConcept C502942594 @default.
• W2130170015 hasConcept C54355233 @default.

• next