The MIG-2/Integrin Interaction Strengthens Cell-Matrix Adhesion and Modulates Cell Motility
2007; Elsevier BV; Volume: 282; Issue: 28 Linguagem: Inglês
10.1074/jbc.m611680200
ISSN1083-351X
AutoresXiaohua Shi, Yan-Qing Ma, Yizeng Tu, Ka Chen, Shan Wu, Koichi Fukuda, Jun Qin, Edward F. Plow, Chuanyue Wu,
Tópico(s)Galectins and Cancer Biology
ResumoIntegrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with β1 and β3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using αIIbβ3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells. Integrin-mediated cell-matrix adhesion plays an important role in control of cell behavior. We report here that MIG-2, a widely expressed focal adhesion protein, interacts with β1 and β3 integrin cytoplasmic domains. Integrin binding is mediated by a single site within the MIG-2 FERM domain. Functionally, the MIG-2/integrin interaction recruits MIG-2 to focal adhesions. Furthermore, using αIIbβ3 integrin-expressing Chinese hamster ovary cells, a well described model system for integrin activation, we show that MIG-2 promotes integrin activation and enhances cell-extracellular matrix adhesion. Although MIG-2 is expressed in many cell types, it is deficient in certain colon cancer cells. Expression of MIG-2, but not of an integrin binding-defective MIG-2 mutant, in MIG-2-null colon cancer cells strengthened cell-matrix adhesion, promoted focal adhesion formation, and reduced cell motility. These results suggest that the MIG-2/integrin interaction is an important element in the cellular control of integrin-mediated cell-matrix adhesion and that loss of this interaction likely contributes to high motility of colon cancer cells. Cell-extracellular matrix (ECM) 3The abbreviations used are: ECM, extracellular matrix; FA, focal adhesion; mAb, monoclonal antibody; Ab, antibody; GST, glutathione S-transferase; GFP, green fluorescent protein; CHO, Chinese hamster ovary; talin-H, talin head domain; BSA, bovine serum albumin; MFI, mean fluorescence intensity(ies). adhesion is a fundamental process that is mediated by transmembrane receptors such as integrins (1Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. 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A. 2004; 101: 4094-4099Crossref PubMed Scopus (112) Google Scholar, 13Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (646) Google Scholar). Integrin extracellular ligand-binding affinity plays an important role in control of initial cell-ECM adhesion. Additionally, integrin-mediated cell-ECM adhesion can be enhanced through interactions with cytoskeletal proteins, a process that has been termed cytoskeletal strengthening (14Lotz M.M. Burdsal C.A. Erickson H.P. McClay D.R. J. Cell Biol. 1989; 109: 1795-1805Crossref PubMed Scopus (341) Google Scholar, 15Yamada K.M. Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. Vol. 1. John Wiley & Sons, Inc., New York2003: 9.0.1-9.0.9Google Scholar, 16McClay D.R. Hertzler P.L. Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. Vol. 1. John Wiley & Sons, Inc., New York2003: 9.2.1-9.2.10Google Scholar). The physical basis underlying the cytoskeletal strengthening of cell-ECM adhesion has been well described (16McClay D.R. Hertzler P.L. Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. Vol. 1. John Wiley & Sons, Inc., New York2003: 9.2.1-9.2.10Google Scholar). However, the molecular interactions that mediate this process remain to be defined. MIG-2 (mitogen-inducible gene-2, also known as kindlin-2) is a widely expressed and evolutionarily conserved cytoplasmic protein (17Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (165) Google Scholar, 18Schaller M.D. J. Cell Biol. 2000; 150: F9-F11Crossref PubMed Google Scholar, 19Mackinnon A.C. Qadota H. Norman K.R. Moerman D.G. Williams B.D. Curr. Biol. 2002; 12: 787-797Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 21Wu C. J. Cell Sci. 2005; 118: 659-664Crossref PubMed Scopus (62) Google Scholar). Genetic studies have shown that Caenorhabditis elegans UNC-112, a homolog of MIG-2, is required for attachment of body-wall muscle cells to the hypodermis (17Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (165) Google Scholar, 19Mackinnon A.C. Qadota H. Norman K.R. Moerman D.G. Williams B.D. Curr. Biol. 2002; 12: 787-797Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Loss of UNC-112 in C. elegans results in an embryonic lethal Pat (paralyzed, arrested elongation at two-fold) phenotype resembling that of α or β integrin loss (17Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (165) Google Scholar, 19Mackinnon A.C. Qadota H. Norman K.R. Moerman D.G. Williams B.D. Curr. Biol. 2002; 12: 787-797Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). In mammalian organisms, MIG-2 has been detected in many cell types, including fibroblasts, muscle cells, endothelial cells, and epithelial cells (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 22Gkretsi V. Zhang Y. Tu Y. Chen K. Stolz D.B. Yang Y. Watkins S.C. Wu C. J. Cell Sci. 2005; 118: 697-710Crossref PubMed Scopus (40) Google Scholar). In these cells, it concentrates at FAs. MIG-2 interacts with migfilin (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), a filamin- and VASP (vasodilator-stimulated phosphoprotein)-binding protein (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 21Wu C. J. Cell Sci. 2005; 118: 659-664Crossref PubMed Scopus (62) Google Scholar, 23Zhang Y. Tu Y. Gkretsi V. Wu C. J. Biol. Chem. 2006; 281: 12397-12407Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Through this interaction, it recruits migfilin to FAs and provides a link from FAs to the actin cytoskeleton (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Although MIG-2 is crucial for recruiting migfilin to FAs, how MIG-2 is recruited to FAs was not known. Structurally, MIG-2 contains an N-terminal region that exhibits no obvious structural motif and a C-terminal FERM domain. Notably, the MIG-2 FERM domain contains a region that shares considerable sequence similarity with the integrin-binding site of the talin FERM domain. Furthermore, kindlin, a protein that shares significant (62%) sequence identity with MIG-2, interacts with β1 and β3 integrin cytoplasmic domains (24Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). However, the functions of the kindlin/integrin interaction remain unknown. Cell-ECM adhesion is intimately involved in the regulation of cell behavior such as cell motility. Theoretical consideration and experimental studies have shown that the relationship between cell-ECM adhesion and motility is biphasic (25Huttenlocher A. Sandborg R.R. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (450) Google Scholar, 26Lauffenburger D.A. Horwitz A.F. Cell. 1996; 84: 359-369Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar). Thus, the regulation of cell-ECM adhesion strength is important in the control of cell motility. Consistent with this, alterations of proteins that are pertinent to the control of cell-ECM adhesion are frequently associated with human diseases such as cancer. Determining the molecular basis underlying the regulation of cell-ECM adhesion and migration is therefore of not only general biological importance but also considerable clinical significance. In this work, we show that MIG-2 interacts with β1 and β3 integrin cytoplasmic domains and functions as an important regulator of integrin activation, cell-ECM adhesion, and migration. Cells, Antibodies, and Other Reagents—Human SK-LMS-1 cells and HT-1080 cells were from American Type Culture Collection. RKO, HT-29, DLD-1, LoVo, and HCT-116 colon cancer cells were from Dr. Lin Zhang (University of Pittsburgh Cancer Institute). Caco-2 colon cancer cells were from Dr. Craig C. Garner (Stanford University). The SK-LMS-1, HT-1080, and RKO cells were cultured in minimum Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, 0.1 mm nonessential amino acids, and 1.0 mm sodium pyruvate. HT-29 and DLD-1 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 2 mm l-glutamine. Caco-2 cells were cultured in Dulbecco's modified Eagle's medium with 20% fetal bovine serum and 2 mm l-glutamine. LoVo and Caco-2 cells were cultured in Ham's F-12 and McCoy's 5A medium supplemented with 10% fetal bovine serum and 2 mm l-glutamine. Mouse anti-MIG-2 monoclonal antibody (mAb) 3A3 has been described (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Mouse anti-kindlin mAb (clone 4A5) was generated using glutathione S-transferase (GST) fusion protein containing human kindlin residues 216–677 as an antigen following previously described protocols (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Function-blocking mouse anti-α2 integrin (clone P1E6) and anti-β1 integrin (clone 6S6) monoclonal antibodies were purchased from Chemicon (Temecula, CA). Mouse anti-vinculin mAb was from Sigma. Rhodamine Red™-conjugated goat anti-mouse IgG antibody (Ab) and horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Cell culture media were from Sigma or Invitrogen. All other chemicals were from Fisher or Sigma. Mutagenesis, DNA Constructs, and Transfection—The vector encoding green fluorescent protein (GFP)-tagged MIG-2 has been described (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Point mutations were introduced into MIG-2 coding sequence as specified in each experiment using a QuikChange™ site-directed mutagenesis system (Stratagene). DNA fragments encoding MIG-2 deletion mutants were generated by PCR. DNA fragments encoding MIG-2 mutants were inserted into the pEGFP-C2 vector (Clontech). All mutations were confirmed by DNA sequencing. Cells were transfected with vectors encoding GFP-tagged wild-type or mutant MIG-2 or GFP alone as a control using Lipofectamine 2000 (Invitrogen). GST Fusion Protein Pulldown Assays—The vectors encoding GST fusion proteins containing the β1A integrin cytoplasmic domain (residues 775–786), the β3 integrin cytoplasmic domain (residues 716–762), or a mutant form of the β3 integrin cytoplasmic domain in which Tyr747 was substituted with Ala were generated as described previously (27Li J. Mayne R. Wu C. J. Cell Biol. 1999; 147: 1391-1397Crossref PubMed Scopus (52) Google Scholar, 28Ma Y.-Q. Yang J. Pesho M.M. Vinogradova O. Qin J. Plow E.F. Biochemistry. 2006; 45: 6656-6662Crossref PubMed Scopus (53) Google Scholar). The vectors were used to transform Escherichia coli DH5α cells. Expression of GST and GST fusion proteins was induced with isopropyl β-d-thiogalactopyranoside. The bacterial cells were lysed with 150 mm NaCl, 10 mm Tris (pH 8.0), and 1 mm EDTA (STE buffer) containing 100 μg/ml lysozyme for 15 min (on ice), followed by sonication in STE buffer containing 5 mm dithiothreitol, 1.5% Sarkosyl, and protease inhibitors. After the cell debris was removed by centrifugation, the lysates were incubated with glutathione-Sepharose beads (Pierce) at 4 °C for 1 h. The glutathione-Sepharose beads were precipitated by centrifugation, washed three times with STE buffer containing 0.1% Triton X-100, and used for GST pulldown assays as we described previously (20Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Brief, Chinese hamster ovary (CHO) cells, SK-LMS-1 cells, or SK-LMS-1 cells transfected with vectors encoding GFP or GFP-tagged wild-type or mutant MIG-2 were lysed with 1% Triton X-100 in 20 mm Tris (pH 7.1) containing 150 mm NaCl, 10 mm Na4P2O7, 2 mm Na3VO4, 100 mm NaF, 10 mm EDTA, and protease inhibitors (lysis buffer). The lysates were incubated with glutathione-Sepharose beads containing GST or GST-integrin cytoplasmic domain fusion proteins for 4 h or longer at 4 °C. The glutathione-Sepharose beads were precipitated by centrifugation, washed four times with lysis buffer, and then analyzed by Western blotting and Coomassie Blue staining as specified in each experiment. Integrin Activation—The effect of MIG-2 on integrin activation was analyzed using CHO cells stably expressing αIIbβ3 integrin and activation-specific anti-αIIbβ3 integrin mAb PAC1 as described (28Ma Y.-Q. Yang J. Pesho M.M. Vinogradova O. Qin J. Plow E.F. Biochemistry. 2006; 45: 6656-6662Crossref PubMed Scopus (53) Google Scholar). Briefly, CHO cells expressing αIIbβ3 integrin were transfected with GFP-tagged MIG-2, the GFP-tagged talin head domain (talin-H; residues 1–429), or GFP. Twenty-four hours after transfection, the cells were harvested; suspended in Hanks' balanced salt solution/bovine serum albumin (BSA); and stained with mAb PAC1 (20 μg/ml) for 30 min at 22 °C, followed by incubation with Alexa Fluor® 633-conjugated goat anti-mouse IgM Ab for 30 min on ice. After washing, the cells were fixed and analyzed using a FACSCalibur flow cytometer. mAb PAC1 binding was analyzed only on a gated subset of cells positive for GFP expression (i.e. GFP- or GFP fusion protein-expressing cells). The mean fluorescence intensity (MFI; generated with CellQuest software) of mAb PAC1 bound to the GFP-MIG-2- or GFP-talin-H-expressing cells was divided by the MFI of mAb PAC1 bound to the control GFP-expressing cells in the same experiment to obtain a relative MFI value. Seven independent experiments were performed, and the relative MFI in each experimental set, GFP-MIG-2 or GFP-talin-H, were compared with the MFI of the GFP control by a paired t test to determine statistical significance. p values <0.05 were considered to be statistically significant. Adenoviral Vectors and Infection—Adenoviral vectors encoding wild-type or mutant MIG-2 were generated using the AdEasy system following a previously described protocol (29Zhang Y. Guo L. Chen K. Wu C. J. Biol. Chem. 2002; 277: 318-326Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 30Guo L. Wu C. FASEB J. 2002; 16: 1298-1300Crossref PubMed Scopus (68) Google Scholar). Briefly, MIG-2 coding sequences were cloned into the NotI/XbaI sites of the pAdTrack-CMV shuttle vector. The resultant plasmids were linearized with PmeI, purified, and mixed with supercoiled pAdEasy-1. The vectors were transferred into E. coli BJ5183 by electroporation using a Bio-Rad Gene Pulser electroporator. Recombinants that were resistant to kanamycin were selected, and recombination was confirmed by PacI digestion. The positive plasmids were then transformed into DH5α by heat shock for large-scale amplification. The plasmid DNAs were digested with PacI, ethanol-precipitated, and used to transfect 293 cells with Lipofectamine PLUS (Invitrogen). The transfectants were harvested ∼10 days after transfection. The cells were lysed by three cycles of freezing in a methanol/dry ice bath and rapid thawing at 37 °C, and the lysates containing the recombinant adenovirus were collected. The control adenoviral expression vector encoding β-galactosidase was kindly provided by Drs. Tong-Chuan He and Bert Vogelstein (Howard Hughes Medical Institute, Johns Hopkins Oncology Center, Baltimore, MD). Adenoviral vectors were purified by CsCl gradient centrifugation as described (31Kolls J. Peppel K. Silva M. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 215-219Crossref PubMed Scopus (212) Google Scholar) and then used to infect cells. The infection efficiency was monitored by the expression of GFP encoded by the viral vectors. The percentage of GFP-positive cells typically reached 80–90% within 1 day after infection. Overexpression of wild-type or mutant MIG-2 in the infected cells was confirmed by Western blotting. FA Localization of GFP-tagged MIG-2—SK-LMS-1 cells were transfected with GFP-tagged wild-type or mutant MIG-2. One day after transfection, the cells were trypsinized, replated on fibronectin-coated coverslips, and cultured for 24 h. The cells were fixed with 4% paraformaldehyde, stained with anti-vinculin mAb and Rhodamine Red™-conjugated anti-mouse IgG Ab, and observed under a Leica DMR fluorescence microscope equipped with GFP and rhodamine filters. Cell-ECM Adhesion—Cell-ECM adhesion was assessed by centrifugation assays either at 4 °C (to measure initial cell-ECM adhesion, which is controlled primarily by integrin ligand-binding activity) or after incubation of the cells at 37 °C (to allow cytoskeletal strengthening) as described previously (16McClay D.R. Hertzler P.L. Bonifacino J.S. Dasso M. Harford J.B. Lippincott-Schwartz J. Yamada K.M. Current Protocols in Cell Biology. Vol. 1. John Wiley & Sons, Inc., New York2003: 9.2.1-9.2.10Google Scholar). For CHO cells, the cells were transfected with expression vectors encoding GFP-MIG-2, GFP-talin-H (as a positive control), or GFP (as a negative control) using Lipofectamine PLUS. Thirty-six hours after DNA transfection, the transfectants (1 × 105 cells/well) were seeded in fibrinogen-coated 96-well plates (Greiner Bio-One). GFP-positive cells were quantified by measuring fluorescence at excitation and emission wavelengths of 485 and 535 nm, respectively, with a GENios Pro fluorescence microplate reader (Tecan). The plates were tightly sealed with sealing films (USA Scientific, Inc.) and centrifuged with a Sorvall RT7 Plus centrifuge equipped with a microplate carrier at 600 rpm for 3 min at 4 °C to facilitate cell settlement. The plates were then inverted and centrifuged at 1000 rpm for 3 min at 4 °C. The transfectants that remained adhered were quantified by measuring fluorescence at excitation and emission wavelengths of 485 and 535 nm, respectively. Cell adhesion was calculated as the fluorescence reading at excitation and emission wavelengths of 485 and 535 nm, respectively, after inverted centrifugation divided by the fluorescence reading at excitation and emission wavelengths of 485 and 535 nm, respectively, before inverted centrifugation. The adhesion of the cells expressing wild-type or mutant MIG-2 was compared with that of the control GFP cells (normalized to 1). Student's t test was used for statistical analyses. p values <0.05 were considered statistically significant. For SK-LMS-1 cells, the cells were infected with the control β-galactosidase adenovirus or adenoviruses encoding wild-type MIG-2 or mutants. One day after infection, the cells (5 × 104/well) were seeded in collagen I-coated 96-well plates (Greiner Bio-One). The GFP-positive adenovirus-infected cells were quantified by measuring fluorescence at excitation and emission wavelengths of 485 and 535 nm, respectively, with the GENios Pro fluorescence microplate reader. The plates were tightly sealed and centrifuged with the Sorvall RT7 Plus centrifuge equipped with a microplate carrier at 600 rpm for 5 min at 4 °C to facilitate cell settlement. To analyze initial cell adhesion, the plates were inverted and centrifuged at 600 rpm for 1 min at 4 °C. To analyze cytoskeletal strengthened cell adhesion, the plates were incubated at 37 °C for 60 min and then inverted and centrifuged at 600 rpm for 12 min at 37 °C. The adenovirus-infected cells that remained adhered were quantified by measuring fluorescence at excitation and emission wavelengths of 485 and 535 nm, respectively. Cell adhesion was calculated as described above. The adhesion of the cells expressing wild-type or mutant MIG-2 was compared with that of the control β-galactosidase cells (normalized to 1). Student's t test was used for statistical analyses. p values <0.05 were considered statistically significant. For RKO cells, the cells were infected with β-galactosidase adenovirus or adenoviruses encoding wild-type or mutant MIG-2. Two days after the infection, the cells (1 × 105/well) were seeded in collagen I-coated 96-well plates, and the GFP-positive adenovirus-infected cells were quantified as described above. The plates were sealed and centrifuged with a Sorvall RTH750 centrifuge at 600 rpm for 5 min at 4 °C. To analyze initial cell adhesion, the plates were inverted and centrifuged at 600 rpm for 1 min at 4 °C. To analyze cytoskeletal strengthened cell adhesion, the plates were incubated at 37 °C for 10 min and then inverted and centrifuged at 600 rpm for 3 min at 37 °C. The amount of the adenovirus-infected cells that remained adhered was quantified by measuring fluorescence at excitation and emission wavelengths of 485 and 535 nm, respectively. Cell adhesion and statistics were calculated as described above. In some experiments, SK-LMS-1 and RKO cells were preincubated with function-blocking mouse anti-β1 integrin mAb (clone 6S6; 6.7 μg/ml IgG), function-blocking mouse anti-α2 integrin mAb (clone P1E6; 6.7 μg/ml IgG), or control mouse IgG Ab (6.7 μg/ml) at 37 °C for 15 min. Integrin-mediated initial cell adhesion was then analyzed as described above. To assess FA formation, SK-LMS-1 or RKO cells infected with the control β-galactosidase adenovirus or adenoviruses encoding wild-type or mutant MIG-2 were plated on coverslips and cultured as specified in each experiment. The cells were fixed with 4% paraformaldehyde, stained with mouse anti-vinculin mAb and Rhodamine Red™-conjugated anti-mouse IgG Ab, and observed under the Leica DMR fluorescence microscope. Cell Migration—Cell migration was analyzed using Transwell cell motility chambers as described (32Bauer J.S. Schreiner C.L. Giancotti F.G. Ruoslahti E. Juliano R.L. J. Cell Biol. 1992; 116: 477-487Crossref PubMed Scopus (116) Google Scholar, 33Fukuda T. Chen K. Shi X. Wu C. J. Biol. Chem. 2003; 278: 51324-51333Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Briefly, the undersurfaces of the 8-mm pore diameter cell motility chambers (BD Biosciences) were coated with 10 μg/ml fibronectin. The cells were trypsinized 24 h after adenoviral infection and washed twice with phosphate-buffered saline containing 1 mg/ml BSA. The cells were suspended in 0.2 ml Dulbecco's modified Eagle's medium containing 1 mg/ml BSA and added to the upper chambers. After incubation at 37 °C in a 5% CO2 and 95% air atmosphere for the periods of time specified in each experiment, the cells on the upper surface of the membrane were removed. The membranes were fixed, and the cells on the undersurface were stained with Gill's hematoxylin III. The cells from five randomly selected microscopic fields were counted. The number of MIG-2- or Q614A/W615A mutant-overexpressing cells that migrated through the membrane pores was compared with the number of control β-galactosidase cells (normalized to 100%). Molecular Modeling—The primary sequences of MIG-2 and talin were aligned using the ClustalW program, which predicted a considerable MIG-2 FERM F3 sequence. A homology model of the MIG-2 FERM F3-free form was constructed using the SWISS-MODEL modeling server (34Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4537) Google Scholar), which adapted the atomic coordinates of the talin FERM domain (Protein Data Bank code 1Y19) as a template. The β1A integrin peptide model was generated using the coordinates of β3 integrin bound to talin F3 (Protein Data Bank code 1MK9), the side chains of which were replaced with the β1A integrin sequences, and then refined using the program CNS (35Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The docking model of the complex between MIG-2 F3 and β1A or β3 integrin was constructed according to the atomic coordinates of the talin F3/β3 integrin interaction observed in the crystallographic asymmetric unit. Model inspection and analysis of the MIG-2 F3 subdomain·β1A integrin complex were performed using the TURBO-FRODO program (36Roussel A. Cambileau C. TURBO-FRODO in Silicon Graphics Geometry. Silicon Graphics, Mountain View, California1991Google Scholar). The figure was generated using the PyMOL program (37DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, San Carlos, CA2002Google Scholar). MIG-2 Interacts with β1 and β3 Integrin Cytoplasmic Domains—To test whether MIG-2 interacts with the β1 integrin cytoplasmic domain, we incubated lysates of MIG-2-expressing mammalian cells with GST or GST fusion protein containing the β1A integrin cytoplasmic domain (residues 775–786; referred to as GST-β1). GST, GST-β1 fusion protein, and associated proteins were precipitated with glutathione-Sepharose beads. Western blot analyses showed that MIG-2 was readily coprecipitated with GST-β1 (Fig. 1A, lane 3) but not with GST (lane 2). The presence of GST and GST-β1 in the precipitates was confirmed by staining the membrane with Coomassie Blue (Fig. 1B). In additional control experiments, no protein bands in the GST-β1 fusion protein preparation were recognized by anti-MIG-2 Ab in the absence of cell lysates (Fig. 1A, lane 4), confirming the specificity of the Western blotting. The β3 integrin tail shares considerable sequence similarity with the β1 integrin tail. To test whether MIG-2 recognizes the β3 integrin tail, we incubated the cell lysates with GST fusion protein containing the β3 integrin tail (residues 716–762; referred to as GST-β3) and analyzed MIG-2 binding by the GST pulldown assay. The results show that MIG-2 was readily coprecipitated with GST-β3 (Fig. 1, C and D, lanes 3) but not with GST (lanes 2). In control experiments, no protein bands in the GST-β3 fusion protein preparation were recognized by anti-MIG-2 Ab in the absence of cell lysates (Fig. 1, C and D, lanes 4). These results suggest that MIG-2 interacts with both β1 and β3 integrin cytoplasmic domains. Structure-based Mutagenesis Identifies a Single Integrin-binding Site within the MIG-2 FERM Domain—MIG-2 contains multiple protein-binding motifs, including a C-terminal FERM domain. To facilitate studies aimed at determining the functions of the MIG-2/integrin interaction, we sought to identify the MIG-2 site(s) that are involved in integrin binding. The F3 subdomain within the MIG-2 FERM domain shares considerable sequence similarity with the F3 subdomain of the talin FERM domain, i
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