Portal de Periódicos da CAPES

Involvement of Nectin in Inactivation of Integrin αvβ3 after the Establishment of Cell-Cell Adhesion

2007; Elsevier BV; Volume: 283; Issue: 1 Linguagem: Inglês

10.1074/jbc.m704195200

1083-351X

Yasuhisa Sakamoto, Hisakazu Ogita, Hitomi Komura, Yoshimi Takai,

Galectins and Cancer Biology

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin αvβ3 is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin αvβ3 becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin αvβ3 during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Iγ90 (PIPKIγ90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin αvβ3 at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIγ is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase μ (PTPμ) effectively dephosphorylated PIPKIγ and thus canceled the PIPKIγ-dependent activation of integrin αvβ3 by blocking the interaction of integrin αvβ3 with talin. Moreover, PTPμ associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIγ90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPμ-induced inactivation of PIPKIγ. Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin αvβ3 is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin αvβ3 becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin αvβ3 during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Iγ90 (PIPKIγ90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin αvβ3 at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIγ is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase μ (PTPμ) effectively dephosphorylated PIPKIγ and thus canceled the PIPKIγ-dependent activation of integrin αvβ3 by blocking the interaction of integrin αvβ3 with talin. Moreover, PTPμ associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIγ90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPμ-induced inactivation of PIPKIγ. Integrin is a key cell-cell adhesion molecule at cell-matrix junctions and comprises heterodimers with α and β subunits (1Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 793-805Crossref PubMed Scopus (1824) Google Scholar). Integrin exhibits intracellular conformational changes between the low and high affinity forms (2Takagi J. Petre B.M. Walz T. Springer T.A. Cell. 2002; 110: 599-611Abstract Full Text Full Text PDF PubMed Scopus (921) Google Scholar). The low affinity form shows weak adhesion activity for extracellular matrix proteins and is inactive, whereas the high affinity form has increased adhesion activity for its extracellular ligands and is active (3Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (388) Google Scholar). It was reported that integrin is essential for the formation of specialized subcellular apparatuses, such as focal complexes and focal adhesions, and for cell movement, proliferation, and differentiation (1Geiger B. Bershadsky A. Pankov R. Yamada K.M. Nat. Rev. Mol. Cell Biol. 2001; 2: 793-805Crossref PubMed Scopus (1824) Google Scholar, 4Watt F.M. EMBO J. 2002; 21: 3919-3926Crossref PubMed Scopus (531) Google Scholar, 5Kinbara K. Goldfinger L.E. Hansen M. Chou F.L. Ginsberg M.H. Nat. Rev. Mol. Cell Biol. 2003; 4: 767-776Crossref PubMed Google Scholar). We recently demonstrated that integrin αvβ3 interacts with Necl-5 at the leading edge of moving cells and that this complex enhances cell movement and proliferation together with platelet-derived growth factor receptor by stimulation of platelet-derived growth factor (6Ikeda W. Kakunaga S. Takekuni K. Shingai T. Satoh K. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2004; 279: 18015-18025Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 7Minami Y. Ikeda W. Kajita M. Fujito T. Amano H. Tamaru Y. Kuramitsu K. Sakamoto Y. Monden M. Takai Y. J. Biol. Chem. 2007; 282: 18481-18496Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Necl-5 is an Ig-like cell adhesion molecule and resembles nectin in its structure: three Ig-like loops at the extracellular region, a single transmembrane domain, and one cytoplasmic region. When moving cells collide with each other, the initial cell-cell contact occurs with the trans-interaction of Necl-5 with nectin-3 (8Ikeda W. Kakunaga S. Itoh S. Shingai T. Takekuni K. Satoh K. Inoue Y. Hamaguchi A. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2003; 278: 28167-28172Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Nectin is an emerging Ig-like cell-cell adhesion molecule that localizes at adherens junctions (AJs) 2The abbreviations used are:AJadherens junctionAbantibodymAbmonoclonal antibodypAbpolyclonal antibodyPIPKIγphosphatidylinositol-phosphate kinase type IγMDCKMadin-Darby canine kidneyPTPprotein-tyrosine phosphataseFAKfocal adhesion kinaseGFPgreen fluorescent proteinLMWlow molecular weightHAhemagglutininDMEMDulbecco's modified Eagle's mediumPIPES1,4-piperazinediethanesulfonic acid 2The abbreviations used are:AJadherens junctionAbantibodymAbmonoclonal antibodypAbpolyclonal antibodyPIPKIγphosphatidylinositol-phosphate kinase type IγMDCKMadin-Darby canine kidneyPTPprotein-tyrosine phosphataseFAKfocal adhesion kinaseGFPgreen fluorescent proteinLMWlow molecular weightHAhemagglutininDMEMDulbecco's modified Eagle's mediumPIPES1,4-piperazinediethanesulfonic acid and is involved in the formation of AJs (9Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (480) Google Scholar). Nectin exerts its cell-cell adhesion activity in a Ca2+-independent manner and consists of four members: nectin-1, nectin-2, nectin-3, and nectin-4 (9Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (480) Google Scholar). However, the trans-interaction of Necl-5 with nectin-3 is tentative, and Necl-5 is down-regulated from the cell surface by clathrin-dependent endocytosis (10Fujito T. Ikeda W. Kakunaga S. Minami Y. Kajita M. Sakamoto Y. Monden M. Takai Y. J. Cell Biol. 2005; 171: 165-173Crossref PubMed Scopus (83) Google Scholar). The down-regulation of Necl-5 impairs the integrin αvβ3- and platelet-derived growth factor receptor-dependent intracellular signaling for cell movement and proliferation, resulting in the reduction of cell movement and proliferation. The phenomenon that moving and proliferating normal cultured cells arrest both movement and proliferation after they grow confluent and form cell-cell junctions has been well known for a long time (11Abercrombie M. Heaysman J.E. Exp. Cell Res. 1953; 5: 111-131Crossref PubMed Scopus (275) Google Scholar, 12Fisher H.W. Yeh J. Science. 1967; 155: 581-582Crossref PubMed Scopus (68) Google Scholar), but its molecular mechanism is poorly understood. The down-regulation of Necl-5 is likely to be at least partly one of the underlying mechanisms of contact inhibition of cell movement and proliferation. On the other hand, nectin-3 dissociated from Necl-5 is retained on the cell surface and subsequently trans-interacts with nectin-1, which most feasibly trans-interacts with nectin-3 among the nectin family members (8Ikeda W. Kakunaga S. Itoh S. Shingai T. Takekuni K. Satoh K. Inoue Y. Hamaguchi A. Morimoto K. Takeuchi M. Imai T. Takai Y. J. Biol. Chem. 2003; 278: 28167-28172Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). This trans-interaction of nectins promotes the recruitment of cadherin, a major cell-cell adhesion molecule at AJs, to the nectin-based cell-cell adhesion sites, eventually establishing AJs (9Takai Y. Nakanishi H. J. Cell Sci. 2003; 116: 17-27Crossref PubMed Scopus (480) Google Scholar, 13Ogita H. Takai Y. IUBMB Life. 2006; 58: 334-343Crossref PubMed Scopus (75) Google Scholar). adherens junction antibody monoclonal antibody polyclonal antibody phosphatidylinositol-phosphate kinase type Iγ Madin-Darby canine kidney protein-tyrosine phosphatase focal adhesion kinase green fluorescent protein low molecular weight hemagglutinin Dulbecco's modified Eagle's medium 1,4-piperazinediethanesulfonic acid adherens junction antibody monoclonal antibody polyclonal antibody phosphatidylinositol-phosphate kinase type Iγ Madin-Darby canine kidney protein-tyrosine phosphatase focal adhesion kinase green fluorescent protein low molecular weight hemagglutinin Dulbecco's modified Eagle's medium 1,4-piperazinediethanesulfonic acid During the nectin-induced formation of cadherin-based AJs, several intracellular signaling molecules including Rap1, Cdc42, and Rac small G proteins are activated, and actin cytoskeleton is reorganized by the trans-interaction of nectin in cooperation with the high affinity form of integrin αvβ3 (14Fukuhara T. Shimizu K. Kawakatsu T. Fukuyama T. Minami Y. Honda T. Hoshino T. Yamada T. Ogita H. Okada M. Takai Y. J. Cell Biol. 2004; 166: 393-405Crossref PubMed Scopus (92) Google Scholar, 15Fukuyama T. Ogita H. Kawakatsu T. Fukuhara T. Yamada T. Sato T. Shimizu K. Nakamura T. Matsuda M. Takai Y. J. Biol. Chem. 2005; 280: 815-825Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 16Kawakatsu T. Ogita H. Fukuhara T. Fukuyama T. Minami Y. Shimizu K. Takai Y. J. Biol. Chem. 2005; 280: 4940-4947Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Inagaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In this process, the activation of protein kinase C and FAK, downstream molecules of integrin αvβ3, is also required (17Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Inagaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 18Ozaki M. Ogita H. Takai Y. Genes Cells. 2007; 12: 651-662Crossref PubMed Scopus (31) Google Scholar). However, after the establishment of AJs, the high affinity form of integrin αvβ3 is converted into the low affinity form that also continues to associate with nectin (17Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Inagaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 18Ozaki M. Ogita H. Takai Y. Genes Cells. 2007; 12: 651-662Crossref PubMed Scopus (31) Google Scholar). Although the molecular mechanism by which integrin αvβ3 is inactivated after the formation of AJs remains to be elucidated, this inactivation seems to be beneficial for the maintenance of AJs, because the sustained activation of integrin renders cells highly motile, which tends to disrupt cell-cell junctions. Integrin is activated by binding of talin to the cytoplasmic tail of integrin β subunit (3Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (388) Google Scholar), which causes the structural change of the integrin α/β dimer from the bent to the extended conformation. This change allows integrin to gain the higher affinity to the extracellular matrix. The binding of talin to integrin is up-regulated by increasing the amount of phosphatidylinositol 4,5-bisphosphate (19Martel V. Racaud-Sultan C. Dupe S. Marie C. Paulhe F. Galmiche A. Block M.R. Albiges-Rizo C. J. Biol. Chem. 2001; 276: 21217-21227Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), which is generated by phosphatidylinositol-phosphate kinases such as phosphatidylinositol-phosphate kinase type Iγ90 (PIPKIγ90). Integrin that is activated in this way induces the activation of c-Src and FAK, both of which phosphorylate and activate PIPKIγ90 (20Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (377) Google Scholar, 21Ling K. Doughman R.L. Iyer V.V. Firestone A.J. Bairstow S.F. Mosher D.F. Schaller M.D. Anderson R.A. J. Cell Biol. 2003; 163: 1339-1349Crossref PubMed Scopus (126) Google Scholar). Moreover, phosphorylated PIPKIγ90 correlates with an increase in its interaction with talin, and this interaction further stimulates the kinase activity of PIPKIγ90 itself (20Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (377) Google Scholar, 22Di Paolo G. Pellegrini L. Letinic K. Cestra G. Zoncu R. Voronov S. Chang S. Guo J. Wenk M.R. De Camilli P. Nature. 2002; 420: 85-89Crossref PubMed Scopus (374) Google Scholar). These combined mechanisms result in the enhancement of phosphatidylinositol 4,5-bisphosphate synthesis and thus the further promotion of talin binding to integrin, suggesting the positive feedback loop of integrin activation. Thus, the phosphorylation state of PIPKIγ is important for the regulation of integrin activation. Based on these lines of evidence, we examined in this study how integrin αvβ3 is inactivated after the nectin-induced formation of AJs by exploring the phosphatase that suppresses the phosphorylation of PIPKIγ and whether nectin actually associates with this phosphatase and regulates its phosphatase activity. Vector Construction—The following expression vectors were kindly provided: GFP-tagged full-length human PIPKIγ90 (pEGFP-PIPKIγ90) was from Dr. P. De Camilli (Yale University, New Heaven, CT), GFP-tagged protein-tyrosine phosphatase μ (PTPμ) (pcDNA-GFP-PTPμ) and PTPμ phosphatase-inactive mutant (pcDNA-GFP-PTPμC/S) were from Dr. S. Brady-Kalnay (Case Western Reserve University, Cleveland, OH), Myc-tagged low molecular weight protein-tyrosine phosphatase (LMW-PTP) (pcDNA3.1-myc-LMW-PTP) was from Dr. T. Konodo (Nagasaki University, Nagasaki, Japan), HA-tagged SHP-1 (pSRα-HA-SHP-1) was from Dr. T. Matozaki (Gunma University, Gunma, Japan), wild-type c-Src (pcDNA3-c-Src-wt) was from Dr. M. Okada (Osaka University, Suita, Japan), and HA-tagged E-cadherin (pCAGGSneo-HA-E-cadherin) was from Dr. M. Ozawa (Kagoshima University, Kagoshima, Japan). Expression vectors for FLAG-tagged nectin-1 (amino acids 27-518, pFLAG-CMV1-nectin-1), FLAG-tagged nectin-2 (amino acids 30-467, pFLAG-CMV1-nectin-2), FLAG-tagged nectin-3 (amino acids 56-549, pCAGIPuro-FLAG-nectin-3), FLAG-tagged nectin-4 (amino acids 29-508, pFLAG-CMV1-nectin-4), FLAG-tagged nectin-3 lacking its cytoplasmic region (amino acids 56-430, pFLAG-CMV1-nectin-3-ΔCP), FLAG-tagged nectin-3 lacking its extracellular region (amino acids 395-549, pFLAG-CMV1-nectin-3-ΔEC), and Myc-tagged nectin-3 (amino acids 56-549, pCAGIPuro-myc-nectin-3) were prepared as described (17Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Inagaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). FLAG-tagged nectin-3 without the C-terminal last four amino acids that is necessary for its binding to afadin (amino acids 56-545, pFLAG-CMV1-nectin-3-ΔC) was constructed by inserting its cDNA fragment into pFLAG-CMV1 vector (Sigma). FLAG-tagged afadin (pCMVF-afadin) and PIPKIγ90 (pCMVF-PIPKIγ90) were also constructed by inserting full-length rat afadin and human PIPKIγ90 cDNA fragments, respectively, into pCMVF vector. cDNA encoding the extracellular region of PTPμ with 10 repeats of His tag (His-PTPμ-EC; amino acids 1-740) was amplified by PCR and inserted into pFLAG-CMV-5 vector (Sigma). Antibodies—The rabbit polyclonal antibody (pAb) against afadin was prepared as described (23Sakisaka T. Nakanishi H. Takahashi K. Mandai K. Miyahara M. Satoh A. Takaishi K. Takai Y. Oncogene. 1999; 18: 1609-1617Crossref PubMed Scopus (78) Google Scholar). Hybridoma cells expressing a mouse anti-Myc mAb (9E10) were obtained from American Type Culture Collection, and the anti-Myc mAb was prepared as described (24Kodama A. Takaishi K. Nakano K. Nishioka H. Takai Y. Oncogene. 1999; 18: 3996-4006Crossref PubMed Scopus (88) Google Scholar). WOW-1 Fab, a rabbit anti-PIPKIγ90, and a rat anti-E-cadherin monoclonal Ab (mAb) (ECCD2) were kind gifts form Dr. S. J. Shatill (University of California San Diego, La Jolla, CA), Dr. Y. Kanaho (University of Tsukuba, Tsukuba, Japan), and Dr. M. Takeichi (RIKEN Center for Developmental Biology, Kobe, Japan), respectively. The following mouse mAbs were purchased from commercial sources; anti-FLAG M2 mAb (Sigma), anti-HA mAb (Berkeley Antibody), anti-phosphotyrosine mAb (4G10; Upstate Biotechnology, Inc.), anti-PTPμ mAb (Chemicon), anti-FAK mAb (Pharmingen), anti-PIPKIγ mAb (Pharmingen), anti-talin mAb (Sigma), and anti-integrin αvβ3 mAb (LM609; Chemicon). The following rabbit pAbs were purchased from commercial sources: anti-FLAG pAb (Sigma), anti-His pAb (Santa Cruz Biotechnology), and anti-integrin β3 pAb (Chemicon). The goat anti-nectin-3 pAb was purchased from Santa Cruz Biotechnology. Cell Lines and Transfection—MDCK cells, HEK293 cells, and L cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. For DNA transfection, Lipofectamine 2000 or Lipofectamine Plus (Invitrogen) was applied following the manufacturer's instructions. Immunofluorescence Microscopy—Immunofluorescence microscopy was performed as described (17Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Inagaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Briefly, the cells were fixed with ice-cold acetone-methanol (1:1) solution for 1 min. After being blocked with 1% bovine serum albumin, the cells were immunostained with the indicated first Abs for 1 h, followed by the incubation with fluorophore-labeled secondary Abs for 30 min. The samples were analyzed by LM510 META confocal microscope (Carl Zeiss). Immunoprecipitation Assay—MDCK and HEK293 cells expressing various combinations of indicated molecules were lysed with Buffer A (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm MgCl2, 1 mm Na3VO4, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 3 μg/ml leupeptin, 5 μg/ml aprotinin). The cell lysates were centrifuged at 100,000 × g at 4 °C for 15 min, and then the supernatant was incubated with the anti-FLAG mAb at 4 °C for 2 h followed by incubation with protein G-Sepharose beads at 4 °C for 2 h. After the beads were extensively washed with Buffer A, the bound proteins were eluted from the beads by boiling with Laemmli buffer for 5 min and subjected to SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar), followed by Western blotting with the indicated Abs. To investigate the association of endogenous PTPμ with nectin-3, MDCK cells cultured on the 0.4-μm pored transwell plate (Corning) were treated with a membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate (Pierce) according to the manufacturer's instructions. After the treatment, the cells were lysed with buffer A, and the cell lysates were incubated with the anti-nectin-3 pAb or the control goat IgG, followed by the incubation with protein G-Sepharose. The immunoprecipitated samples were then analyzed by Western blotting. In Vitro Binding of PTPμ and Nectin-3—For the preparation of the purified protein of His-PTPμ-EC, HEK293 cells were transfected with pFLAG-CMV-5-PTPμ-EC. At 48 h after the transfection, the culture supernatant containing soluble His-PTPμ-EC was collected and then was applied to nickel-Sepharose 6 fast flow beads (GE Healthcare) equilibrated with Buffer B (25 mm Tris-HCl, pH 8.0, 200 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 20 mm imidazole at pH 8.0). After the beads were extensively washed with Buffer B, bound His-PTPμ-EC was eluted with Buffer C (25 mm Tris-HCl, pH 8.0, 200 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 500 mm imidazole at pH 8.0). The protein concentration of His-PTPμ-EC was determined with bovine serum albumin as a reference protein on SDS-PAGE. The extracellular region of nectin-3 fused to IgG Fc (Nef-3) was prepared as described (26Honda T. Shimizu K. Kawakatsu T. Yasumi M. Shingai T. Fukuhara A. Ozaki-Kuroda K. Irie K. Nakanishi H. Takai Y. Genes Cells. 2003; 8: 51-63Crossref PubMed Scopus (80) Google Scholar). To examine the direct binding of His-PTPμ-EC and Nef-3, His-PTPμ-EC (6 pmol) was immobilized on nickel-Sepharose beads, and Nef-3 (60 pmol) was incubated with these beads or nickel-Sepharose beads alone as a control in 0.3 ml of Buffer B containing 0.5 mg/ml bovine serum albumin for 1 h. After the beads were extensively washed with Buffer B, the bound proteins were eluted with Buffer C. The eluate was then subjected to SDS-PAGE, followed by Western blotting. Bound Nef-3 was determined by the anti-human IgG Fc pAb conjugated with horseradish peroxidase (GE Healthcare). Separation of Cytoplasmic and Cytoskeletal Fractions—Triton X-100-soluble (cytoplasmic) and -insoluble (cytoskeletal) fractions were prepared as described previously (27Carragher N.O. Levkau B. Ross R. Raines E.W. J. Cell Biol. 1999; 147: 619-630Crossref PubMed Scopus (211) Google Scholar). Briefly, MDCK cells were lysed with Triton X-100 lysis buffer (20 mm Tris-HCl, pH 7.4, 1% Triton X-100, 5 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 3 μg/ml leupeptin, 5 μg/ml aprotinin, and 1 mm Na3VO4) at 4 °C for 1 h. Triton X-100-insoluble and -soluble extracts were separated by centrifugation at 15,000 × g at 4 °C for 5 min. The cytoskeletal pellet was washed twice with Triton X-100-free lysis buffer, and the proteins were extracted using radioimmune precipitation assay buffer (10 mm Tris-HCl, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 3 μg/ml leupeptin, 5 μg/ml aprotinin, and 1 mm Na3VO4). Sucrose Density Gradient Centrifugation—The assay for isolation of plasma membrane fraction was performed as described previously (28Yamamoto Y. Mandai K. Okabe N. Hoshino T. Nakanishi H. Takai Y. Oncogene. 2002; 21: 2545-2554Crossref PubMed Scopus (15) Google Scholar). Briefly, the MDCK cells were washed with phosphate-buffered saline and then sonicated in Buffer D (10 mm HEPES-NaOH at pH 7.5, 100 mm KCl, 1 mm MgCl2, and 25 mm NaHCO3) on ice for 15 s six times at 3-min intervals. The homogenate was centrifuged at 1,000 × g at 4 °C for 5 min. The supernatant was diluted with Buffer D into 5 mg/ml of protein, and 0.2 ml was applied on a 4.8 -ml continuous sucrose density gradient (10-50% sucrose in Buffer D), followed by centrifugation at 100,000 × g at 4 °C for 1 h with a swing rotor (P55ST2; Hitachi). After the centrifugation, fractions of 0.3 ml each were collected. Each fraction was subjected to SDS-PAGE, followed by Western blotting with the anti-E-cadherin and anti-talin mAbs. Assessment for Integrin αvβ3 Activity—MDCK cells cultured on 18-mm coverslips in a 12-well dish were used for the Ca2+ switch assay as described previously (18Ozaki M. Ogita H. Takai Y. Genes Cells. 2007; 12: 651-662Crossref PubMed Scopus (31) Google Scholar). Briefly, the cells were washed with phosphate-buffered saline and incubated in serum-free DMEM (Normal Ca2+ medium) for 1 h. Next, the cells were incubated in serum-free DMEM containing 5 mm EGTA (low Ca2+ medium) for 3 h. The cells were then incubated in serum-free DMEM (normal Ca2+ medium) for indicated period. To detect the high affinity form of integrin αvβ3, we used His-tagged recombinant WOW-1 Fab as described previously (29Katsumi A. Naoe T. Matsushita T. Kaibuchi K. Schwartz M.A. J. Biol. Chem. 2005; 280: 16546-16549Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Briefly, the cells were incubated with His-tagged recombinant WOW-1 Fab for 30 min before the end of the Ca2+ assay. The cells were then washed twice with DMEM and were lysed with Laemmli buffer. The amount of WOW-1 bound to the high affinity form of integrin αvβ3 was detected by Western blotting with the anti-His pAb. Assay for PTPμ Phosphatase Activity—The PTPμ phosphatase activity was assessed using a Universal tyrosine phosphatase assay kit (Takara Bio) as previously described (30Feiken E. van Etten I. Gebbink M.F. Moolenaar W.H. Zondag G.C. J. Biol. Chem. 2000; 275: 15350-15356Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Briefly, HEK293 cells transiently expressing Myc-nectin-3 or HA-E-cadherin were cultured in confluent and lysed with Lysis buffer attached to this kit and then centrifuged at 100,000 × g at 4 °C for 15 min. The supernatant was precleared by protein G-Sepharose beads, and the precleared supernatant was incubated with the anti-PTPμ mAb at 4 °C for 2 h, followed by incubation with protein G-Sepharose beads at 4 °C for 2 h. After the beads were extensively washed with Buffer E (0.5% Tween 20, 50 mm PIPES, pH 7.0) three times, these beads were suspended into PTP buffer attached to this kit, and the samples were subjected to the phosphatase assay according to the manufacturer's instructions. A paired Student t test was performed for statistical analysis. Knockdown of PIPKIγ and PTPμ—To knock down PIPKIγ and PTPμ, double-stranded 25-nucleotide RNA duplexes (Stealth™; Invitrogen) for PIPKIγ (duplex 1, 5′-UCUUGUAGGUGGUUUCACCAGAUGC-3′; duplex 2, 5′-UGAACCUGAAGUCCUGGAAGUGGUG-3′; duplex 3, 5′-UUCUGGUUGAGGUUCAUGUAGUAGC-3′) and PTPμ (duplex 1, 5′-AUUAAAUGAAGCUAUGAAUCGCCGG-3′; duplex 2, 5′-UUCACACUAACAUUGGUAUAUGGUG-3′; and duplex 3, 5′-UUCAUAAGCCGGCAUAGACGGUGCU-3′), respectively, were transfected into MDCK cells using Amaxa Nucleofection kit T. The knockdown of each protein was confirmed by Western blotting. Activation of Integrin αvβ3 by PIPKIγ at Cell-Cell Junctions—We previously showed that after the achievement of AJs, integrin αvβ3 becomes inactive and localizes at cell-cell adhesion sites as well as focal adhesions (17Sakamoto Y. Ogita H. Hirota T. Kawakatsu T. Fukuyama T. Yasumi M. Inagaki M. Takai Y. J. Biol. Chem. 2006; 281: 19631-19644Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 18Ozaki M. Ogita H. Takai Y. Genes Cells. 2007; 12: 651-662Crossref PubMed Scopus (31) Google Scholar). Consistent with this, integrin αvβ3 was concentrated at the cell-cell adhesion sites and co-localized with E-cadherin in confluent MDCK cells, whereas talin was distributed throughout the cytoplasm and did not co-localize with E-cadherin (Fig. 1A), indicating the different localization of integrin αvβ3 from talin. Because talin is involved in the final step of the activation of integrin by directly binding to the cytoplasmic tail of integrin β3 subunit (31Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (965) Google Scholar), this different localization of integrin αvβ3 from talin represents the accumulation of the low affinity form of integrin αvβ3 at AJs in confluent MDCK cells. However, when GFP-PIPKIγ90 was transfected into MDCK cells, talin as well as GFP-PIPKIγ90 was preferentially targeted to the plasma membrane of the cell-cell adhesion sites where the immunofluorescence signal for integrin αvβ3 was concentrated (Fig. 1B, arrowheads), leading to the notion that PIPKIγ90 induces the reactivation of integrin αvβ3 through talin. Interestingly, E-cadherin-based AJs were partially disrupted, probably because of this reactivation of integrin αvβ3 (Fig. 1B, arrow), which was not observed in GFP-transfected (Fig. 1A) or untransfected MDCK cells. These results indicate the critical role of PIPKIγ in integrin αvβ3 reactivation that causes the instability of AJs. Conversely, the inactivation of PIPKIγ seems to be at least one of the important underlying mechanisms in the inactivation of integrin αvβ3 after the establishment of AJs. We further examined by knockdown of PIPKIγ whether endogenous PIPKIγ is indeed involved in the association of integrin with talin. The expression of PIPKIγ was markedly reduced in MDCK cells using siRNA against PIPKIγ (Fig. 1C). Although integrin β3 and talin clustered well and co-localized at focal adhesions of wild-type MDCK cells, this clustering or co-localization was not observed in PIPKIγ knockdown MDCK cells (Fig. 1D), indicating the necessity of PIPKIγ for the association of integrin with talin even at the endogenous level. Identification of PTPμ as a Phosphatase for PIPKIγ—It was reported that the kinase activity of PIPKIγ90 is enhanced by its tyrosine phosphorylation (20Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (377) Google Scholar, 21Ling K. Doughman R.L. Iyer V.V. Firestone A.J. Bairstow S.F. Mosher D.F. Schaller M.D. Anderson R.A. J. Cell Biol. 2003; 163: 1339-1349Crossref PubMed Scopus (126) Google Scholar, 2