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Originally published In Press as doi:10.1074/jbc.M704195200 on October 26, 2007

J. Biol. Chem., Vol. 283, Issue 1, 496-505, January 4, 2008
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Involvement of Nectin in Inactivation of Integrin {alpha}vβ3 after the Establishment of Cell-Cell Adhesion*

Yasuhisa Sakamoto{ddagger}, Hisakazu Ogita{ddagger}, Hitomi Komura{ddagger}, and Yoshimi Takai{ddagger}§1

From the {ddagger}Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita, Osaka 565-0871, Japan and the §Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan

Received for publication, May 22, 2007 , and in revised form, September 20, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}vβ3 is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin {alpha}vβ3 becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin {alpha}vβ3 during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type I{gamma}90 (PIPKI{gamma}90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin {alpha}vβ3 at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKI{gamma} is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase µ (PTPµ) effectively dephosphorylated PIPKI{gamma} and thus canceled the PIPKI{gamma}-dependent activation of integrin {alpha}vβ3 by blocking the interaction of integrin {alpha}vβ3 with talin. Moreover, PTPµ associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKI{gamma}90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPµ-induced inactivation of PIPKI{gamma}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin is a key cell-cell adhesion molecule at cell-matrix junctions and comprises heterodimers with {alpha} and β subunits (1). Integrin exhibits intracellular conformational changes between the low and high affinity forms (2). 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 (3). 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 (1, 4, 5). We recently demonstrated that integrin {alpha}vβ3 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 (6, 7). 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 (8). Nectin is an emerging Ig-like cell-cell adhesion molecule that localizes at adherens junctions (AJs)2 and is involved in the formation of AJs (9). 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 (9). 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 (10). The down-regulation of Necl-5 impairs the integrin {alpha}vβ3- 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 (11, 12), 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 (8). 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 (9, 13).

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 {alpha}vβ3 (14-17). In this process, the activation of protein kinase C and FAK, downstream molecules of integrin {alpha}vβ3, is also required (17, 18). However, after the establishment of AJs, the high affinity form of integrin {alpha}vβ3 is converted into the low affinity form that also continues to associate with nectin (17, 18). Although the molecular mechanism by which integrin {alpha}vβ3 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 (3), which causes the structural change of the integrin {alpha}/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 (19), which is generated by phosphatidylinositol-phosphate kinases such as phosphatidylinositol-phosphate kinase type I{gamma}90 (PIPKI{gamma}90). Integrin that is activated in this way induces the activation of c-Src and FAK, both of which phosphorylate and activate PIPKI{gamma}90 (20, 21). Moreover, phosphorylated PIPKI{gamma}90 correlates with an increase in its interaction with talin, and this interaction further stimulates the kinase activity of PIPKI{gamma}90 itself (20, 22). 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 PIPKI{gamma} is important for the regulation of integrin activation.

Based on these lines of evidence, we examined in this study how integrin {alpha}vβ3 is inactivated after the nectin-induced formation of AJs by exploring the phosphatase that suppresses the phosphorylation of PIPKI{gamma} and whether nectin actually associates with this phosphatase and regulates its phosphatase activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Vector Construction—The following expression vectors were kindly provided: GFP-tagged full-length human PIPKI{gamma}90 (pEGFP-PIPKI{gamma}90) 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 (pSR{alpha}-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-{Delta}CP), FLAG-tagged nectin-3 lacking its extracellular region (amino acids 395-549, pFLAG-CMV1-nectin-3-{Delta}EC), and Myc-tagged nectin-3 (amino acids 56-549, pCAGIPuro-myc-nectin-3) were prepared as described (17). 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-{Delta}C) was constructed by inserting its cDNA fragment into pFLAG-CMV1 vector (Sigma). FLAG-tagged afadin (pCMVF-afadin) and PIPKI{gamma}90 (pCMVF-PIPKI{gamma}90) were also constructed by inserting full-length rat afadin and human PIPKI{gamma}90 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 (23). 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 (24). WOW-1 Fab, a rabbit anti-PIPKI{gamma}90, 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%20Biotechnology">Upstate Biotechnology, Inc.), anti-PTPµ mAb (Chemicon), anti-FAK mAb (Pharmingen), anti-PIPKI{gamma} mAb (Pharmingen), anti-talin mAb (Sigma), and anti-integrin {alpha}vβ3 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 (17). 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 x 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 (25), 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 (26).

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 (27). 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 x 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 (28). 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 x 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 x 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 {alpha}vβ3 Activity—MDCK cells cultured on 18-mm coverslips in a 12-well dish were used for the Ca2+ switch assay as described previously (18). 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 {alpha}vβ3, we used His-tagged recombinant WOW-1 Fab as described previously (29). 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 {alpha}vβ3 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 (30). 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 x 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 PIPKI{gamma} and PTPµ—To knock down PIPKI{gamma} and PTPµ, double-stranded 25-nucleotide RNA duplexes (StealthTM; Invitrogen) for PIPKI{gamma} (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.


Figure 1
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  		FIGURE 1.
Involvement of PIPKI{gamma} in the recruitment of talin to the cell-cell adhesion sites and activation of integrin {alpha}vβ3 in MDCK cells. A, no recruitment of talin to the cell-cell adhesion sites in GFP-transfected MDCK cells. At 24 h after the transfection of GFP, confluent MDCK cells were immunostained with the indicated Abs. B, recruitment of talin to the cell-cell adhesion sites and disruption of cell-cell junctions in PIPKI{gamma}90-transfected MDCK cells. At 24 h after the transfection of GFP-PIPKI{gamma}90, confluent MDCK cells were immunostained with the indicated Abs. Arrowheads, talin targeted to the plasma membrane of cell-cell junctions; Arrow, disrupted cell-cell junctions. Scale bars, 10 µm. C, knockdown of PIPKI{gamma}. The cell lysates from wild-type or siRNA-transfected MDCK cells were immunoprecipitated and immunoblotted with the anti-PIPKI{gamma} Ab. Actin was also immunoblotted for the loading control. The sequence of each siRNA against PIPKI{gamma} was indicated under "Experimental Procedures." Because siRNA 2 (#2) most effectively reduced the expression of PIPKI{gamma} in MDCK cells, this siRNA was used in the following experiments. WT, wild type; KD, knockdown. D, inhibition of the interaction of integrin β3 with talin by knockdown of PIPKI{gamma} in MDCK cells. At 48 h after the transfection of siRNA, the cells were immunostained with the anti-integrin β3 and anti-talin Abs. Scale bars, 10 mm. The results shown in this figure are representative of three independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Activation of Integrin {alpha}vβ3 by PIPKI{gamma} at Cell-Cell Junctions—We previously showed that after the achievement of AJs, integrin {alpha}vβ3 becomes inactive and localizes at cell-cell adhesion sites as well as focal adhesions (17, 18). Consistent with this, integrin {alpha}vβ3 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 {alpha}vβ3 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 (31), this different localization of integrin {alpha}vβ3 from talin represents the accumulation of the low affinity form of integrin {alpha}vβ3 at AJs in confluent MDCK cells.

However, when GFP-PIPKI{gamma}90 was transfected into MDCK cells, talin as well as GFP-PIPKI{gamma}90 was preferentially targeted to the plasma membrane of the cell-cell adhesion sites where the immunofluorescence signal for integrin {alpha}vβ3 was concentrated (Fig. 1B, arrowheads), leading to the notion that PIPKI{gamma}90 induces the reactivation of integrin {alpha}vβ3 through talin. Interestingly, E-cadherin-based AJs were partially disrupted, probably because of this reactivation of integrin {alpha}vβ3 (Fig. 1B, arrow), which was not observed in GFP-transfected (Fig. 1A) or untransfected MDCK cells. These results indicate the critical role of PIPKI{gamma} in integrin {alpha}vβ3 reactivation that causes the instability of AJs. Conversely, the inactivation of PIPKI{gamma} seems to be at least one of the important underlying mechanisms in the inactivation of integrin {alpha}vβ3 after the establishment of AJs.

We further examined by knockdown of PIPKI{gamma} whether endogenous PIPKI{gamma} is indeed involved in the association of integrin with talin. The expression of PIPKI{gamma} was markedly reduced in MDCK cells using siRNA against PIPKI{gamma} (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 PIPKI{gamma} knockdown MDCK cells (Fig. 1D), indicating the necessity of PIPKI{gamma} for the association of integrin with talin even at the endogenous level.


Figure 2
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  		FIGURE 2.
PTPµ-induced dephosphorylation of PIPKI{gamma}. A, reduced amount of phosphorylation of PIPKI{gamma}90 by PTPµ. The cell lysates of HEK293 cells co-transfected with indicated kinds of phosphatases as well as FLAG-PIPKI{gamma}90 and c-Src were immunoprecipitated (IP) with the anti-FLAG mAb. The immunoprecipitants were subjected to Western blotting with the anti-FLAG and anti-phosphotyrosine mAbs. B, co-immunoprecipitation of endogenous PTPµ with PIPKI{gamma} in MDCK cells. The cell lysates from MDCK cells were immunoprecipitated with the anti-PIPKI{gamma} Ab or control IgG, followed by Western blotting with the anti-PTPµ and anti-PIPKI{gamma} Abs. C, knockdown of PTPµ. The cell lysates from wild-type or siRNA-transfected MDCK cells were used for Western blotting with the anti-PTPµ mAb. Actin was also immunoblotted (IB) for the loading control. The sequence of each siRNA against PTPµ was indicated under "Experimental Procedures." Because siRNA 1 (#1) most effectively reduced the expression of PTPµ in MDCK cells, this siRNA was used in the following experiments. WT, wild type; KD, knockdown. D, enhanced phosphorylation of PIPKI{gamma} in PTPµ knockdown MDCK cells. The cell lysates from wild-type or PTPµ knockdown MDCK cells were immunoprecipitated with the anti-PIPKI{gamma} mAb, and the immunoprecipitates were subjected to SDS-PAGE, followed by Western blotting with the anti-phosphotyrosine and anti-PIPKI{gamma} Abs to assess the phosphorylation of PIPKI{gamma}. The results shown in this figure are representative of three independent experiments.

 
Identification of PTPµ as a Phosphatase for PIPKI{gamma}—It was reported that the kinase activity of PIPKI{gamma}90 is enhanced by its tyrosine phosphorylation (20-22), resulting in the increased binding of talin to integrin and consequent integrin activation. Thus, the phosphorylation state of PIPKI{gamma}90 is likely to be closely correlated with the regulation of integrin activation. We then examined the implication of the tyrosine phosphatase in the dephosphorylation and inhibition of PIPKI{gamma}90. To explore which phosphatases most effectively dephosphorylate PIPKI{gamma}90, HEK293 cells were co-transfected with FLAG-PIPKI{gamma}90, c-Src, and several tyrosine phosphatases including SHP-1, PTPµ, and LMW-PTP, and the phosphatase-induced decrease in PIPKI{gamma}90 phosphorylation was monitored. In the presence of SHP-1, the phosphorylation of PIPKI{gamma}90 was slightly reduced (Fig. 2A). As compared with SHP-1, PTPµ more markedly decreased the phosphorylation level of PIPKI{gamma}90. In contrast, LMW-PTP did not attenuate PIPKI{gamma}90 phosphorylation. This result indicates that PTPµ is the most promising candidate for the inhibitor of PIPKI{gamma}. Then we confirmed the association of endogenous PTPµ with PIPKI{gamma} in MDCK cells (Fig. 2B). To further specify the role of PTPµ in the dephosphorylation of PIPKI{gamma}, we knocked down PTPµ in MDCK cells and examined whether the phosphorylation level of PIPKI{gamma} is affected by the expression of PTPµ. The expression of PTPµ was markedly reduced in MDCK cells using siRNA against PTPµ (Fig. 2C). The tyrosine phosphorylation of PIPKI{gamma} was actually enhanced in PTPµ knockdown MDCK cells (Fig. 2D). These results indicate that PTPµ specifically acts as a tyrosine phosphatase for PIPKI{gamma} in MDCK cells.

Inhibitory Effect of PTPµ on the PIPKI{gamma}-dependent Recruitment of Talin and Activation of Integrin {alpha}vβ3—We next investigated whether PTPµ is involved in the inhibition of the PIPKI{gamma}-dependent assembly of talin and the inactivation of integrin {alpha}vβ3 at the cell-cell adhesion sites in MDCK cells. Co-transfection of FLAG-PIPKI{gamma}90 and GFP-PTPµ into MDCK cells clearly canceled the PIPKI{gamma}90-dependent translocation of talin to the cell-cell adhesion sites and prevented the disruption of AJs, whereas the phosphatase-inactive mutant of GFP-PTPµ (GFP-PTPµC/S) did not exert such inhibitory effects (Fig. 3A). These results indicate that PTPµ inhibits the PIPKI{gamma}-induced translocation of talin and its binding to integrin {alpha}vβ3 at the cell-cell adhesion sites.

The involvement of endogenous PTPµ in the localization of talin was further certified biochemically in MDCK cells by the knockdown of PTPµ. The subcellular localization of talin as well as FAK, a binding protein of integrin (32), was changed from the cytoplasmic to the cytoskeletal membrane fraction by knockdown of PTPµ (Fig. 3B). In addition, when the total cell lysates were subjected to sucrose density gradient centrifugation, the shift of talin to high density fractions where the membrane marker E-cadherin exists was observed in PTPµ knockdown MDCK cells (Fig. 3C, lanes 3-6). These results provide another line of evidence that PTPµ suppresses the translocation of talin to the plasma membrane.

To gain the distinct evidence for the involvement of PTPµ in the inactivation of integrin {alpha}vβ3, the high affinity form of integrin {alpha}vβ3 was monitored using WOW-1 Fab during the formation of cell-cell junctions induced by the Ca2+ switch assay. WOW-1 Fab specifically detects the high affinity form of integrin {alpha}vβ3 (33). As compared with wild-type MDCK cells, the high affinity form of integrin {alpha}vβ3 in PTPµ knockdown MDCK cells was increased at 0.5 and 1.5 h after the reculture with normal Ca2+ medium (Fig. 3D). Taken together, these results indicate that PTPµ perturbs the interaction of talin with integrin {alpha}vβ3 and inactivates integrin {alpha}vβ3 by the inhibition of PIPKI{gamma}.

Interaction of PTPµ with Nectin—We next examined the relationship between PTPµ and the nectin-afadin system in the inactivation of integrin {alpha}vβ3, because nectin and integrin {alpha}vβ3 physically and functionally associate together (17). In MDCK cells, PTPµ co-localized with nectin-3 and afadin endogenously at cell-cell junctions (Fig. 4A). The association of endogenous PTPµ with nectin-3 in MDCK cells was also confirmed by co-immunoprecipitation assay by chemically cross-linking cell surface proteins (Fig. 4B). Moreover, PTPµ was co-immunoprecipitated with not only nectin-3 but also other nectin family members including nectin-1, nectin-2, and nectin-4 in HEK293 cells ectopically expressing GFP-PTPµ with FLAG-nectin-1, FLAG-nectin-2, FLAG-nectin-3, or FLAG-nectin-4, when each FLAG-nectin molecule was immunoprecipitated with the anti-FLAG mAb (Fig. 4C, panel a). However, another tyrosine phosphatase LMW-PTP was not co-immunoprecipitated with FLAG-nectin-3 (Fig. 4C, panel b), suggesting that the co-immunoprecipitation of PTPµ with nectin is not nonspecific. In addition, PTPµ was not co-immunoprecipitated with afadin in HEK293 cells overexpressing these molecules (Fig. 4C, panel c). These results indicate that PTPµ associates with nectin at the cell-cell adhesion sites.


Figure 3
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  		FIGURE 3.
Negative effect of PTPµ on the PIPKI{gamma}-dependent recruitment of talin and the activation of integrin {alpha}vβ3. A, suppression of the PIPKI{gamma}90-dependent recruitment of talin by PTPµ. MDCK cells were transfected with FLAG-PIPKI{gamma}90 alone or co-transfected with FLAG-PIPKI{gamma}90 and GFP-PTPµ or GFP-PTPµC/S. GFP-PTPµC/S is a dominant negative mutant of PTPµ. At 24 h after transfection, confluent MDCK cells were immunostained with the anti-FLAG pAb and anti-talin mAb. Scale bars, 10 µm. B, increased recruitment of talin to the cytoskeletal fraction in PTPµ knockdown MDCK cells. The cell lysates from wild-type or PTPµ knock-down MDCK cells were separated into Triton X-100-soluble (cytoplasmic) and Triton X-100-insoluble (cytoskeletal) fractions. The samples were subjected to SDS-PAGE, followed by Western blotting with the anti-talin and anti-FAK Abs. WT, wild type; KD, knockdown. C, increased recruitment of talin to membrane fraction in PTPµ knockdown MDCK cells. The cell lysates from wild-type or PTPµ knockdown MDCK cells were used for sucrose density gradient centrifugation. The fractions were collected from bottom to top of the centrifugation tubes, followed by Western blotting with the anti-talin and anti-E-cadherin Abs. D, necessity of PTPµ for the inactivation of integrin {alpha}vβ3 after the formation of cell-cell junctions. The Ca2+ switch assay was performed in wild-type or PTPµ knockdown MDCK cells as described under "Experimental Procedures." The cells were incubated with His-tagged WOW-1 Fab for 30 min before the end of the Ca2+ switch assay. The cells were then lysed with Laemmli buffer, and WOW-1 Fab-bound integrin {alpha}vβ3 was determined with the anti-His pAb. Actin was also immunoblotted (IB) for the loading control. The results shown in this figure are representative of three independent experiments.

 
We further examined the mode of interaction of nectin with PTPµ. To test this, GFP-PTPµ was co-expressed with FLAG-tagged nectin-3 lacking its extracellular region (FLAG-nectin-3-{Delta}EC), FLAG-tagged nectin-3 lacking its cytoplasmic region (FLAG-nectin-3-{Delta}CP), or FLAG-tagged nectin-3 without the C-terminal tail of four amino acids that is necessary for binding to afadin (FLAG-nectin-3-{Delta}C) in HEK293 cells. When each nectin mutant was immunoprecipitated with the anti-FLAG mAb, GFP-PTPµ was co-immunoprecipitated with FLAG-nectin-3-{Delta}CP, but not FLAG-nectin-3-{Delta}EC, indicating the interaction of nectin-3 with PTPµ through their extracellular regions (Fig. 5A). We also found co-immunoprecipitation of FLAG-nectin-3-{Delta}C with GFP-PTPµ. This suggests no requirement of the nectin-afadin binding for the interaction of nectin with PTPµ.

We next prepared the recombinant proteins of the His-tagged extracellular region of PTPµ (His-PTPµ-EC) and the extracellular region of nectin-3 fused to IgG Fc (Nef-3) to investigate the direct binding of PTPµ and nectin-3. When Nef-3 was incubated with His-PTPµ-EC immobilized on nickel beads, the interaction of His-PTPµ-EC with Nef-3 was detected (Fig. 5B). These results indicate that PTPµ and nectin-3 physically interact with each other through their extracellular regions at cell-cell junctions.

To examine whether the interaction of nectin with PTPµ affects the localization of PTPµ at the cell-cell adhesion sites, GFP-PTPµ was transfected into L cells expressing full-length nectin-3 (nectin-3-L cells) or nectin-3-{Delta}EC (nectin-3-{Delta}EC-L cells) as well as wild-type L cells. The assembly of GFP-PTPµ at the cell-cell adhesion was markedly increased in nectin-3-L cells compared with wild-type L cells (Fig. 5C). Such an increase in the assembly of GFP-PTPµ at the cell-cell adhesion was not observed in nectin-3-{Delta}EC-L cells. Thus, the interaction of nectin with PTPµ is important for the effective recruitment of PTPµ to the cell-cell adhesion sites where integrin {alpha}vβ3 and PIPKI{gamma}90 also localize.


Figure 4
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  		FIGURE 4.
Association of PTPµ with nectin. A, co-localization of endogenous PTPµ with the nectin-afadin complex. Confluent MDCK cells were immunostained with the indicated Abs. Scale bars, 10 µm. B, co-immunoprecipitation (IP) of endogenous PTPµ with nectin-3. The cell lysates from MDCK cells pretreated with cell surface cross-linker were immunoprecipitated with the anti-nectin-3 pAb, followed by Western blotting with the anti-PTPµ and anti-nectin-3 Abs. C, immunoprecipitation assay with phosphatases and nectin family members or afadin. Panel a, co-immunoprecipitation of PTPµ with the nectin family members. The cell lysates of HEK293 cells transfected with the indicated combinations of GFP-PTPµ and FLAG-nectin molecules were immunoprecipitated with the anti-FLAG mAb. The immunoprecipitants were subjected to Western blotting with the anti-PTPµ and anti-FLAG mAbs. Panel b, no co-immunoprecipitation of LMW-PTP with nectin-3. The cell lysates of HEK293 cells transfected with Myc-LMW-PTP and FLAG-nectin-3 were immunoprecipitated with the anti-FLAG mAb, followed by Western blotting with the anti-Myc and anti-FLAG mAbs. Panel c, no co-immunoprecipitation of PTPµ with afadin. The cell lysates of HEK293 cells transfected with GFP-PTPµ and FLAG-afadin were immunoprecipitated with the anti-FLAG mAb, followed by Western blotting with the anti-PTPµ and anti-FLAG mAbs. The results shown in this figure are representative of three independent experiments. IB, immunoblotting.

 
Enhancement of PTPµ Phosphatase Activity by Trans-interacting Nectin—We finally examined whether the trans-interaction of nectin actually enhances the phosphatase activity of PTPµ for PIPKI{gamma}90. When HEK293 cells ectopically expressing FLAG-PIPKI{gamma}90 and c-Src with or without GFP-PTPµ and Myc-nectin-3 were cultured in confluence to form the trans-interaction of nectin-3, the c-Src-induced phosphorylation of PIPKI{gamma}90 was reduced by co-transfection of PTPµ (Fig. 6A). Intriguingly, nectin-3 remarkably increased the dephosphorylation of PIPKI{gamma}90 in the presence of PTPµ. This nectin-induced dephosphorylation of PIPKI{gamma}90 was not observed when GFP-PTPµ was not transfected. In contrast, when HEK293 cells expressing FLAG-PIPKI{gamma}90 and c-Src with or without GFP-PTPµ and HA-E-cadherin were cultured in confluence, the phosphorylation of PIPKI{gamma}90 was not affected by the trans-interaction of E-cadherin, although there is a report that E-cadherin associates with PTPµ (34). Moreover, we confirmed that the trans-interaction of nectin, but not E-cadherin, significantly raised the phosphatase activity of PTPµ (Fig. 6B). Taken together, these results indicate that the trans-interaction of nectin preferentially reduces the phosphorylation of PIPKI{gamma}90 mediated by the nectin-induced activation of PTPµ, eventually resulting in the inactivation of integrin {alpha}vβ3 after the establishment of AJs.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Nectin and the high affinity form of integrin {alpha}vβ3 cooperatively play a pivotal role in the formation of AJs by activating signaling molecules that are necessary for the formation of AJs (13). However, after the achievement of AJs, the high affinity form of integrin {alpha}vβ3 is converted into the low affinity form that continues to localize at AJs. The molecular mechanism for how integrin {alpha}vβ3 becomes inactive during the formation of AJs has not been elucidated yet to date. In this manuscript, we successfully proposed the molecular mechanism by which trans-interacting nectin also functions to inactivate integrin {alpha}vβ3 at mature AJs through the PTPµ-mediated dephosphorylation of PIPKI{gamma}90. The schematic representation of this molecular mechanism is depicted in Fig. 7.


Figure 5
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  		FIGURE 5.
Physical interaction of PTPµ with nectin-3 through their extracellular regions and its involvement in the recruitment of PTPµ to the nectin-based cell-cell adhesion sites. A, co-immunoprecipitation of PTPµ with the cytoplasmic region-deleted mutant of nectin-3. The cell lysates of HEK293 cells transfected with GFP-PTPµ and the indicated FLAG-nectin-3 mutants (nectin-3-{Delta}CP, nectin-3 without its cytoplasmic region; nectin-3-{Delta}EC, nectin-3 without its extracellular region; nectin-3-{Delta}C, nectin-3 lacking C-terminal four amino acids that is necessary for binding of nectin to afadin) were immunoprecipitated (IP) with the anti-FLAG mAb. The immunoprecipitants were subjected to Western blotting with the anti-PTPµ and anti-FLAG mAbs. B, direct interaction of PTPµ with nectin-3. The recombinant protein of His-PTPµ-EC was immobilized on nickel-Sepharose beads and incubated with Nef-3. After the incubation, the eluates were subjected to SDS-PAGE, followed by Western blotting with the anti-human IgG Fc for the detection of Nef-3 and anti-His Abs. C, recruitment of PTPµ to the cell-cell adhesion sites dependent on the association of PTPµ with nectin-3. Wild-type L cells or L cells ectopically expressing nectin-3 or nectin-3-{Delta} EC were transfected with GFP-PTPµ and then stained for F-actin with phalloidin. Scale bars, 10 µm. The results shown in this figure are representative of three independent experiments. IB, immunoblotting.

 


Figure 6
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  		FIGURE 6.
Nectin-induced dephosphorylation of PIPKI{gamma}90 and up-regulation of the phosphatase activity of PTPµ. A, increased dephosphorylation of PIPKI{gamma}90 by the trans-interaction of nectin, but not E-cadherin. Confluent HEK293 cells expressing FLAG-PIPKI{gamma}90 and c-Src with or without GFP-PTPµ, Myc-nectin-3, and HA-E-cadherin were lysed and immunoprecipitated (IP) with the anti-FLAG mAb, followed by Western blotting with the anti-FLAG and anti-phosphotyrosine (pY) mAbs. B, enhancement of PTPµ phosphatase activity by nectin. The cell lysates from HEK293 cells expressing Myc-nectin-3 or HA-E-cadherin were immunoprecipitated with the anti-PTPµ mAb. Untransfected HEK293 cells were used as a control. The phosphatase activity of PTPµ in the immunoprecipitants were analyzed using tyrosine phosphatase assay kit (Takara Bio). The data shown in this graph are the relative PTPµ phosphatase activity as compared with the value of the control, which is expressed as 1. *, p < 0.05. The results shown in this figure are representative of three independent experiments. IB, immunoblotting.

 
PTPµ is one of the receptor type protein-tyrosine phosphatases expressing in several epithelial cells and endothelial cells (35). PTPµ contains a MAM (Merpin/A5/PTPµ) domain, an Ig-like domain, four fibronectin type III repeats in its extracellular region, and two phosphatase domains in its cytoplasmic region. PTPµ itself trans-interacts through its MAM and Ig-like domains and localizes at AJs (36-38). It was previously demonstrated that PTPµ interacts with the cytoplasmic region of cadherin, β-catenin, and p120ctn and reduces the phosphorylation of these molecules (39, 40). In addition to this, we found here that nectin physically associates with PTPµ through their extracellular region and increases the phosphatase activity of PTPµ. This increase is somewhat small, but statistically significant, indicating the essential involvement of nectin in the up-regulation of the PTPµ phosphatase activity. Moreover, the assembly of PTPµ at the cell-cell adhesion sites is dependent on the association of PTPµ with nectin, because the signal intensity of GFP-PTPµ at cell-cell junctions are higher in L fibroblasts expressing full-length nectin-3 than those expressing nectin-3-{Delta}EC that is incapable of interacting with PTPµ. Notably, this nectin-mediated PTPµ assembly does not depends on cadherin, because L fibroblasts do not express any cadherins. However, it remains unknown whether these transmembrane proteins, nectin, cadherin, and PTPµ form a ternary complex and how these proteins communicate with each other to increase the phosphatase activity of PTPµ at AJs. A detailed investigation to address these issues would be needed in the future.


Figure 7
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  		FIGURE 7.
A schematic model of inactivation of integrin {alpha}vβ3 by trans-interaction of nectin after the formation of cell-cell junctions. The details are described under "Discussion."

 
There are several reports that PIPKI{gamma}90 is involved in the regulation of integrin activation by generating phosphatidylinositol 4,5-bisphosphate and targeting talin to integrin and that the kinase activity of PIPKI{gamma}90 is up-regulated by its phosphorylation (19, 20, 22). Thus, the phosphorylation level of PIPKI{gamma}90 seems to be important for the regulation of integrin activation. c-Src and FAK are shown to phosphorylate PIPKI{gamma}90 (20, 21), but little is known regarding how PIPKI{gamma}90 is dephosphorylated. We demonstrated here that PIPKI{gamma}90 is one of the substrates of PTPµ and that among several phosphatases, PTPµ most efficiently dephosphorylates PIPKI{gamma}90 in a trans-interacting nectin-dependent manner. Actually, PTPµ inhibits the PIPKI{gamma}90-induced recruitment of talin to the cell-cell adhesion sites and the reactivation of integrin {alpha}vβ3. Thus, PTPµ and PIPKI{gamma} are involved in the dynamic regulation of integrin {alpha}vβ3 activation/inactivation during the formation of AJs.

Balanced and controlled phosphorylation of cell-cell adhesion molecules and signaling molecules related to AJs are essential for the formation and maintenance of AJs. Tyrosine phosphorylation of cadherins and their binding proteins catenins as well as nectin-induced phosphorylation of intracellular signaling molecules including c-Src effectively promote the formation of AJs (13, 41). In contrast, continuous phosphorylation of E-cadherin leads to its ubiquitination, which enhances its endocytosis and the disassembly of AJs (42). Consistent with this, knockdown of PTPµ impairs the barrier function in human lung microvascular endothelia because of the improper tyrosine phosphorylation state of VE-cadherin (43). It is of note that we newly show the contribution of PTPµ to the stability of AJs by regulating the phosphorylation state of PIPKI{gamma} and inhibiting integrin {alpha}vβ3 in addition to its role in dephosphorylation of cadherins and catenins for the maintenance of AJs.

The necessity of PIPKI{gamma} for the formation of AJs was recently reported (44). In that study, PIPKI{gamma} interacts with E-cadherin and the µ subunit of the clathrin adaptor protein complex and facilitates the E-cadherin transport to the plasma membrane through the adaptor protein complex to efficiently form AJs. Down-regulation of PIPKI{gamma} by RNA interference impairs the E-cadherin target to the plasma membrane and inhibits the formation of AJs. Combined with our findings that overexpression of PIPKI{gamma}90 in MDCK cells disrupts E-cadherin-based AJs by the reactivation of integrin {alpha}vβ3, the proper amount of PIPKI{gamma} and its controlled kinase activation and inactivation are critical for the formation and maintenance of AJs. Because the detailed mechanisms for the prolonged maintenance of AJs remain to be elucidated, further extensive studies are necessary in the future.


   FOOTNOTES
 
* This work was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (2006 and 2007). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan. Tel.: 81-78-382-5400; Fax: 81-78-382-5419; E-mail: ytakai{at}med.kobe-u.ac.jp.

2 The abbreviations used are: AJ, adherens junction; Ab, antibody; mAb, monoclonal antibody; pAb, polyclonal antibody; PIPKI{gamma}, phosphatidylinositol-phosphate kinase type I{gamma}; MDCK, Madin-Darby canine kidney; PTP, protein-tyrosine phosphatase; FAK, focal adhesion kinase; GFP, green fluorescent protein; LMW, low molecular weight; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; PIPES, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank Drs. S. J. Shatill, Y. Kanaho, M. Takeichi, P. De Camilli, S. Brady-Kalnay, T. Konodo, T. Matozaki, M. Okada, and M. Ozawa for generous gifts of reagents.



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 RESULTS
 DISCUSSION
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