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Volume 271, Number 38, Issue of September 20, 1996 pp. 23363-23367
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of IQGAP as a Putative Target for the Small GTPases, Cdc42 and Rac1*

(Received for publication, June 17, 1996)

Shinya Kuroda Dagger , Masaki Fukata §, Kenta Kobayashi Dagger , Masato Nakafuku Dagger , Nobuo Nomura , Akihiro Iwamatsu par and Kozo Kaibuchi Dagger ''

From the Dagger  Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-01, § Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734,  Kazusa DNA Research Institute, 1532-3 Yanauchino, Kisarazu 292, and par  Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama 236, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES

ABSTRACT

Cdc42 and Rac1 have been implicated in the regulation of various cell functions such as cell morphology, polarity, and cell proliferation. We have partially purified a Cdc42- and Rac1-associated protein with molecular mass of about 170 kDa (p170) from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTPgamma S)·glutathione S-transferase (GST)-Cdc42 and GTPgamma S·GST-Rac1 but not with the GDP·GST-Cdc42, GDP·GST-Rac1, or GTPgamma S·GST-RhoA). We identified p170 as an IQGAP, which is originally identified as a putative Ras GTPase-activating protein. Recombinant IQGAP specifically interacted with GTPgamma S·Cdc42 and GTPgamma S·Rac1. The C-terminal fragment of IQGAP was responsible for their interactions. IQGAP was specifically immunoprecipitated with dominant-active Cdc42Val12 or Rac1Val12 from the COS7 cells expressing Cdc42Val12 or Rac1Val12, respectively. Immunofluorescence analysis revealed that IQGAP was accumulated at insulin- or Rac1-induced membrane ruffling areas. This accumulation of IQGAP was blocked by the microinjection of the dominant-negative Rac1Asn17 or Cdc42Asn17. Moreover, IQGAP was accumulated at the cell-cell junction in MDCK cells, where alpha -catenin and ZO-1 were localized. These results suggest that IQGAP is a novel target molecule for Cdc42 and Rac1.

INTRODUCTION

Cdc42 and Rac1, the members of the small GTPase Rho family, are shown to regulate the cytoskeleton through the actin filament (reviewed in Refs. 1 and 2). Cdc42 and Rac1 are implicated in filopodia (3, 4) and lamellipodia (4, 5) formation, respectively, in Swiss 3T3 cells. Rac1 is shown to be involved in platelet-derived growth factor-induced membrane ruffling (5) and insulin-induced one in KB cells (6, 7). Rac1 is also shown to stimulate phosphatidylinositol 4-phosphate 5-kinase activity and actin-uncapping in human platelets (8) and arachidonic acid release in Swiss 3T3 cells or in Rat-1 cells (9). Cdc42 and Rac1 are shown to regulate the activities of c-Jun N-terminal kinase and p38 (10, 11, 12), members of mitogen-activated protein kinases. In T cells, Cdc42 is shown to regulate the polarization of both actin and microtubules toward antigen-presenting cells (13). In Drosophila, ectopic expression of dominant-active Cdc42 inhibits the dendrite and axon growth of neural cells and normal muscle formation (14), and that of dominant-negative Cdc42 inhibits the apico-basal elongation of wing disc epithelial cells (15). Ectopic expression of dominant-active Rac inhibits only axon growth (14) and assembly of actin at adherence junction of wing disc epithelial cells (15), and that of dominant-negative Rac causes disruption of cell shape changes in the lateral epidermis (16). Ectopic expression of dominant-active Rac1 in mice results in the reduction of Purkinje cell axon terminals (17).

Cdc42 and Rac1 have two interconvertible forms: GDP-bound inactive forms and GTP-bound active forms (reviewed in Refs. 1 and 18) and their GTP-bound forms interact with their target molecules and exert their biological functions. The target molecules of Cdc42 and Rac1 have been identified to be serine/threonine kinase PAK (19, 20, 21), phosphatidylinositol 3'-kinase (22, 23), and WASP1 (24, 25); the target molecule of Cdc42 is ACK (26), and the target molecule of Rac1 in neutrophils is p67 phox (27, 28). To understand the pleiotropic functions of Cdc42 and Rac1, it is important to identify their novel targets.

We have previously purified target molecules for Rho by the Rho affinity column chromatography and identified them as protein kinase N (29), Rho-kinase (30), and myosin-binding subunit of myosin phosphatase (31). In the present study, we purified a putative target for Cdc42 and Rac1 with a molecular mass of 170 kDa and identified it as an IQGAP, which is originally isolated as a Ras GAP family (32, 33).

EXPERIMENTAL PROCEDURES

Materials and Chemicals

Anti-alpha -catenin antibody and anti-ZO-1 antibody were kindly provided by Drs. A. Nagafuchi and S. Tsukita (Kyoto University, Kyoto, Japan), and Drs. M. Itoh and S. Tsukita (Kyoto University), respectively. KB cells and MDCK cells were provided from Health Science Research Resources Bank (Osaka, Japan). C3 exoenzyme was kindly provided by Dr. Ohashi (Nihon Shering, Osaka, Japan). Other materials and chemicals were obtained from commercial sources.

Plasmid Construction

pGEX2T-Cdc42, pGEX2T-Rac1, and pGEX2TRhoA were constructed as described previously (29, 34). Cdc42Val12 and Rac1Val12, or Cdc42Asn17 and Rac1Asn17 cDNAs were generated by the site-directed mutagenesis of Gly12 and Thr17 to Val12 and Asn17, respectively. Recombinant wild-type and mutant small GTPases were expressed as GST-fusion proteins and purified as described (29). For microinjection, they were cleaved with thrombin, purified to remove the GST, and concentrated (5, 35). For expression in COS7 cells, pEF-BOS-HA-small GTPases were constructed as described (29, 36). To obtain the IQGAP (521-914 amino acids) as an antigen, a cDNA fragment corresponding to the IQGAP fragment was subcloned into pGEX4T-2. For in vitro translation of IQGAP, the cDNA fragments corresponding to the indicated IQGAP fragments were subcloned into pBluescript KS(-) having a sequence encoding a myc epitope tag (MEQKLISEEDL).

GST-Cdc42 and GST-Rac1 Affinity Column Chromatography

The affinity purification was performed essentially as described (29). Briefly, bovine brain cytosol was passed through glutathione beads to remove endogenous GST. Then, the pass fraction was loaded on glutathione beads containing respective GST-small GTPases preloaded with guanine nucleotides as described (29, 37). After washing the columns, bound proteins were coeluted with respective GST-small GTPases by the addition of reduced glutathione.

Interaction of in Vitro Translated IQGAP with Small GTPases

The interactions of indicated fragments of in vitro translated IQGAP with GST-small GTPases were examined as described (38). Briefly, indicated fragments of in vitro translated IQGAP were mixed with affinity beads coated with the respective guanine nucleotide-bound GST-small GTPases. After washing the beads, the bound proteins were coeluted with respective small GTPases by the addition of glutathione. The eluates were resolved by SDS-PAGE, and radioactivities were detected using the bioimaging analyzer BAS2000 (Fujix, Tokyo, Japan).

Overlay Assay

The overlay assay was performed as described (19, 30). Briefly, an aliquot of the eluate fraction of GTPgamma S·GST-Cdc42 affinity column was subjected to SDS-PAGE and blotted onto nitrocellulose membrane. After the denaturation with guanidinium hydrochloride and the subsequent renaturation, the membrane was probed with [35S]GTPgamma S·GST-small GTPases. The bound small GTPases to IQGAP were visualized using bioimaging analyzer.

Coimmunoprecipitation of IQGAP with HA-Small GTPases

COS7 cells were transfected with pEF-BOS-HA-small GTPases by use of the DEAE-dextran method (39). Immunoprecipitation of HA-tagged small GTPases by anti-HA antibody was performed as described (29). The immunocomplex was subjected to SDS-PAGE, followed by immunoblotting using anti-IQGAP antibody.

Microinjection and Immunofluorescence Analysis

KB cells were cultured, seeded, and starved for 36 h as described (6). Microinjection of small GTPases (1 mg/ml) or C3 (200 µg/ml) followed by the stimulation of insulin was performed as described (6). MDCK cells were cultured and seeded as described (40). Immunofluorescence analysis with anti-IQGAP antibody was carried out essentially as described (41).

Other Procedures

The peptide sequence of p170 was determined as described (37, 42). Anti-IQGAP polyclonal antibody was raised by standard procedures using GST-IQGAP (521-914 amino acids) as an antigen.

RESULTS AND DISCUSSION

To identify Cdc42- and Rac1-interacting molecules, the bovine brain cytosol was loaded onto a GST-Cdc42 affinity column. The proteins bound to the affinity column were coeluted with GST-Cdc42 by the addition of glutathione. A protein with molecular mass of about 170 kDa (p170) was detected in the glutathione-eluate from GTPgamma S·GST-Cdc42 affinity column but not from GST or GDP·GST-Cdc42 affinity column (Fig. 1). Less amount of p170 was detected in the eluate from the GTPgamma S·GST-Rac1 affinity column but not from the GDP·GST-Rac1 affinity column. To further confirm the specificity of the interaction, affinity column chromatography using GST-RhoA was performed (29). p170 was not eluted from the GTPgamma S·GST-RhoA affinity column. p170 was eluted from neither GST-Ha-Ras nor GST-RalA affinity columns (data not shown). Proteins with molecular masses of 62 kDa (p62), 90 kDa (p90), and 110 kDa (p110) were also detected in both of the eluates from GTPgamma S·GST-Rac1 and GTPgamma S·GST-Cdc42 affinity columns. p62 was recognized by anti-PAK antibody (data not shown). Proteins with molecular masses of 122 kDa (p122) and 140 kDa (p140) were specifically detected in the eluate of GTPgamma S·GST-Rac1. Identifications of p90, p110, p122, and p140 are currently under investigation.2

Fig. 1. Purification of Cdc42- or Rac1-interacting proteins. The bovine brain cytosol was loaded onto a glutathione-agarose column containing the indicated GST-small GTPases. The bound proteins were coeluted with the respective GST-small GTPases by the addition of glutathione. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining. Lane 1, GDP·GST-Cdc42; lane 2, GTPgamma S·GST-Cdc42; lane 3, GDP·GST-Rac1; lane 4, GTPgamma S·GST-Rac1; lane 5, GDP·GST-RhoA; lane 6, GTPgamma S·GST-RhoA; lane 7, GST. An arrowhead denotes the position of p170. Arrows from the top denote the positions of p140, p122, p110, and p90, respectively. The results shown are representative of three independent experiments.
[View Larger Version of this Image (88K GIF file)]

To identify the p170, it was subjected to amino acid sequencing as described (42). Three peptide sequences derived from p170 were determined. The amino acid sequences of the peptides are YGEQVDYYK, IFYPETTDIYDRK, and RLIVDVIRFQPGETLTEILE. All of the three peptide sequences obtained were identical to the deduced amino acid sequence of human IQGAP, which is identified as a Ras GAP family (32, 33). Furthermore, p170 was recognized by anti-IQGAP antibody (data not shown). The molecular weight of IQGAP is calculated to be 189,249, which is almost the same as the apparent molecular mass of p170 estimated by SDS-PAGE. We, therefore, concluded that p170 was a bovine counterpart of human IQGAP and hereafter refer to it as IQGAP.

To examine whether recombinant IQGAP interacts with GTPgamma S·Cdc42 or GTPgamma S·Rac1, the affinity beads coated with GST-small GTPases were mixed with in vitro translated IQGAP. After washing the affinity beads, GST-small GTPases were eluted by the addition of glutathione. In vitro translated IQGAP coeluted strongly with GTPgamma S·GST-Cdc42 or GTPgamma S·GST-Rac1 (Fig. 2A). It was slightly retained on and eluted from the GDP·GST-Cdc42 affinity beads, but it was not detected in the eluate of GDP·GST-Rac1, GST-RhoA, or GST affinity beads.

Fig. 2. Interactions of in vitro translated IQGAP and affinity-purified IQGAP with activated Cdc42 and Rac1. A and B, in vitro translated IQGAP was produced and its interaction with GST-small GTPases was determined as described under ``Experimental Procedures.'' A, with in vitro translated IQGAP (1-1657 amino acids). B, with in vitro translated C-terminal IQGAP (764-1657 amino acids). Lanes are the same as those in the legend of Fig. 1. Arrowheads in A and B denote the positions of the in vitro translated IQGAP (1-1657 amino acids) and the in vitro translated C-terminal IQGAP (764-1657 amino acids), respectively. C, the interaction of affinity-purified IQGAP with [35S]GTPgamma S·GST-small GTPases was examined using an overlay assay method as described under ``Experimental Procedures.'' The membrane was probed with [35S]GTPgamma S·GST-Cdc42 (lane 1), [35S]GTPgamma S·GST-Rac1 (lane 2), or [35S]GTPgamma S·GST-RhoA (lane 3). An arrowhead denotes the position of the affinity-purified IQGAP. The results shown are representative of three independent experiments.
[View Larger Version of this Image (28K GIF file)]

To determine the binding domain of IQGAP to Cdc42 and Rac1, the N-terminal (1-863 amino acids) and the C-terminal (764-1657 amino acids) fragments of IQGAP were in vitro translated, and their interactions with Cdc42 and Rac1 were examined. The C-terminal fragment of IQGAP interacted with GTPgamma S·Cdc42 or GTPgamma S-Rac1 (Fig. 2B), whereas the N-terminal fragment did not (data not shown). Both fragments interacted with neither GST, GDP·Cdc42, nor GDP·Rac1.

Furthermore, direct interaction of the purified IQGAP with GTPgamma S·GST-Cdc42 or GTPgamma S·GST-Rac1 was examined using the overlay assay method (19, 30). [35S]GTPgamma S·GST-Cdc42 and [35S]GTPgamma S·GST-Rac1 bound to the purified IQGAP, whereas [35S]GTPgamma S·GST-RhoA did not (Fig. 2C). The consensus sequence of the Cdc42- or Rac1-binding domain of target proteins, such as PAK or WASP, has been determined and was termed as CRIB (25, 43). There is no CRIB domain in the fragment (764-1657 amino acids). Therefore, Cdc42 or Rac1 interacts with at least two distinct target interfaces.

We next examined whether IQGAP interacts with Cdc42 or Rac1 in vivo. Cdc42Val12 and Rac1Val12, structurally equivalent to RasVal12 (18), are thought to be a constitutively GTP-bound form in vivo as a result of defective GTPase activity (3, 4, 5), whereas Cdc42Asn17 and Rac1Asn17, structurally equivalent to RasAsn17 (44), are thought to be a constitutively GDP-bound form in vivo (3, 4, 5) as a result of preferential binding to GDP relative to GTP and to specifically interfere in the activation of endogenous respective small GTPases. When HA-tagged Cdc42Val12, Cdc42, Cdc42Asn17, Rac1Val12, or RhoAVal14 was transiently transfected into COS7 cells and HA-small GTPases were immunoprecipitated by anti-HA antibody, IQGAP was coimmunoprecipitated with Cdc42Val12 or Rac1Val12 (Fig. 3). It coimmunoprecipitated with neither Cdc42, Cdc42Asn17, nor RhoAVal14. Almost the same amounts of the HA-small GTPases were precipitated (data not shown). Therefore, it is most likely that IQGAP specifically interacts with GTP·Cdc42 and GTP·Rac1 in vivo.

Fig. 3. In vivo association of IQGAP with activated Cdc42 and Rac1 in COS7 cells. pEF-BOS-HA-small GTPases were transfected into COS7 cells and incubated for 48 h. After the incubation, HA-tagged small GTPases were immunoprecipitated by anti-HA antibody (12CA5). The immunoprecipitates were washed and subjected to SDS-PAGE, followed by immunoblotting with anti-IQGAP antibody. Lane 1, Cdc42Val12; lane 2, Cdc42; lane 3, Cdc42Asn17; lane 4, Rac1Val12; lane 5, RhoAVal14; lane 6, mock. An arrowhead denotes the position of IQGAP. The results shown are representative of three independent experiments.
[View Larger Version of this Image (12K GIF file)]

IQGAP was originally identified as a Ras GAP, but the recombinant IQGAP did not show any GAP activity toward Ras and Rho (33). We also attempted to detect GAP activity using affinity-purified IQGAP toward Ha-Ras, Ki-Ras, R-Ras, RalA, Cdc42, Rac1, and RhoA but failed (data not shown). We cannot exclude the possibility that the purified IQGAP lost its activity. Further study is necessary to clarify the function of the GAP domain of IQGAP.

Insulin is shown to induce membrane ruffling in KB cells (45, 46), and the insulin-induced membrane ruffling is blocked by the prior microinjection of Rac1Asn17 into the cells but not by that of C3 (6). Microinjection of GTPgamma S·Rac1 is shown to induce the membrane ruffling in KB cells (6). Then, we examined the localization of IQGAP in KB cells. When KB cells were stimulated with insulin, membrane ruffling was induced, and IQGAP accumulated at the membrane ruffling area (Fig. 4, A and B). The insulin-induced membrane ruffling and IQGAP accumulation at the membrane ruffling area were blocked by prior microinjection of Rac1Asn17 (Fig. 4, C and D). The prior microinjection of Cdc42Asn17 also blocked the insulin-induced membrane ruffling and the IQGAP accumulation in some injected cells (Fig. 4, E and F), whereas both processes weakly occurred in other injected cells. Cdc42Asn17 may not completely block insulin-dependent activation of endogenous Rac1. The prior microinjection of C3 did not block the insulin-induced membrane ruffling and IQGAP accumulation (Fig. 4, G and H). Microinjection of Rac1Val12 induced membrane ruffling and IQGAP accumulation at the membrane ruffling area (Fig. 4, I and J). That of Cdc42Val12 induced filopodia formation, but IQGAP was not accumulated at the filopodia (data not shown). It should be noted that IQGAP was accumulated at the cell-cell junction of KB cells.

Fig. 4. Specific localization of IQGAP at the insulin- or Rac1-induced membrane ruffling area in KB cells. KB cells were starved for 36 h, and microinjection of the indicated proteins was performed. Thirty minutes after the microinjection, cells were stimulated with 1 µ insulin for 5 min, fixed, and stained with anti-IQGAP antibody. A, C, E, G, and I, phase-contrast microscopy. B, D, F, H, and J, fluorescence microscopy. A and B, with microinjection of buffer alone; C and D, with microinjection of Rac1Asn17; E and F, with microinjection of Cdc42Asn17; G and H, with microinjection of C3; I and J, with microinjection of Rac1val12. Arrows in I and J indicate the Rac1val12-injected cells. The results shown are representative of three independent experiments. Bar, 20 µm. All photographs were taken with the same magnification.
[View Larger Version of this Image (64K GIF file)]

To further examine the IQGAP accumulation at the cell-cell junction, we examined the localization of IQGAP in MDCK cells. IQGAP was specifically accumulated at the cell-cell junction (Fig. 5, B and E). IQGAP showed similar distribution as alpha -catenin and ZO-1, which are marker molecules of adherence junction and tight junction, respectively, at the cell-cell junction (Fig. 5, C and F). It seems that IQGAP is well colocalized with alpha -catenin, whereas IQGAP is not completely colocalized with ZO-1. It remains to be clarified whether IQGAP is localized at the adherence junction or tight junction in MDCK cells.3 This observation suggests that IQGAP regulates the cell-cell junction, or that the cell-cell junctional signal leads to the IQGAP-accumulation.3 This accumulation was not affected by the addition of insulin or by microinjection of various small GTPases. It is possible that Rac1 or Cdc42 is not necessary for the localization of IQGAP at the cell-cell junction once IQGAP is complexed with the cell-cell junctional cytoskeleton, or that activation of Rac1 or Cdc42 at the cell-cell junction is blocked by neither Rac1Asn17 nor Cdc42Asn17. When actin filament was visualized with rhodamine-labeled phalloidin, IQGAP was observed in association with cortical actin at the membrane ruffling area in KB cells (Fig. 6).

Fig. 5. Specific localization of IQGAP at the cell-cell junction in MDCK cells. MDCK cells were cultured and seeded as described (40). Forty-eight hours after seeding, cells were fixed and doubly stained with anti-IQGAP antibody and anti-alpha -catenin antibody or anti-ZO-1 antibody. A and D, phase-contrast microscopy. B, C, E, and F, fluorescence microscopy. B and E, with anti-IQGAP antibody; C, with anti-alpha -catenin antibody; F, with anti-ZO-1 antibody. Fluorescein-labeled anti-rat IgG antibody for alpha -catenin, fluorescein-labeled anti-mouse IgG antibody for ZO-1, and Texas Red-labeled anti-rabbit IgG antibody for IQGAP were used as secondary antibody. The results shown are representative of three independent experiments. Bar, 20 µm. All photographs were taken with the same magnification.
[View Larger Version of this Image (62K GIF file)]

Fig. 6. Colocalization of IQGAP with actin filament at the insulin-induced membrane ruffling area in KB cells. KB cells were starved for 36 h and stimulated with 1 µ insulin. Five minutes after the stimulation, cells were fixed and doubly stained with anti-IQGAP antibody and rhodamine-labeled phalloidin. A, phase-contrast microscopy. B and C, fluorescence microscopy. B, with anti-IQGAP antibody. C, with rhodamine-labeled phalloidin. The results shown are representative of three independent experiments. Bar, 20 µm. All photographs were taken with the same magnification.
[View Larger Version of this Image (32K GIF file)]

We here identified the Cdc42- or Rac1-interacting p170 protein as IQGAP. We showed that recombinant IQGAP directly interacts with active forms of Rac1 and Cdc42 in vitro and in vivo. Moreover, we found that IQGAP is accumulated at the insulin- or Rac1-induced membrane ruffling area, and that the insulin-induced membrane ruffling and the IQGAP accumulation at the membrane ruffling area is blocked by the prior microinjection of Rac1Asn17 or Cdc42Asn17. Our results indicate that IQGAP is the target for Rac1 and Cdc42 and suggest that IQGAP is involved in the insulin- and Rac1-induced membrane ruffling.

The targets for Cdc42 and Rac1 are identified as PAK (19, 20, 21), phosphatidylinositol 3'-kinase (22, 23), and WASP (24, 25). Among these targets, the localization of WASP is determined (25). WASP was clustered and associated with actin filament when WASP was transiently expressed in cells (25), but it was not observed that WASP was accumulated at the membrane ruffling area. WASP is expressed exclusively in cells of hematopoietic lineage (47, 48). PAK (19, 21) and phosphatidylinositol 3'-kinase (49) are ubiquitously expressed, but there is no evidence obtained thus far that these molecules are accumulated at the membrane ruffling area. We showed that IQGAP is accumulated at the insulin- or Rac1-induced membrane ruffling area. On the basis of these observations, it is likely that the target molecules for Rac1 and Cdc42, including IQGAP, have distinct roles, and that IQGAP functions in concert with other targets, resulting in the exertion of pleiotropic functions of Rac1 and Cdc42.

FOOTNOTES

*   This study was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports and Culture, Japan, and by a grant from the Yamanouchi Foundation for Research on Metabolic Disease. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
''   To whom correspondence should be addressed. Tel.: 81-7437-2-5440; Fax: 81-7437-2-5449; E-mail: kaibuchi{at}bs.aist-nara.ac.jp.
1   The abbreviations used are: WASP, Wiskott-Aldrich syndrome protein; GAP, GTPase-activating protein; GST, glutathione S-transferase; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-(3-O-thio)triphosphate; CRIB, Cdc42/Rac1 interactive binding region.
2   Identifications of p90, p110, p122, and p140 will be described elsewhere.
3   A detailed analysis will be described elsewhere.

Acknowledgments

We thank Dr. A. Nagafuchi and Dr. S. Tsukita (Kyoto University) for providing anti-alpha -catenin antibody, Dr. M. Itoh and Dr. S. Tsukita (Kyoto University) for providing anti-ZO-1 antibody, and Dr. S. Tsukita for helpful discussion.

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