JACOP, a Novel Plaque Protein Localizing at the Apical Junctional Complex with Sequence Similarity to Cingulin
2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês
10.1074/jbc.m402616200
ISSN1083-351X
AutoresHiroe Ohnishi, Takuo Nakahara, Kyoko Furuse, Hiroyuki Sasaki, Shöichiro Tsukita, Mikio Furuse,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThe apical junctional complex is composed of various cell adhesion molecules and cytoplasmic plaque proteins. Using a monoclonal antibody that recognizes a chicken 155-kDa cytoplasmic antigen (p155) localizing at the apical junctional complex, we have cloned a cDNA of its mouse homologue. The full-length cDNA of mouse p155 encoded a 148-kDa polypeptide containing a coiled-coil domain with sequence similarity to cingulin, a tight junction (TJ)-associated plaque protein. We designated this protein JACOP (junction-associated coiled-coil protein). Immunofluorescence staining showed that JACOP was concentrated in the junctional complex in various types of epithelial and endothelial cells. Furthermore, in the liver and kidney, JACOP was also distributed along non-junctional actin filaments. Upon immunoelectron microscopy, JACOP was found to be localized to the undercoat of TJs in the liver, but in some tissues, its distribution was not restricted to TJs but extended to the area of adherens junctions. Overexpression studies have revealed that JACOP was recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts. These findings suggest that JACOP is involved in anchoring the apical junctional complex, especially TJs, to actin-based cytoskeletons. The apical junctional complex is composed of various cell adhesion molecules and cytoplasmic plaque proteins. Using a monoclonal antibody that recognizes a chicken 155-kDa cytoplasmic antigen (p155) localizing at the apical junctional complex, we have cloned a cDNA of its mouse homologue. The full-length cDNA of mouse p155 encoded a 148-kDa polypeptide containing a coiled-coil domain with sequence similarity to cingulin, a tight junction (TJ)-associated plaque protein. We designated this protein JACOP (junction-associated coiled-coil protein). Immunofluorescence staining showed that JACOP was concentrated in the junctional complex in various types of epithelial and endothelial cells. Furthermore, in the liver and kidney, JACOP was also distributed along non-junctional actin filaments. Upon immunoelectron microscopy, JACOP was found to be localized to the undercoat of TJs in the liver, but in some tissues, its distribution was not restricted to TJs but extended to the area of adherens junctions. Overexpression studies have revealed that JACOP was recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts. These findings suggest that JACOP is involved in anchoring the apical junctional complex, especially TJs, to actin-based cytoskeletons. Tight junctions (TJs), 1The abbreviations used are: TJ, tight junction; mAb, monoclonal antibody; pAb, polyclonal antibody; aa, amino acid; PBS, phosphate-buffered saline; HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JACOP, junction-associated coiled-coil protein; GST, glutathione S-transferase; Ab, antibody; DIG, digoxigenin. the most apical component of the junctional complex, play crucial roles in the epithelial and endothelial barrier function (1Farquhar M.G. Palade G.E. J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2144) Google Scholar, 2Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 3Gumbiner B. J. Cell Biol. 1993; 123: 1631-1633Crossref PubMed Scopus (341) Google Scholar, 4Anderson J.M. van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar, 5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2060) Google Scholar). TJs create the circumferential seal around cells and work as barriers against the free diffusion of solutes through the paracellular pathway (1Farquhar M.G. Palade G.E. J. Cell Biol. 1963; 17: 375-412Crossref PubMed Scopus (2144) Google Scholar, 2Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 3Gumbiner B. J. Cell Biol. 1993; 123: 1631-1633Crossref PubMed Scopus (341) Google Scholar, 4Anderson J.M. van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar, 5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2060) Google Scholar). The strength and selectivity of the TJ barrier vary among cell types depending on their physiological requirement of paracellular transport (2Schneeberger E.E. Lynch R.D. Am. J. Physiol. 1992; 262: L647-L661PubMed Google Scholar, 3Gumbiner B. J. Cell Biol. 1993; 123: 1631-1633Crossref PubMed Scopus (341) Google Scholar, 4Anderson J.M. van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar, 5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2060) Google Scholar). Furthermore, the biogenesis and barrier function of TJs are influenced by various intracellular signaling systems (4Anderson J.M. van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar). Recent progresses in the identification and characterization of TJ-constituting proteins have enabled the analysis of the molecular basis of the structure and the functional regulation of the TJ barrier. TJ strands, the intramembrane part of TJs observed by freeze-fracture electron microscopy, mainly consist of at least two types of integral membrane proteins, claudins and occludin, both of which have four membrane-spanning regions (6Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2143) Google Scholar, 7Furuse M. Fujita K. Hiiragi T. Fujimoto K. Tsukita S. J. Cell Biol. 1998; 141: 1539-1550Crossref PubMed Scopus (1730) Google Scholar). JAMs (junctional adhesion molecules), members of the immunoglobulin superfamily, are also included in or localized very close to TJ strands (8Martin-Padura I. Lostaglio S. Schneemann M. Williams L. Romano M. Fruscella P. Panzeri C. Stoppacciaro A. Luco L. Villa A. Simmons D. Dejana E. J. Cell Biol. 1998; 142: 117-127Crossref PubMed Scopus (1158) Google Scholar, 9Itoh M. Sasaki H. Furuse M. Ozaki H. Kita T. Tsukita S. J. Cell Biol. 2001; 154: 491-497Crossref PubMed Scopus (321) Google Scholar). Among these proteins, claudins, which comprise a multi-gene family containing >20 members, have been shown to contribute directly to the structure and barrier function of TJs (5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2060) Google Scholar). Similar to other intercellular junctions, TJs also contain various cytoplasmic plaque proteins that are thought to play important roles in the formation and regulation of the TJ barrier by providing a scaffold for various proteins and linking adhesion molecules to cytoskeletons (4Anderson J.M. van Itallie C.M. Am. J. Physiol. 1995; 269: G467-G475Crossref PubMed Google Scholar, 5Tsukita S. Furuse M. Itoh M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 285-293Crossref PubMed Scopus (2060) Google Scholar). Among TJ-associated plaque proteins, ZO-1, ZO-2, and ZO-3 have been well characterized, belonging to the membrane-associated guanylate kinase family (10Stevenson B.R. Siliciano J.D. Mooseker M.S. Goodenough D.A. J. Cell Biol. 1986; 103: 755-766Crossref PubMed Scopus (1289) Google Scholar, 11Gumbiner B. Lowenkopf T. Apatira D. Proc. Natl. Acad. Sci. U. S. A. 1991; 8: 3460-3464Crossref Scopus (430) Google Scholar, 12Haskins J. 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Cell Biol. 2003; 160: 729-740Crossref PubMed Scopus (171) Google Scholar), suggesting that TJs are involved in various cellular functions other than the barrier function such as cell polarization, protein transport, cell growth regulation, and so forth. Along this line, we continued to identify novel TJ constituents by raising mAbs against a junction-enriched fraction isolated from the liver (37Hirase T. Furuse M. Tsukita S. Eur. J. Cell Biol. 1997; 72: 174-181PubMed Google Scholar). Here, we describe the cDNA cloning and characterization of JACOP, a novel cytoplasmic plaque protein localizing at the apical junctional complex including TJs and AJs. This novel molecule showed significant sequence similarity to cingulin. Cloning of Mouse JACOP cDNA and Mouse Cingulin cDNA—A TJ- and AJ-enriched plasma membrane fraction was isolated from chick liver as described previously (37Hirase T. Furuse M. Tsukita S. Eur. J. Cell Biol. 1997; 72: 174-181PubMed Google Scholar). Peripheral membrane proteins were extracted from this membrane fraction with 1.5 m sodium trichloroacetate for 2 h at 4 °C. After ultracentrifugation at 100,000 × g for 1 h at 4 °C, the supernatant was recovered and dialyzed against PBS, pH 7.4. This was followed by centrifugation at 20,000 × g for 20 min at 4 °C. Chicken p155/JACOP protein was precipitated from the supernatant with protein G-Sepharose 4B beads (Amersham Biosciences) with which E14 mAb was covalently coupled by the use of dimethyl pimerimide (Pierce). The precipitated proteins were eluted from the beads with the SDS-PAGE sample buffer, separated by SDS-PAGE, and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad). After staining with Coomassie Brilliant Blue R-250, the protein band of p155/JACOP on the polyvinylidene difluoride membrane was excised and subjected to amino acid sequence analysis by the in-gel digestion method described by Rosenfeld et al. (38Rosenfeld J. Capdevielle J. Guillemot J.C. Ferrara P. Anal. Biochem. 1992; 203: 173-179Crossref PubMed Scopus (1132) Google Scholar) in which four distinct peptide sequences (Peptides 1–4 in Fig. 1B) were determined. A homology search using the GenBank™/EMBL/DDBJ data base identified a mouse EST clone (GenBank™ accession number AA144597) encoding a polypeptide, showing significant identity with Peptide 4 (Fig. 1B). Using two primers, 5′-TACTATGGCTGGAGTGT-3′ and 5′-CTGCAGCTGCTCATTCA-3′ designed from the sequence information in this mouse EST, a 348-base DNA fragment was amplified by PCR from mouse lung cDNA prepared from mouse lung total RNA with Superscript II reverse transcriptase (Invitrogen). This fragment was used as a template to generate a digoxigenin (DIG)-labeled hybridization probe with the DIG High Prime labeling kit (Roche Applied Science), and hybridization screening was performed using a Lambda ZAP mouse lung cDNA library (Clontech). Among the positive clones, cl.9Z of 2.9 kb was the longest and included the termination codon but the 5′ part of the cDNA containing the initiation codon was not found in this cDNA library. Using a hybridization probe designed from the 5′ sequence of cl.9Z, a Lambda ZAP F9 mouse teratocarcinoma cDNA library was screened and we successfully obtained a cDNA clone (cl.1–3), which contained the initiation codon and overlapped with cl.9Z. Based on the sequence information of these cDNA clones, DNA fragments that cover the full-length protein were obtained from F9 cDNA by PCR with appropriate primers and were ligated in pBluescript SK(-) to form the full-length JACOP cDNA using standard methods of molecular biology. Mouse cingulin cDNA was obtained by PCR based on the sequence information from a mouse EST (GenBank™ accession number BC042459). Using the primer pairs 5′-GAATCCGGGAGCACTGATCTGGAC-3′/5′-AGTCTGAATTGGATCACTTGTAGG-3′ and 5′-AGCATAGCCAGAGTCCCGATTCTG-3′/5′-AGACATCTTCTGCCTCTCAGCCTC-3′, two overlapping DNA fragments whose combination covers the whole open reading frame were amplified by PCR from cDNA prepared from mouse small intestine total RNA with Superscript II reverse transcriptase. Nucleotide sequences of these two fragments (1.7 and 2.4 kb, respectively) were checked not to contain mutations responsible for the amino acid replacement. They were then linked together through the SphI site in pBluescript SK(-) to generate cDNA encoding the full-length mouse cingulin. Compared with BC042459, the cloned cingulin cDNAs lacked 24 nucleotides encoding MVSPAST (aa 338–345 in BC042459), probably because of alternative splicing. Generation of Expression Constructs—Using PCR and standard methods of molecular biology, an HA epitope tag (YPYDVPDYA) with a three-glycine linker was bound to the N or C terminus of full-length JACOP. The C-terminal deletion constructs encoding aa 1–367 and 1–581 of JACOP with the N-terminal HA tag (j1–367 and j1–581, respectively) were produced by PCR using the N-terminal HA-tagged full-length JACOP construct as a template. An N-terminal deletion construct encoding aa 582–1298 of JACOP with a C-terminal HA tag (j582–1298) was produced by PCR using the C-terminal HA-tagged full-length JACOP construct as a template. DNA fragments for HA-tagged full-length JACOP and deletion constructs were subcloned into a mammalian expression vector, pCAGGSneodelEcoRI (39Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4617) Google Scholar). Northern Blotting—Total RNAs of various mouse tissues were prepared with TRIzol reagents (Invitrogen) based on the manufacturer's instructions, separated by 1.0% agarose-formaldehyde gel electrophoresis, and blotted onto the nylon membrane. A ∼800-bp DIG-labeled DNA probe was generated with the DIG-PCR amplification kit (Roche Applied Science) using two primers, 5′-TTTGGCGAATACCAACACGTA-3′ and 5′-TTTCTTGGTTTCAGAATAGGC-3′, and full-length cDNA of JACOP as a template. Northern hybridization was performed in buffer containing 5× SSC, 50% formamide, 0.1% N-lauroyl sarcosine, 0.02% SDS, and 2% blocking reagent (Roche Applied Science) at 68 °C for 12 h. The membrane was washed with 2× SSC containing 0.1% SDS at room temperature for 30 min and with 0.1× SSC containing 0.1% SDS at 68 °C for 45 min and incubated with alkaline phosphatase-conjugated anti-DIG antibody (Roche Applied Science). For the detection of hybridized probe, the membrane was soaked in CSPD chemiluminescence substrate (Roche Applied Science) and exposed to x-ray film, which was processed for imaging. As a loading control, the membrane was used for re-hybridization with a DIG-labeled probe of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antibodies—Bacterial expression constructs of the GST fusion protein containing aa 493–633 of JACOP and the maltose-binding protein fusion protein containing the identical region of JACOP were generated by subcloning the corresponding DNA fragment amplified by PCR into pGEX4T-1 (Amersham Biosciences) and pMAL-c2 (New England Biolabs), respectively. The GST-JACOP fusion protein then was produced in Escherichia coli. DH5-α was purified with glutathione-Sepharose 4B beads and subcutaneously injected into rabbits to raise anti-JACOP pAb. Anti-JACOP pAb was used for experiments after affinity purification. The obtained rabbit serum was incubated with nitrocellulose membranes blotted with the maltose-binding protein-JACOP fusion protein produced in E. coli, and the bound antibodies were eluted with 0.2 m glycine buffer, pH 2.8, followed by neutralization to pH 7.5 with 2 m Tris-HCl, pH 9.5. To produce rat anti-mouse cingulin mAb, an expression construct of the GST fusion protein containing aa 216–419 of mouse cingulin was generated by subcloning the corresponding DNA fragment amplified by PCR into pGEX4T-1. The GST-cingulin fusion protein was produced in E. coli, purified, and injected into rats. mAb production was performed as described previously (6Furuse M. Hirase T. Itoh M. Nagafuchi A. Yonemura S. Tsukita S. Tsukita S. J. Cell Biol. 1993; 123: 1777-1788Crossref PubMed Scopus (2143) Google Scholar). Mouse anti-afadin monoclonal antibody (40Sakisaka 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) was kindly provided by Dr. Yoshimi Takai (Osaka University Graduate School of Medicine, Osaka, Japan). Mouse anti-ZO-1 monoclonal antibody (T8–754) was generated and characterized as reported previously (41Itoh M. Yonemura S. Nagafuchi A. Tsukita S. Tsukita S. J. Cell Biol. 1991; 115: 1449-1462Crossref PubMed Scopus (206) Google Scholar). Mouse anti-HA tag mAb and rat anti-HA tag mAb were purchased from Covance and Roche Applied Science, respectively. Actin filaments were labeled with Alexa 488-phalloidin (Molecular Probes). Cell Culture and Transfection—Eph4 cells (a gift from Dr. Reichmann, Institute Suisse de Recherches, Lausanne, Switzerland), L cells, and NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. DNA transfection was performed using LipofectAMINE with Plus reagent (Invitrogen) according to the manufacturer's instructions. After 48 h in culture, cells were analyzed by immunoblotting or immunostaining. To obtain stable transfectants of Eph4 cells expressing j582–1298, cells were replated after 48 h after transfection and cultured in the presence of 500 μg/ml G418 for 2 weeks. SDS-PAGE and Western Blotting—SDS-PAGE (10%) was performed according to the method of Laemmli (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and the gels were stained with a silver-staining kit (Wako Pure Chemicals). For Western blotting, proteins resolved by SDS-PAGE were electrophoretically transferred from gels to nitrocellulose filters. Filters were incubated with a 1:1000 dilution of anti-JACOP rabbit serum or culture supernatant of anti-cingulin monoclonal antibody followed by biotinylated secondary antibodies and streptavidin-conjugated alkaline phosphatase. Nitro blue tetrazolium and bromochloroindoryl phosphate were used as substrates for the detection of alkaline phosphatase. Immunofluorescence Microscopy—To analyze the distribution of JACOP, various mouse tissues were cut in pieces and embedded in O.C.T. compound using liquid nitrogen. Frozen sections (∼5-μm thick) were cut in a cryostat, mounted on glass slides, air-dried, and fixed in 95% ethanol at 4 °C for 30 min followed by 100% acetone at room temperature for 1 min. They were then washed with PBS, incubated with 1% bovine serum albumin/PBS for 10 min, and finally incubated with primary antibodies at room temperature for 30 min. After being washed with PBS, samples were incubated for 30 min with secondary antibodies. For the immunostaining of cultured cells, cells plated on glass coverslips were fixed with 1% formaldehyde in PBS for 10 min at room temperature. They were then permeabilized with 0.2% Triton X-100 in PBS for 5 min and washed three times with PBS. After blocking with 1% bovine serum albumin/PBS for 10 min, the samples were treated with primary antibodies, washed with PBS, and finally incubated with secondary antibodies. As secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 647 goat anti-mouse IgG, Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes), Cy3-conjugated donkey anti-rat IgG, and Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch laboratories) were used. After being washed with PBS, samples were mounted in 90% glycerol-PBS containing 0.1% para-phenylenediamine and 1% n-propylgalate. Specimens were observed using a Zeiss Axio-phot photomicroscope or Zeiss LSM510 confocal laser-scanning microscope (Carl Zeiss). Immunoelectron Microscopy—Immunoelectron microscopy using the silver-enhancement immunogold method was performed as described previously with some modifications (43Mizoguchi A. Kim S. Ueda T. Kikuchi A. Yorifuji H. Hirokawa N. Takai Y. Biochem. Biophys. Res. Commun. 1990; 202: 1235-1243Crossref Scopus (93) Google Scholar). Frozen sections (∼10-μm thick) of tissues fixed with 2% paraformaldehyde were incubated with anti-JACOP pAb followed by a second Ab coupled with 1.4-nm gold particles (Nanoprobes Inc.). The sample-bound gold particles were silver-enhanced with the HQ-silver enhancement kit (Nanoprobes Inc.) at 25 °C for 8 min. After washes with distilled water, samples were post-fixed with 1% osmium oxide in 100 mm phosphate buffer, pH 7.3. They were dehydrated through a graded series of ethanol (50, 60, 70, 80, 90, 95, and 100%) and propylene oxide and embedded in epoxy resin. From these samples, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and then observed with a Hitachi H7500 electron microscope. Purification and cDNA Cloning of JACOP—We previously reported the identification of a novel 155-kDa protein (p155) localizing at epithelial and endothelial cell-cell junctions using the monoclonal antibody E14, which was produced in a rat immunized with a cell-cell junction-enriched plasma membrane fraction isolated from chick liver (37Hirase T. Furuse M. Tsukita S. Eur. J. Cell Biol. 1997; 72: 174-181PubMed Google Scholar). To further characterize p155, we attempted to clone its cDNA. Using protein G beads covalently coupled with E14 monoclonal antibody, the chicken p155 protein was purified from the peripheral membrane proteins extracted from the cell-cell junction-enriched plasma membrane fraction (Fig. 1A). From the protease-digested chicken p155 protein, amino acid sequences of four peptides were determined, one of which corresponded to a peptide sequence encoded by a mouse EST in the data base with high homology (Fig. 1B, Peptide 4). The full-length cDNA of this EST was cloned by hybridization screening of cDNA libraries of mouse lung and mouse F9 teratocarcinoma cells. The entire open reading frame of the obtained cDNA clone encodes 1,298 amino acid residues with a calculated molecular mass of ∼150 kDa, which is comparable with the molecular mass of p155 observed on SDS-PAGE (Fig. 1C). Furthermore, the amino acid sequence predicted from the obtained cDNA contains sequences homologous to the remaining three chicken peptides, indicating that we certainly cloned the mouse homologue of p155 (Fig. 1, B and C). Coils, Paircoil, and Multicoil programs predicted an extended dimeric coiled-coil structure in the C-terminal half (Fig. 2A). The N-terminal half was not predicted to contain a coiled-coil and was likely to assume a globular structure. Downstream of the coiled-coil domain, there was another small domain. We referred to these three domains as the head, rod, and tail from the N terminus (Fig. 2B) and designated this novel protein JACOP (junction-associated coiled-coil protein). A homology search of the databases revealed that the C-terminal half of the amino acid sequence shared similarity with the α-helical coiled-coil domain of various proteins, among which cingulin, a cytoplasmic component of TJs, was most related to JACOP (Fig. 2B). The coiled-coil domains of JACOP and mouse cingulin were 39% identical at the amino acid level (Fig. 2B). In addition, JACOP and cingulin had two highly homologous regions in their head domains, confirming the structural similarity between these two proteins (Fig. 2C). The JACOP gene appeared to be conserved among mammalian species. The human and rat (GenBank™ accession numbers NM_032866 and XM_236385, respectively) homologues of JACOP were 85 and 94% identical to mouse JACOP at the amino acid level, respectively (data not shown). To examine the tissue distribution of JACOP, Northern blot analysis was performed using mRNAs prepared from various mouse tissues. As shown in Fig. 3, a single band of ∼8 kb was detected in most tissues including non-epithelial ones. JACOP mRNA was detected in large amounts in the kidney and lung, whereas only a trac
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