A new 400-kD protein from isolated adherens junctions: its localization at the undercoat of adherens junctions and at microfilament bundles such as stress fibers and circumferential bundles

In the previous study, we succeeded in isolating the cell-to-cell adherens junctions from rat liver (Tsukita, S., and S. Tsukita. 1989. J. Cell Biol. 108:31-41.). In this study, we have obtained mAbs specific to the 400-kD protein, which was identified as one of the major constituents of the undercoat of isolated adherens junctions. Immune blot analyses showed that this protein occurs in various types of tissues. Immunofluorescence microscopy and immune electron microscopy have revealed that this protein is distributed not only at the undercoat of adherens junctions but also along actin bundles associated with the junction in nonmuscle cells: stress fibers in cultured fibroblasts and circumferential bundles in epithelial cells. The partially purified protein molecule looks like a slender rod approximately 400 nm in length. By virtue of its molecular shape, we have named this protein 'tenuin' (from Latin 'tenuis', thin or slender).     

Isolation of cell-to-cell adherens junctions from rat liver

A new isolation procedure for cell-to-cell adherens junctions has been developed using rat liver. From the bile canaliculi-enriched fraction obtained by homogenization of the liver and sucrose gradient centrifugation, the fraction rich in adherens junction was recovered by detergent treatment followed by sucrose gradient centrifugation. Light and electron microscopy revealed that this final fraction was mainly composed of the belt-like adherens junctions with their associated short actin filaments. Biochemical and immunological analyses have shown that vinculin is highly enriched in this fraction. Considering that vinculin is known to be localized in the cell-to-cell adherens junctions, we can conclude that we have succeeded in isolating the cell-to-cell adherens junctions. Furthermore, the constituents of the undercoat (dense layer underlying the membrane) of adherens junctions were selectively extracted from the fraction rich in junctions. Upon SDS electrophoresis of this extract, 10 polypeptides including vinculin, alpha-actinin, and actin were dominant. The results obtained are discussed with special reference to the molecular organization of the undercoats of cell-to-cell adherens junctions.     

A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites

Recently we developed an isolation procedure for the cell-to-cell adherens junctions (AJ; cadherin-based junctions) from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41). In this study, using the isolated AJ, we have obtained two mAbs specific to the 220-kD undercoat-constitutive protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this 220-kD protein was highly concentrated at the undercoat of cell-to-cell AJ in various types of tissues and that this protein was located in the immediate vicinity of the plasma membrane in the undercoat of AJ. In the cells lacking typical cell-to-cell AJ, such as fibroblasts, the 220-kD protein was immunofluorescently shown to be coconcentrated with cadherin molecules at cell-cell adhesion sites. These localization analyses appeared to indicate the possible direct or indirect association of the 220-kD protein with cadherin molecules. Furthermore, it was revealed that the 220-kD protein and alpha-spectrin were coimmunoprecipitated with the above mAbs in both the isolated AJ and the brain. The affinity-purified 220-kD protein molecule looked like a spherical particle, and its binding site on the spectrin molecule was shown to be in the position approximately 10-20 nm from the midpoint of spectrin tetramer by low-angle rotary-shadowing electron microscopy. Taking all these results together with biochemical and immunological comparisons, we are persuaded to speculate that the 220-kD protein is a novel member of the ankyrin family. However, the possibility cannot be excluded that the 220-kD protein is an isoform of beta-spectrin. The possible roles of this 220-kD protein in the association of cadherin molecules with the spectrin-based membrane skeletons at the cadherin-based cell-cell adhesion sites are discussed.     

Radixin is a novel member of the band 4.1 family

Radixin is an actin barbed-end capping protein which is highly concentrated in the undercoat of the cell-to-cell adherens junction and the cleavage furrow in the interphase and mitotic phase, respectively (Tsukita, Sa., Y. Hieda, and Sh. Tsukita. 1989 a.J. Cell Biol. 108:2369-2382; Sato, N., S. Yonemura, T. Obinata, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 113:321-330). To further understand the structure and functions of the radixin molecule, we isolated and sequenced the cDNA clones encoding mouse radixin. Direct peptide sequencing of radixin and immunological analysis with antiserum to a fusion protein were performed to confirm that the protein encoded by these clones is identical to radixin. The composite cDNA is 4,241 nucleotides long and codes for a 583-amino acid polypeptide with a calculated molecular mass of 68.5 kD. Sequence analysis has demonstrated that mouse radixin shares 75.3% identity with human ezrin, which was reported to be a member of the band 4.1 family. We then isolated the cDNA encoding mouse ezrin. Sequence analysis and Northern blot analysis revealed that radixin and ezrin are similar but distinct (74.9% identity), leading us to conclude that radixin is a novel member of the band 4.1 family. In erythrocytes the band 4.1 protein acts as a key protein in the association of short actin filaments with a plasma membrane protein (glycophorin), together with spectrin. Therefore, the sequence similarity between radixin and band 4.1 protein described in this study favors the idea that radixin plays a crucial role in the association of the barbed ends of actin filaments with the plasma membrane in the cell-to-cell adherens junction and the cleavage furrow.     

Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis [published erratum appears in J Cell Biol 1991 Sep;114(5):1101-3]

Radixin is a barbed end-capping actin-modulating protein which was first identified in isolated cell-to-cell adherens junctions from rat liver (Tsukita, Sa., Y. Hieda, and Sh. Tsukita, 1989. J. Cell Biol. 108:2369-2382). In the present study, we have analyzed the distribution of radixin in dividing cells. For this purpose, an mAb specific for radixin was obtained using chicken gizzard radixin as an antigen. By immunofluorescence microscopy with this mAb and a polyclonal antibody obtained previously, it was clearly shown in rat fibroblastic cells (3Y1 cells) that radixin was highly concentrated at the cleavage furrow during cytokinesis. Radixin appeared to accumulate rapidly at the cleavage furrow at the onset of furrowing, continued to be concentrated at the furrow during anaphase and telophase, and was finally enriched at the midbody. This concentration of radixin at the cleavage furrow was detected in all other cultured cells we examined: bovine epithelial cells (MDBK cells), mouse myeloma cells (P3 cells), rat kangaroo Ptk2 cells, mouse teratocarcinoma cells, and chicken fibroblasts. Furthermore, it became clear that the epitope for the mAb was immunofluorescently masked in the cell-to-cell adherens junctions. Together, these results lead us to conclude that radixin is present in the undercoat of the cell-to-cell adherens junctions and that of the cleavage furrow, although their respective molecular architectures are distinct. The possible roles of radixin at the cleavage furrow are discussed with special reference to the molecular mechanism of the actin filament-plasma membrane interaction at the furrow.     

The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy

We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.

The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.     

Rabbit alveolar macrophages contain a Ca2+-sensitive, 41,000-dalton protein which reversibly blocks the "barbed" ends of actin filaments but does not sever them

A 41,000-dalton Ca2+-sensitive actin-modulating protein has been purified from rabbit alveolar macrophages using ion exchange and gel filtration chromatography. On sodium dodecyl-polyacrylamide gel electrophoresis, this macrophage protein migrates more rapidly than actin and fails to cross-react with polyclonal anti-actin antibody. It has a Stokes radius of 3.0 nm and an isoelectric point of 6.6. In the presence of micromolar Ca2+ this 41,000-Da protein: reduces the viscosity of polymerized actin, nucleates actin filament assembly, causes a nearly instantaneous increase in fluorescence intensity of subcritical concentrations of pyrenyl-actin (estimated KD of the pyrene actin-macrophage protein complex, 5 X 10(-8) M), increases the critical concentration of actin by 0.65 microM (molar ratios of protein/actin, 1/100-1/10), blocks actin monomer depolymerization from the "barbed" filament ends, and does not sever preformed actin filaments. The ability of this protein to block filament ends is rapidly and completely inhibited by lowering free calcium ion concentration below the micromolar range.     

Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction

A novel actin filament (F-actin)–binding protein with a molecular mass of ~205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.     

Effects of macrophage profilin on actin in the presence and absence of acumentin and gelsolin

We purified profilin from rabbit alveolar macrophages and documented its structural and functional similarity to profilins isolated from other cells. The KD for formation of the macrophage profilin-actin complex was 3.0 +/- 0.8 microM (mean +/- S.D.). The affinity of this protein for actin did not change significantly in the presence of various concentrations of KCl and MgCl2, profilin-actin complex concentration being strictly dependent on the critical actin monomer concentration and free profilin concentration. We also examined profilin's interactions with actin in the presence of acumentin, a macrophage protein which inhibits actin monomer exchange at the "pointed" ends of actin filaments. Low concentrations of this protein caused substantial decreases in estimated profilin-actin complex concentration. The macrophage gelsolincalcium ion complex which blocks exchange at the "barbed" end of actin filaments, when added to profilin and actin solutions in substoichiometric concentrations, caused large increases in estimated profilin-actin complex concentration. The changes in calculated profilin-actin complex concentration induced by these two actin-modulating proteins were too large to be explained solely by their effects on critical actin monomer concentration.     

Purification and characterization of zyxin, an 82,000-dalton component of adherens junctions

We describe here the purification and characterization of a recently identified adherens junction protein that has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels (Beckerle, M. C. (1986) J. Cell Biol. 103, 1679-1687). The 82-kDa protein was isolated from avian smooth muscle by a low ionic strength alkaline pH extraction followed by ammonium sulfate fractionation. Sequential chromatographic separation using DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite resins results in a purified 82-kDa protein. The 82-kDa protein has a Stokes radius of 5.6 nm and a relative sedimentation coefficient of 3.0 S. The calculated native molecular mass of the protein based on its hydrodynamic properties is 69 kDa, and the derived frictional ratio (f/fo) is 2.1. The protein does not focus discretely by isoelectric-focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis; there are numerous isoelectric point variants in the range of 6.4-7.2, with the average isoelectric point being 6.9. The 82-kDa protein is phosphorylated in vivo and appears to be a cytoplasmic component of adherens junctions. The properties of the 82-kDa protein distinguish it from other known adherens junction proteins of this molecular mass. In fibroblasts, the 82-kDa protein is found in adhesion plaques as well as along actin-containing stress fibers near where they terminate at sites of cell-substratum adhesion. It is also found in the cell-cell adherens junctions of pigmented retinal epithelial cells and the dense plaques of smooth muscle cells. Since the 82-kDa protein is found at both cell-substratum and cell-cell adherens junctions, we propose to call it zyxin, meaning a joining, to indicate that it is found at regions where extracellular ligands are structurally and functionally joined to the cytoskeleton.     

Modification of epithelial cell barrier permeability and intercellular junctions by Clostridium sordellii lethal toxins

Clostridium sordellii lethal toxin (LT) is a glucosyltransferase which inactivates small GTPases from the Rho and Ras families. In the present work, we studied the effects of two variants, LT82 and LT9048, on the integrity of epithelial cell barrier using polarized MCCD (Mouse Cortical Collecting Duct) and MDCK (Madin-Darby Canine Kidney) cells. Our results demonstrate for the first time that LTs have very limited effects on tight junctions. In contrast, we show that both toxins modified the paracellular permeability within 2-4 h. Concomitantly LT82 and LT9048 induced a disorganization of basolateral actin filaments, without modifying apical actin. Both toxins mainly altered adherens junctions by removing E-cadherin-catenin complexes from the membrane to the cytosol. Similar effects on adherens junctions have been observed with other toxins, which directly or indirectly depolymerize actin. Thereby, Rac, a common substrate of both LTs, might play a central role in LT-dependent adherens junction alteration. Here, we show that adherens junction perturbation induced by LTs results neither from a direct effect of toxins on adherens junction proteins nor from an actin-independent Rac pathway, but rather from a Rac-dependent disorganization of basolateral actin cytoskeleton. This further supports that a dynamic equilibrium of cortical actin filaments is essential for functional E-cadherin organization in epithelia.     

A novel actin filament-capping protein from sea urchin eggs: a 20,000-molecular-weight protein-actin complex

A novel protein factor which reduced the low-shear viscosity of rabbit skeletal muscle actin was purified from a 0.6 M KCl-extract of an insoluble fraction of sea urchin eggs by ammonium sulfate fractionation, gel filtration column chromatography, DNase I column chromatography, and hydroxylapatite column chromatography. This protein factor was shown to be a one-to-one complex of a 20,000-molecular-weight protein and egg actin. This protein complex accelerated the initial rate of actin polymerization, but reduced the steady-state viscosity of F-actin. It inhibited at substoichiometric amounts the elongation of actin filaments on sonicated F-actin fragments and depolymerization of F-actin induced by dilution. In addition, it increased the critical concentration of actin for polymerization. All these effects of this protein complex on actin could be explained by the "capping the barbed end" of the actin filament by the complex. The 20,000-molecular-weight protein which was separated from actin also possessed the barbed end-capping activities, but differed from the complex in that it did not accelerate the polymerization of actin.     

Regulated tyrosine phosphorylation at the tips of growth cone filopodia

Several types of evidence suggest that protein-tyrosine phosphorylation is important during the growth of neuronal processes, but few specific roles, or subcellular localizations suggestive of such roles, have been defined. We report here a localization of tyrosine-phosphorylated protein at the tips of growth cone filopodia. Immunocytochemistry using a mAb to phosphorylated tyrosine residues revealed intense staining of the tips of most filopodia of Aplysia axons growing slowly on a polylysine substrate, but of few filopodia of axons growing rapidly on a substrate coated with Aplysia hemolymph, which has growth-promoting material. Cytochalasin D, which causes F-actin to withdraw rapidly from the growth cone, caused the tyrosine-phosphorylated protein to withdraw rapidly from filopodia, suggesting that the protein associates or interacts with actin filaments. Phosphotyrosine has previously been found concentrated at adherens junctions, where bundles of actin filaments terminate, but video-enhanced contrast-differential interference contrast and confocal interference reflection microscopy demonstrated that the filopodial tips were not adherent to the substrate. Acute application of either hemolymph or inhibitors of protein-tyrosine kinases to neurons on polylysine resulted in a rapid loss of intense staining at filopodial tips concomitant with a lengthening of the filopodia (and their core bundles of actin filaments). These results demonstrate that tyrosine-phosphorylated protein can be concentrated at the barbed ends of actin filaments in a context other than an adherens junction, indicate an association between changes in phosphorylation and filament dynamics, and provide evidence for tyrosine phosphorylation as a signaling mechanism in the filopodium that can respond to environmental cues controlling growth cone dynamics.     

Isolation and partial characterization of a 110-kD dimer actin-binding protein

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.     

A 45,000-mol-wt protein-actin complex from unfertilized sea urchin egg affects assembly properties of actin

A one-to-one complex of a 45,000-mol-wt protein and actin was purified from unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, by means of DNase l-Sepharose affinity and gel filtration column chromatographies. Effects of the complex on the polymerization of actin were studied by viscometry, spectrophotometry, and electron microscopy. The results are summarized as follows: (a) The initial rate of actin polymerization is inhibited at a very low molar ratio of the complex to actin. (b) Acceleration of the initial rate of polymerization occurs at a relatively high, but still substoichiometric, molar ratio of the complex to actin. (c) Annealing of F-actin fragments is inhibited by the complex. (d) The complex prevents actin filaments from depolymerizing. (e) Growth of the actin filament is inhibited at the barbed end. In all cases except b, a molar ratio of less than 1:100 of the 45,000-mol-wt protein-actin complex to actin is sufficient to produce these significant effects. These results indicate that the 45,000-mol-wt protein-actin complex from the sea urchin egg regulates the assembly of actin by binding to the barbed end (preferred end or rapidly growing end) of the actin filament. The 45,000-mol-wt protein-actin complex can thus be categorized as a capping protein.     

JACOP, a novel plaque protein localizing at the apical junctional complex with sequence similarity to cingulin

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.     

Restricted tissue distribution of a 37-kD possible adherens junction protein

A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.     

Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.