Masayuki Ozawa

The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species.

Uvomorulin belongs to the group of Ca2+‐dependent cell adhesion molecules, which are integral membrane proteins with several structural features in common. In particular, the cytoplasmic part of these proteins is highly conserved in different species, suggesting a common biological function. To test this assumption we transfected a uvomorulin full‐length cDNA into uvomorulin‐negative mouse NIH 3T3 and L cells. Immunoprecipitations with anti‐uvomorulin antibodies detected, in addition to uvomorulin, three independent proteins of 102, 88 and 80 kd which are of host origin and which form complexes with uvomorulin. Using cDNA constructs coding for uvomorulin with cytoplasmic or extracellular deletions it is shown that the 102, 88 and 80 kd proteins complex with the cytoplasmic domain of uvomorulin. Peptide pattern analysis revealed that these three proteins are identical in different mouse cells. When uvomorulin cDNA was introduced into cell lines from other species, such as human HeLa and avian fibroblasts, the expressed uvomorulin was also associated with endogenous 102, 88 and 80 kd proteins and, moreover, each of these proteins showed structural similarities to the respective mouse molecule. A panel of antibodies specific for known cytoplasmic proteins of mol. wts similar to those of the three proteins did not react with any of the described components. This suggests that the 102, 88 and 80 kd proteins constitute a new group of proteins for which we propose the nomenclature of catenin alpha, beta and gamma respectively. The characterization of these proteins provides a first molecular basis for a possible cytoplasmic anchorage of uvomorulin to the cytoskeleton.

Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity

Na+,K(+)-ATPase has distinctly different distributions in mesenchymal cells, where it has an unrestricted distribution over the entire cell surface, compared with polarized epithelial cells, where it is restricted to the basal-lateral membrane domain. The generation of this restricted distribution is important in mesenchyme to epithelia conversion in development and the function of transporting epithelia, but the mechanisms involved are unknown. Here we show that expression of the epithelial CAM uvomorulin in transfected fibroblasts is sufficient to induce a redistribution of Na+,K(+)-ATPase to sites of uvomorulin-mediated cell-cell contacts, similar to that in polarized epithelial cells. This restricted distribution of Na+,K(+)-ATPase occurs in the absence of tight junctions but coincides with the reorganization of the membrane cytoskeleton. The results indicate a direct role for CAMs as inducers of cell surface polarity of selective cytoplasmic and membrane proteins.

Single amino acid substitutions in one Ca2+ binding site of uvomorulin abolish the adhesive function

We show that a synthetic peptide corresponding to the sequence of one putative Ca2+ binding motif of the cell adhesion molecule uvomorulin is able to complex Ca2+. This function is abolished if the first Asp in the peptide is replaced by Lys. Accordingly, we expressed in L cells mutant uvomorulin with a replacement of Asp to Lys or Ala. Mutant protein was resistant to Ca2+/trypsin under mild conditions but became susceptible at or near the site of replacement at higher concentrations, leaving the remaining Ca2+ binding domains protected. Remarkably, in cell aggregation assays both mutant uvomorulins failed to mediate cell adhesiveness, demonstrating that a single amino acid substitution in one Ca2+ binding site inactivates the adhesive function.

The transcription factor Snail downregulates the tight junction components independently of E-cadherin downregulation

Snail, a transcriptional repressor of E-cadherin expression, is involved in epithelial-mesenchymal transitions during development. We demonstrate that Snail activity is not restricted to E-cadherin downregulation. Expression of tight junction proteins, including claudin-1, occludin and ZO-1, was downregulated in MDCK cells exogenously expressing Snail protein. Although occludin mRNA levels were downregulated by Snail expression, the transcription of claudin-1 and ZO-1 were unaffected. Reporter assays using the claudin-1 promoter region revealed that promoter activity was not affected by Snail overexpression. Decreased synthesis of claudin-1 protein was observed, however, suggesting that Snail may act in translation initiation. Snail expression also altered the splicing pattern of p120. The levels of mRNA encoding the epithelial variant decreased, while the fibroblastic mRNA form increased. Although ectopic E-cadherin expression resulted in a downregulation of Snail-induced fibronectin expression, fibroblastic morphology was affected only minimally; the expression of tight junctional proteins remained at low levels. These results indicate that Snail is involved in both the direct transcriptional repression of genes, such as E-cadherin and occludin, and post-transcriptional events, including downregulation of claudin-1. These data support the idea that Snail is a transcription factor possessing pleiotropic activities.

Altered cell adhesion activity by pervanadate due to the dissociation of alpha-catenin from the E-cadherin.catenin complex.

Leukemia cells (K562) that grow as non-adhesive single cells and have no endogenous cadherin were transfected with an E-cadherin expression vector, and cell clones stably expressing E-cadherin on their surface were established. The expression of E-cadherin induced the up-regulation of catenins, and E-cadherin became associated with catenins. The transfected cells grew as floating aggregates. Cell aggregation was Ca2+-dependent and was inhibited by E-cadherin antibodies. The aggregates dissociated into single cells on the addition of pervanadate. Pervanadate caused a dramatic augmentation of the phosphorylation of E-cadherin, β-catenin, and γ-catenin (plakoglobin), but α-catenin was not detectably phosphorylated. After pervanadate treatment, β-catenin and γ-catenin migrated more slowly on gel electrophoresis, suggesting changes in their conformations due to eventual changes in their phosphorylation levels. In the treated cells, a significant amount of α-catenin was dissociated from the E-cadherin·catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with α-catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of α-catenin from the complex underlies the observed E-cadherin dysfunction.

P120CTN BINDS TO THE MEMBRANE-PROXIMAL REGION OF THE E-CADHERIN CYTOPLASMIC DOMAIN AND IS INVOLVED IN MODULATION OF ADHESION ACTIVITY

Cadherins are transmembrane glycoproteins involved in Ca2+-dependent cell-cell adhesion. Previously, we showed that the conserved membrane-proximal region of the E-cadherin cytoplasmic domain negatively regulates adhesion activity. In this report, we provide several lines of evidence that p120ctn is involved in this negative regulation. p120ctn binds to the membrane-proximal region of the nonfunctional carboxyl-terminally deleted E-cadherin protein. An additional internal deletion in this region prevented the association with p120ctn and activated the protein, as seen in an aggregation assay. Furthermore, the nonfunctional E-cadherin can be activated through coexpression of p120ctn proteins with amino-terminal deletions, which eliminate several potential serine/threonine phosphorylation sites but do not affect the ability to bind to cadherins. Finally, we show that staurosporine, a kinase inhibitor, induces an increased electrophoretic mobility of p120ctn bound to E-cadherin polypeptides, activates the nonfunctional E-cadherin protein, and converts the wild-type E-cadherin and an E-cadherin-α-catenin chimeric protein from a cytochalasin D-sensitive to a cytochalasin D-insensitive state. Together, these results indicate that p120ctn is a modulator of E-cadherin-mediated cell adhesion.

Increased midkine gene expression in human gastrointestinal cancers.

Midkine (MK) is a product of a retinoic acid‐responsive gene, and is a novel growth differentiation factor. We examined the expression of the MK gene in specimens of 47 surgically removed human carcinomas of the gastrointestinal organs, namely, gastric, colorectal, hepatocellular, pancreatic, esophageal, ampullary duodenal and bile duct carcinomas. In most cases, the MK mRNA level was higher in cancer specimens than in the corresponding non‐cancerous tissues. Furthermore, MK mRNA was more highly expressed in the colon adenocarcinoma lesion than in the adenoma lesions, in the two familial polyposis cases. While MK mRNA was not detected in the normal liver, it became detectable in cirrhotic tissues in 2 of 4 cases, and its expression was increased in the cancerous tissues. Thus, the increase of MK mRNA level is a phenomenon seen in many human gastrointestinal carcinomas. The increased expression of the MK gene in gastric carcinoma was significantly more prominent in well and moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas and signet ring cell carcinomas.

Presenilin-1 binds cytoplasmic epithelial cadherin, inhibits cadherin/p120 association, and regulates stability and function of the cadherin/catenin adhesion complex

Here we show that presenilin-1 (PS1), a protein involved in Alzheimers disease, binds directly to epithelial cadherin (E-cadherin). This binding is mediated by the large cytoplasmic loop of PS1 and requires the membrane-proximal cytoplasmic sequence 604–615 of mature E-cadherin. This sequence is also required for E-cadherin binding of protein p120, a known regulator of cadherin-mediated cell adhesion. Using wild-type and PS1 knockout cells, we found that increasing PS1 levels suppresses p120/E-cadherin binding, and increasing p120 levels suppresses PS1/E-cadherin binding. Thus PS1 and p120 bind to and mutually compete for cellular E-cadherin. Furthermore, PS1 stimulates E-cadherin binding to β- and γ-catenin, promotes cytoskeletal association of the cadherin/catenin complexes, and increases Ca2+-dependent cell–cell aggregation. Remarkably, PS1 familial Alzheimer disease mutant ΔE9 increased neither the levels of cadherin/catenin complexes nor cell aggregation, suggesting that this familial Alzheimer disease mutation interferes with cadherin-based cell–cell adhesion. These data identify PS1 as an E-cadherin-binding protein and a regulator of E-cadherin function in vivo.

CD151 regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin cytoskeletal reorganization.

CD151, a member of the tetraspanin family proteins, tightly associates with integrin α3β1 and localizes at basolateral surfaces of epithelial cells. We found that overexpression of CD151 in A431 cells accelerated intercellular adhesion, whereas treatment of cells with anti-CD151 mAb perturbed the integrity of cortical actin filaments and cell polarity. E-Cadherin puncta formation, indicative of filopodia-based adhesion zipper formation, as well as E-cadherin anchorage to detergent-insoluble cytoskeletal matrix, was enhanced in CD151-overexpressing cells. Levels of GTP-bound Cdc42 and Rac were also elevated in CD151-overexpressing cells, suggesting the role of CD151 in E-cadherin–mediated cell–cell adhesion as a modulator of actin cytoskeletal reorganization. Consistent with this possibility, engagement of CD151 by the substrate-adsorbed anti-CD151 mAb induced prominent Cdc42-dependent filopodial extension, which along with E-cadherin puncta formation, was strongly inhibited by calphostin C, a protein kinase C (PKC) inhibitor. Together, these results indicate that CD151 is involved in epithelial cell–cell adhesion as a modulator of PKC- and Cdc42-dependent actin cytoskeletal reorganization.

Increased Internalization of p120-uncoupled E-cadherin and a Requirement for a Dileucine Motif in the Cytoplasmic Domain for Endocytosis of the Protein

E-cadherin is a member of the cadherin family of Ca2+-dependent cell-cell adhesion molecules. E-cadherin associates with β-catenin at the membrane-distal region of its cytosolic domain and with p120 at the membrane-proximal region of its cytoplasmic domain. It has been shown that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. Further, p120 knockdown by small interference RNA resulted in dose-dependent elimination of cell surface E-cadherin. Consistent with these observations, we found that selective uncoupling of p120 from E-cadherin by introduction of amino acid substitutions in the p120-binding site increased the level of E-cadherin endocytosis. The increased endocytosis was clathrin-dependent, because it was blocked by expression of a dominant-negative form of dynamin or by hypertonic shock. A dileucine motif in the juxtamembrane cytoplasmic domain is required for E-cadherin endocytosis, because substitution of these residues to alanine resulted in impaired internalization of the protein. The alanine substitutions in the p120-uncoupled construct reduced endocytosis of the protein, indicating that this motif was dominant to p120 binding in the control of E-cadherin endocytosis. Therefore, these results are consistent with the idea that p120 regulates E-cadherin endocytosis by masking the dileucine motif and preventing interactions with adaptor proteins required for internalization.