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Masayuki Ozawa - One of the best experts on this subject based on the ideXlab platform.

  • Identification of regions of α-Catenin required for desmosome organization in epithelial cells
    International journal of molecular medicine, 2005
    Co-Authors: Tamotsu Taniguchi, Mutsumi Miyazaki, Yayoi Miyashita, Terukatsu Arima, Masayuki Ozawa
    Abstract:

    Alpha-Catenin, a cadherin-associated protein, links cadherin/beta-Catenin and cadherin/plakoglobin complexes to the actin cytoskeleton. This protein is required for the function of cadherins, cell adhesion molecules. We transfected an Alpha-Catenin-deficient colon carcinoma line, which cannot organize desmosomes, with a series of Alpha-Catenin mutant constructs. We examined the formation of desmosomes in these cells by immunofluorescence staining using anti-desmoglein and anti-desmoplakin antibodies. The results demonstrated that either the middle or the carboxy-terminal region of Alpha-Catenin was required for desmosome formation. Immunoblot analysis revealed that the amounts of desmoglein and desmoplakin did not differ significantly between cells that were capable of forming desmosomes and those that failed to form desmosomes. Cell surface biotinylation revealed that desmoglein was retained intracellularly in cells that could not organize desmosomes. The internal domain binds vinculin and Alpha-actinin, actin-binding proteins, while the carboxy-terminal domain has the ability to bind and bundle actin filaments. These results indicate that the interaction of Alpha-Catenin and actin functions in the assembly of desmosomes in epithelial cells.

  • The reduced expression of e-cadherin, Alpha-Catenin and gamma-Catenin but not beta-Catenin in human lung cancer.
    Oncology Reports, 1999
    Co-Authors: Hironobu Toyoyama, Kensuke Nuruki, Hiroki Ogawa, Masayuki Yanagi, Hidehiko Matsumoto, Hiroo Nishijima, Tetsurou Shimotakahara, Takashi Aikou, Masayuki Ozawa
    Abstract:

    : Cadherins are Ca2+-dependent cell-cell adhesion molecules, and are involved in the formation and maintenance of the histo-architecture. Using a combination of biochemical and immunohistochemical methods, we analyzed the expression of cadherin-Catenin complexes in 37 non-small cell lung carcinomas. In 19 cases, decreased expression of E-cadherin protein was observed. In 12 of them, decreased expression of Alpha-Catenin protein was also observed. Thus, decreased expression of Alpha-Catenin was apparently preceded by decreased expression of E-cadherin. In no cases was decreased expression of beta-Catenin observed. In the 12 cases in which mRNA expression was analyzed by Northern blot analysis, decreased expression of mRNAs for E-cadherin and Alpha-Catenin was observed in 11 and 9 cases, respectively. In cases with reduced E-cadherin and Alpha-Catenin expression, immunohistochemistry revealed two types of staining pattern for the proteins. In the first type, almost all the cells in a tumor were stained weakly (homogeneous pattern). In the second type, different percentages of cells were stained strongly, the rest being almost negative for the staining (heterogeneous pattern).

  • Altered cell adhesion activity by pervanadate due to the dissociation of Alpha-Catenin from the E-cadherin.Catenin complex.
    The Journal of biological chemistry, 1998
    Co-Authors: Masayuki Ozawa, Rolf Kemler
    Abstract:

    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, beta-Catenin, and gamma-Catenin (plakoglobin), but Alpha-Catenin was not detectably phosphorylated. After pervanadate treatment, beta-Catenin and gamma-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 Alpha-Catenin was dissociated from the E-cadherin.Catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with Alpha-Catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of Alpha-Catenin from the complex underlies the observed E-cadherin dysfunction.

  • The fourth armadillo repeat of plakoglobin (gamma-Catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cadherin Dsg2, and the tumor suppressor APC protein.
    Journal of biochemistry, 1995
    Co-Authors: Masayuki Ozawa, Hirokazu Terada, Claudio Pedraza
    Abstract:

    Plakoglobin is a member of a protein family with a repeated amino acid motif, the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Plakoglobin has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to complex with APC, the tumor suppressor gene product. Recently we isolated a cDNA clone encoding plakoglobin lacking the fourth armadillo repeat of the original 13-repeat protein [Ozawa et al. (1995) J. Biochem. 118, 836-840]. In this study, we established an in vitro assay system to study the molecular interaction of plakoglobin with cadherins, the APC gene product, and Alpha-Catenin. Establishment of the system and cloning of an alternate form of plakoglobin cDNA allowed us to examine the biological activity of plakoglobin lacking the fourth armadillo repeat. Experiments with the bacterially expressed 12-repeat plakoglobin revealed that the protein binds to E-cadherin, desmoglein (Dsg2), and APC with lower affinity than the 13-repeat form does. Consistent with the observation that the affinity of Alpha-Catenin for these two alternate forms was similar, we found amino acid residues 104 to 145 of plakoglobin, the residues present in both isoforms, are sufficient for its binding to Alpha-Catenin.

  • The uvomorulin-anchorage protein Alpha Catenin is a vinculin homologue.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Kurt Herrenknecht, Masayuki Ozawa, Christoph Eckerskorn, Friedrich Lottspeich, Martin Lenter, Rolf Kemler
    Abstract:

    Abstract The cytoplasmic region of the Ca(2+)-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed Alpha, beta, and gamma Catenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against Alpha Catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that Alpha Catenin is complexed with other cadherins. In immunofluorescence tests, Alpha Catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk- cells, Alpha Catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of Alpha Catenin at membrane areas of cell contacts. We also have cloned and sequenced murine Alpha Catenin. The deduced amino acid sequence reveals a significant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.

Akira Nagafuchi - One of the best experts on this subject based on the ideXlab platform.

  • α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin
    Molecular biology of the cell, 2001
    Co-Authors: Shigekazu Yokoyama, Morito Monden, Akira Nagafuchi, Kouichi Tachibana, Hiroyuki Nakanishi, Yasunori Yamamoto, Kenji Irie, Kenji Mandai, Yoshimi Takai
    Abstract:

    ZO-1 is an actin filament (F-actin)-binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through beta- and Alpha-Catenins as one of many F-actin-binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin-binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin-beta-Catenin complex through Alpha-Catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-Catenin and nectin-afadin systems, and Alpha-Catenin-deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only Alpha-Catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of Alpha-Catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with Alpha-Catenin but also with the nectin-afadin system.

  • Co-expression of E-cadherin and α-Catenin molecules in colorectal cancer
    Surgery today, 1999
    Co-Authors: Tatsushi Kitagawa, Shoichiro Tsukita, Akira Nagafuchi, Koichi Matsumoto, Hiroshi Suzuki
    Abstract:

    Immunohistochemical staining for epithelial (E)-cadherin and Alpha-Catenin was performed using frozen sections taken from fresh operative specimens, by the avidin-biotin-peroxidase complex method. Tumors were classified into three types according to the expression modality. Cancer cells with expression at the cell-cell boundaries were defined as normal; when the expression was positive, but not concentrated at the cell-cell boundaries, they were defined as cytoplasmic; and when the tumor showed no staining, they were defined as lost. The relationship between these three expression types and the clinicopathological features of colorectal cancer was investigated. In all 50 normal mucosa samples, E-cadherin and Alpha-Catenin were coexpressed normally. The expression type of E-cadherin and Alpha-Catenin was normal in 11 and 13 of the cancer tissue specimens, respectively, cytoplasmic in 26 and 29, respectively, and lost in 13 and 8, respectively. Cytoplasmic or lost expression was observed in cancer demonstrating an advanced clinical stage (E-cadherin, P = 0.0065; Alpha-Catenin, P = 0.0069), advanced tumor penetration (P = 0.0003, P = 0.0001), undifferentiated tumor histology (P = 0.0196, P = 0.0343), widespread lymph node involvement (P = 0.0204, P = 0.0340), and liver metastasis (P = 0.0063, P = 0.0299). In conclusion, the expression type of E-cadherin is significantly correlated to that of Alpha-Catenin, and the loss of their expression indicates the metastatic potentiality of colorectal cancer.

  • dynamics of connexins e cadherin and Alpha Catenin on cell membranes during gap junction formation
    Journal of Cell Science, 1997
    Co-Authors: Kazushi Fujimoto, Shoichiro Tsukita, Akira Nagafuchi, Akio Kuraoka, Akiko Ohokuma, Yosaburo Shibata
    Abstract:

    We examined the dynamics of connexins, E-cadherin and Alpha-Catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or Alpha-Catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-Catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

  • A Truncated β-Catenin Disrupts the Interaction between E-Cadherin and α-Catenin: A Cause of Loss of Intercellular Adhesiveness in Human Cancer Cell Lines
    Cancer research, 1994
    Co-Authors: Tsukasa Oyama, Shoichiro Tsukita, Akira Nagafuchi, Atsushi Ochiai, Shingo Akimoto, Yae Kanai, Tatsuya Oda, Kazuyoshi Yanagihara, Sayumi Shibamoto, Fumiaki Ito
    Abstract:

    Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with Catenins, a group of cytoplasmic proteins including Alpha- and beta-Catenins. While Alpha-Catenin has been demonstrated to be crucial for cadherin function, the role of beta-Catenin is not yet fully understood. In this study, we analyzed the cadherin-Catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-Catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-Catenin gene. On the other hand, these cells expressed E-cadherin, Alpha-Catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-Catenin but not with Alpha-Catenin, and antibodies against beta-Catenin did not copurify Alpha-Catenin. However, the recombinant fusion protein containing wild-type beta-Catenin precipitated Alpha-Catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with Alpha-Catenin, and that this defect results from the mutation in beta-Catenin. Thus, it is most likely that the association between E-cadherin and Alpha-Catenin is mediated by beta-Catenin, and that this process is blocked by NH2-terminal deletion in beta-Catenin. These findings indicate that genetic abnormality of beta-Catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.

  • The roles of Catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-Alpha Catenin fusion molecules.
    The Journal of cell biology, 1994
    Co-Authors: Akira Nagafuchi, S Ishihara, Shoichiro Tsukita
    Abstract:

    The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called Alpha and beta Catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and Alpha Catenin, nE Alpha, nE Alpha N, and nE Alpha C, where the intact, amino-terminal and carboxy-terminal half of Alpha Catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE Alpha and nE Alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE Alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-Catenin complex, the mechanical association of Alpha Catenin, especially its carboxy-terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE Alpha molecules during cytokinesis was quite different from that of intact E-cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE Alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE Alpha was not associated with endogenous beta Catenin in transfectants, the difference in the nature of cell adhesion between nE Alpha and intact E-cadherin transfectants may be explained by the function of beta Catenin. The possible functions of beta Catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.

Shoichiro Tsukita - One of the best experts on this subject based on the ideXlab platform.

  • Co-expression of E-cadherin and α-Catenin molecules in colorectal cancer
    Surgery today, 1999
    Co-Authors: Tatsushi Kitagawa, Shoichiro Tsukita, Akira Nagafuchi, Koichi Matsumoto, Hiroshi Suzuki
    Abstract:

    Immunohistochemical staining for epithelial (E)-cadherin and Alpha-Catenin was performed using frozen sections taken from fresh operative specimens, by the avidin-biotin-peroxidase complex method. Tumors were classified into three types according to the expression modality. Cancer cells with expression at the cell-cell boundaries were defined as normal; when the expression was positive, but not concentrated at the cell-cell boundaries, they were defined as cytoplasmic; and when the tumor showed no staining, they were defined as lost. The relationship between these three expression types and the clinicopathological features of colorectal cancer was investigated. In all 50 normal mucosa samples, E-cadherin and Alpha-Catenin were coexpressed normally. The expression type of E-cadherin and Alpha-Catenin was normal in 11 and 13 of the cancer tissue specimens, respectively, cytoplasmic in 26 and 29, respectively, and lost in 13 and 8, respectively. Cytoplasmic or lost expression was observed in cancer demonstrating an advanced clinical stage (E-cadherin, P = 0.0065; Alpha-Catenin, P = 0.0069), advanced tumor penetration (P = 0.0003, P = 0.0001), undifferentiated tumor histology (P = 0.0196, P = 0.0343), widespread lymph node involvement (P = 0.0204, P = 0.0340), and liver metastasis (P = 0.0063, P = 0.0299). In conclusion, the expression type of E-cadherin is significantly correlated to that of Alpha-Catenin, and the loss of their expression indicates the metastatic potentiality of colorectal cancer.

  • Expression of E-Cadherin and α-Catenin in Patients with Colorectal Carcinoma: Correlation with Cancer Invasion and Metastasis
    American journal of clinical pathology, 1999
    Co-Authors: Junji Gofuku, Hitoshi Shiozaki, Shigeo Matsui, Shoichiro Tsukita, Shigeyuki Tamura, Masatoshi Inoue, Yuichiro Doki, Toshimasa Tsujinaka, Nobuteru Kikkawa, Morito Monden
    Abstract:

    Cadherins form complexes with groups of cytoplasmic proteins, such as Alpha-, beta-, and gamma-Catenins, that link the cadherin molecule to the cytoskeleton. In this study, we conducted an immunohistochemical investigation of E-cadherin and Alpha-Catenin expression in 100 tissue samples obtained from colorectal cancer patients undergoing surgical treatment. Reduced expression of Alpha-Catenin was observed in 56 (56%) of the cases and found to be significantly correlated with the depth of invasion of the patients' colorectal cancer and its metastasis to lymph nodes and liver. In contrast, E-cadherin expression was reduced in 29 (29%) of the cases and was not significantly correlated with either depth of invasion or metastasis. Although the levels of expression of these proteins were positively correlated, coexpression pattern analysis showed that invasion and metastasis were correlated with a reduction of Alpha-Catenin expression regardless of the status of E-cadherin staining. Thus, to predict tumor invasion and metastasis in colorectal adenocarcinoma, it is useful to investigate not just the expression of E-cadherin but also the expression of Alpha-Catenin.

  • dynamics of connexins e cadherin and Alpha Catenin on cell membranes during gap junction formation
    Journal of Cell Science, 1997
    Co-Authors: Kazushi Fujimoto, Shoichiro Tsukita, Akira Nagafuchi, Akio Kuraoka, Akiko Ohokuma, Yosaburo Shibata
    Abstract:

    We examined the dynamics of connexins, E-cadherin and Alpha-Catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or Alpha-Catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-Catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

  • A Truncated β-Catenin Disrupts the Interaction between E-Cadherin and α-Catenin: A Cause of Loss of Intercellular Adhesiveness in Human Cancer Cell Lines
    Cancer research, 1994
    Co-Authors: Tsukasa Oyama, Shoichiro Tsukita, Akira Nagafuchi, Atsushi Ochiai, Shingo Akimoto, Yae Kanai, Tatsuya Oda, Kazuyoshi Yanagihara, Sayumi Shibamoto, Fumiaki Ito
    Abstract:

    Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with Catenins, a group of cytoplasmic proteins including Alpha- and beta-Catenins. While Alpha-Catenin has been demonstrated to be crucial for cadherin function, the role of beta-Catenin is not yet fully understood. In this study, we analyzed the cadherin-Catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-Catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-Catenin gene. On the other hand, these cells expressed E-cadherin, Alpha-Catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-Catenin but not with Alpha-Catenin, and antibodies against beta-Catenin did not copurify Alpha-Catenin. However, the recombinant fusion protein containing wild-type beta-Catenin precipitated Alpha-Catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with Alpha-Catenin, and that this defect results from the mutation in beta-Catenin. Thus, it is most likely that the association between E-cadherin and Alpha-Catenin is mediated by beta-Catenin, and that this process is blocked by NH2-terminal deletion in beta-Catenin. These findings indicate that genetic abnormality of beta-Catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.

  • The roles of Catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-Alpha Catenin fusion molecules.
    The Journal of cell biology, 1994
    Co-Authors: Akira Nagafuchi, S Ishihara, Shoichiro Tsukita
    Abstract:

    The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called Alpha and beta Catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and Alpha Catenin, nE Alpha, nE Alpha N, and nE Alpha C, where the intact, amino-terminal and carboxy-terminal half of Alpha Catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE Alpha and nE Alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE Alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-Catenin complex, the mechanical association of Alpha Catenin, especially its carboxy-terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE Alpha molecules during cytokinesis was quite different from that of intact E-cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE Alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE Alpha was not associated with endogenous beta Catenin in transfectants, the difference in the nature of cell adhesion between nE Alpha and intact E-cadherin transfectants may be explained by the function of beta Catenin. The possible functions of beta Catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.

Rolf Kemler - One of the best experts on this subject based on the ideXlab platform.

  • Protein kinase CKII regulates the interaction of beta-Catenin with Alpha-Catenin and its protein stability.
    Journal of cell science, 2002
    Co-Authors: Rolf Kemler
    Abstract:

    beta-Catenin is a multi-functional cellular component and a substrate for several protein kinases. Here we investigated the interaction of protein kinase CKII (casein kinase II) and beta-Catenin. We show that CKII phosphorylates the N-terminal region of beta-Catenin and we identified Ser29, Thr102, and Thr112 as substrates for the enzyme. We provide evidence that CKII regulates the cytoplasmic stability of beta-Catenin and acts synergistically with GSK-3beta in the multi-protein complex that controls the degradation of beta-Catenin. In comparing wild-type and Ser/Thr-mutant beta-Catenin, a decreased affinity of the mutant protein to Alpha-Catenin was observed. Moreover, kinase assays in vitro demonstrate a CKII-dependent increase in the binding of wild-type beta-Catenin with Alpha-Catenin. In line with that, cells expressing Ser/Thr-mutant beta-Catenin exhibit an increased migratory potential, which correlates with an enhanced cytosolic localization and a reduced association with the cytoskeleton of the mutant protein. From these results we conclude that CKII regulates the function of beta-Catenin in the cadherin adhesion complex as well as its cytoplasmic stability.

  • Altered cell adhesion activity by pervanadate due to the dissociation of Alpha-Catenin from the E-cadherin.Catenin complex.
    The Journal of biological chemistry, 1998
    Co-Authors: Masayuki Ozawa, Rolf Kemler
    Abstract:

    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, beta-Catenin, and gamma-Catenin (plakoglobin), but Alpha-Catenin was not detectably phosphorylated. After pervanadate treatment, beta-Catenin and gamma-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 Alpha-Catenin was dissociated from the E-cadherin.Catenin complex. Aggregates of cells expressing an E-cadherin chimeric molecule covalently linked with Alpha-Catenin were not dissociated on pervanadate treatment, supporting the idea that the dissociation of Alpha-Catenin from the complex underlies the observed E-cadherin dysfunction.

  • A specific domain in Alpha-Catenin mediates binding to beta-Catenin or plakoglobin
    Journal of Cell Science, 1997
    Co-Authors: Otmar Huber, M. Krohn, Rolf Kemler
    Abstract:

    The E-cadherin-Catenin adhesion complex has been the subject of many structural and functional studies because of its importance in development, normal tissue function and carcinogenesis. It is well established that the cytoplasmic domain of E-cadherin binds either beta-Catenin or plakoglobin, which both can assemble Alpha-Catenin into the complex. Recently we have identified an Alpha-Catenin binding site in beta-Catenin and plakoglobin and postulated, based on sequence analysis, that these protein-protein interactions are mediated by a hydrophobic interaction mechanism. Here we have now identified the reciprocal complementary binding site in Alpha-Catenin which mediates its interaction with beta-Catenin and plakoglobin. Using in vitro association assays with C-terminal truncations of Alpha-Catenin expressed as recombinant fusion proteins, we found that the N-terminal 146 amino acids are required for this interaction. We then identified a peptide of 27 amino acids within this sequence (amino acid positions 117–143) which is necessary and sufficient to bind beta-Catenin or plakoglobin. As shown by mutational analysis, hydrophobic amino acids within this binding site are important for the interaction. The results described here, together with our previous work, give strong support for the idea that these proteins associate by hydrophobic interactions of two Alpha-helices.

  • Catenins in Xenopus embryogenesis and their relation to the cadherin-mediated cell-cell adhesion system
    Development (Cambridge England), 1993
    Co-Authors: Stephan Q. Schneider, Kurt Herrenknecht, Rolf Kemler, Stefan Butz, Peter Hausen
    Abstract:

    In the course of an analysis of cell-cell adhesion in the Xenopus embryo, antibodies directed against Alpha- and beta-Catenin were applied to investigate their relation to the cadherins occurring early in this system. The results demonstrate that Alpha- and beta-Catenin are provided maternally and increase in amount throughout embryogenesis. Immunoprecipitations indicate that both of the Catenins are complexed to U-cadherin in the early phase of embryogenesis and to E-cadherin, when it appears during gastrulation. An excess of Alpha-Catenin occurs in free form in the early embryo, whereas all of the beta-Catenin seems to be complexed to cadherin. Synthesis of the two components throughout early embryogenesis and their binding to newly synthesized cadherins were demonstrated by metabolic labelling. The spatial distribution of Alpha-Catenin was analysed by immunohistology. During cleavage Alpha-Catenin is deposited evenly along the plasma membranes within the embryo, while the cell peripheries at the surface of the embryo remain devoid of Alpha-Catenin. At later stages, the pattern of Alpha-Catenin distribution becomes more complex. Quantitative differences in the intensity of staining along the plasma membranes in the different regions of the embryo can be distinguished. Particularly the appearance of E-cadherin in the gastrula ectoderm is accompanied by conspicuous depositions of Alpha-Catenin along the respective plasma membranes in this layer. All cells in the later embryo, apart from the neural crest cells, carry Alpha-Catenin on their plasma membranes indicating the universal character of cadherin-mediated cell-cell adhesion in the Xenopus embryo.

  • The uvomorulin-anchorage protein Alpha Catenin is a vinculin homologue.
    Proceedings of the National Academy of Sciences of the United States of America, 1991
    Co-Authors: Kurt Herrenknecht, Masayuki Ozawa, Christoph Eckerskorn, Friedrich Lottspeich, Martin Lenter, Rolf Kemler
    Abstract:

    Abstract The cytoplasmic region of the Ca(2+)-dependent cell-adhesion molecule (CAM) uvomorulin associates with distinct cytoplasmic proteins with molecular masses of 102, 88, and 80 kDa termed Alpha, beta, and gamma Catenin, respectively. This complex formation links uvomorulin to the actin filament network, which seems to be of primary importance for its cell-adhesion properties. We show here that antibodies against Alpha Catenin also immunoprecipitate complexes that contain human N-cadherin, mouse P-cadherin, chicken A-CAM (adherens junction-specific CAM; also called N-cadherin) or Xenopus U-cadherin, demonstrating that Alpha Catenin is complexed with other cadherins. In immunofluorescence tests, Alpha Catenin is colocalized with cadherins at the plasma membrane. However, in cadherin-negative Ltk- cells, Alpha Catenin is found uniformly distributed in the cytoplasm, suggesting some additional biological function(s). Expression of uvomorulin in these cells results in a concentration of Alpha Catenin at membrane areas of cell contacts. We also have cloned and sequenced murine Alpha Catenin. The deduced amino acid sequence reveals a significant homology to vinculin. Our results suggest the possibility of a new vinculin-related protein family involved in the cytoplasmic anchorage of cell-cell and cell-substrate adhesion molecules.

Manna Wang - One of the best experts on this subject based on the ideXlab platform.

  • Abstract LB-021: Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking Alpha-Catenin expression
    Molecular and Cellular Biology Genetics, 2017
    Co-Authors: Manna Wang, Xiangkai Ning, Ang Chen, Hongyan Huang, Qiang Sun, Zhaolie Chen, Xiaoning Wang
    Abstract:

    Majority of internal lumens in our bodies are covered with a layer of epithelial cells, whose integrity is critical for the organs to function properly. The integrity of epithelial tissues depends on intact adherens junctions (AJs), which is a multiple-components complex comprising cadherins, the transmembrane adhesion receptors, and their cytoplasmic binding proteins such as p120-Catenin and b-Catenin etc. Functional AJs is coupled with actin filaments through linker molecules, of which a-Catenin and EPLIN are best characterized. Actin polymerization and actomyosin contraction regulated by Rho GTPases and their effectors play important role in AJs maintenance and remodeling. Aberrations, structural or functional, in AJs were associated with a number of pathological conditions, such as infection, inflammation and tumors and the like. Recent studies indicated that AJs mediated the formation of cell-in-cell structures (CICs). CICs refer to the cellular structures formed between viable cells, in which one or more cells exist inside other ones. Early records on CICs could be dated back to last century, when pathologists identified this type of unusual structures in human tumor samples10. Recent progress showed that cell-in-cell structures are rather complex than initially described, and could be classified into homotypic or heterotypic CICs based on the cells involved. Heterotypic CICs are usually formed by penetration of lymphocytes into tumor cells through processes like emperitosis. Homotypic CICs are formed between cells from same type, for example, epithelial cells inside epithelial cells. Mechanisms like entosis and homotypic cell cannibalism (HoCC) are responsible for this type of CICs formation. Once formed, CICs usually result in death of the internalized cells, which lead to the conception that CICs formation is a process of cell death. Limited researches identified extensive involvement of CICs in several important biological processes including development, immune homeostasis and tumor development and evolution etc. Recently, we and others found that formation of homotypic CICs by entosis was dependent on intact AJs and polarized actomyosin contraction. Tumor cells lacking epithelial cadherins (E and P-cadherin) failed to form CICs, moreover, re-expression of E- or P-cadherin could efficiently induce CICs in these cells, suggesting that disrupting AJs is a mechanism whereby tumor cells escape entosis-mediated CICs formation. In this work, we found that tumor cells deficient of a-Catenin, a key component of functional AJs, also displayed impaired CICs formation, which could be fixed by restored expression of a -Catenin. Therefore, tumor cells could escape entotic CICs formation by targeting multiple AJs components including E-/P-cadherin and a-Catenin, and CICs formation by entosis may constitute a novel mechanism underlying the tumor suppressive function imposed by a -Catenin. Note: This abstract was not presented at the meeting. Citation Format: Manna Wang, Xiangkai Ning, Ang Chen, Hongyan Huang, Qiang Sun, Zhaolie Chen, Li Ma, Xiaoning Wang. Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking Alpha-Catenin expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-021. doi:10.1158/1538-7445.AM2017-LB-021

  • Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking Alpha-Catenin expression.
    Scientific reports, 2015
    Co-Authors: Manna Wang, Xiangkai Ning, Ang Chen, Hongyan Huang, Changxi Zhou, Sanchun Lan, Qiwei Wang
    Abstract:

    Impaired formation of homotypic cell-in-cell structures in human tumor cells lacking Alpha-Catenin expression