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The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.

ABSTRACT: Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.

SUBMITTER: Woodfield RJ

PROVIDER: S-EPMC1222233 | biostudies-other | 2001 Dec

REPOSITORIES: biostudies-other

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p85? phosphoinositide 3-kinase subunit regulates tumor progression.

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X-ray structure of the SH3 domain of the phosphoinositide 3-kinase p85? subunit.

Project description:Src-homology 3 (SH3) domains are involved in extensive protein-protein interactions and constitute key elements of intracellular signal transduction. Three-dimensional structures have been reported for SH3 domains of various proteins, including the 85 kDa regulatory subunit (p85) of phosphoinositide 3-kinase. However, all of the latter structures are of p85 isoform ? and no crystal structure of the SH3 domain of the equally important isoform ? has been reported to date. In this structural communication, the recombinant production, crystallization and X-ray structure determination at 2.0 Å resolution of the SH3 domain of human p85? is described. The structure reveals a compact ?-barrel fold very similar to that of p85?. However, binding studies with two classes of proline-rich ligand peptides demonstrate that the ligand-binding specificity differs slightly between the SH3 domains of human p85? and p85?, despite their high structural similarity.

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| S-EPMC26139 | biostudies-literature

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Project description:ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

| S-EPMC1219642 | biostudies-other

Transcriptional impairment of beta-catenin/E-cadherin complex is not associated with beta-catenin mutations in colorectal carcinomas.

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| S-EPMC2377055 | biostudies-other

Wnt/frizzled-2 signaling induces aggregation and adhesion among cardiac myocytes by increased cadherin-beta-catenin complex.

Project description:Wingless is known to be required for induction of cardiac mesoderm in Drosophila, but the function of Wnt family proteins, vertebrate homologues of wingless, in cardiac myocytes remains unknown. When medium conditioned by HEK293 cells overexpressing Wnt-3a or -5a was applied to cultured neonatal cardiac myocytes, Wnt proteins induced myocyte aggregation in the presence of fibroblasts, concomitant with increases in beta-catenin and N-cadherin in the myocytes and with E- and M-cadherins in the fibroblasts. The aggregation was inhibited by anti-N-cadherin antibody and induced by constitutively active beta-catenin, but was unaffected by dominant negative and dominant positive T cell factor (TCF) mutants. Thus, increased stabilization of complexed cadherin-beta-catenin in both cell types appears crucial for the morphological effect of Wnt on cardiac myocytes. Furthermore, myocytes overexpressing a dominant negative frizzled-2, but not a dominant negative frizzled-4, failed to aggregate in response to Wnt, indicating frizzled-2 to be the predominant receptor mediating aggregation. By contrast, analysis of bromodeoxyuridine incorporation and transcription of various cardiogenetic markers showed Wnt to have little or no impact on cell proliferation or differentiation. These findings suggest that a Wnt-frizzled-2 signaling pathway is centrally involved in the morphological arrangement of cardiac myocytes in neonatal heart through stabilization of complexed cadherin- beta-catenin.

| S-EPMC2185559 | biostudies-literature

Autophosphorylation of serine 608 in the p85 regulatory subunit of wild type or cancer-associated mutants of phosphoinositide 3-kinase does not affect its lipid kinase activity.

Project description:BACKGROUND: The ?-isoform of the Type 1A Phosphoinositide 3-kinases (PI3K?) has protein kinase activity as well as phosphoinositide lipid kinase activity. The best described substrate for its protein kinase activity is its regulatory subunit, p85?, which becomes phosphorylated on Serine 608. Phosphorylation of Serine 608 has been reported to down-regulate its lipid kinase activity. RESULTS: We have assessed whether oncogenic mutants of PI3K?, which have up-regulated lipid kinase activity, have altered levels of Serine 608 phosphorylation compared to wild type PI3K?, and whether differential phosphorylation of Serine 608 contributes to increased activity of oncogenic forms of PI3K? with point mutations in the helical or the kinase domains. Despite markedly increased lipid kinase activity, protein kinase activity was not altered in oncogenic compared to wild type forms of PI3K?. By manipulating levels of phosphorylation of Serine 608 in vitro, we found no evidence that the protein kinase activity of PI3K? affects its phosphoinositide lipid kinase activity in either wild-type or oncogenic mutants of PI3K?. CONCLUSIONS: Phosphorylation of p85? S608 is not a significant regulator of wild-type or oncogenic PI3K? lipid kinase activity.

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Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85? Homodimer.

Project description:Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85?, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85? dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85? that is functionally equivalent to native p85?. Analytical ultracentrifugation studies showed that p85? undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85? using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85? dimer. Our study provides new insight into the structure and assembly of the p85? homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

| S-EPMC4683262 | biostudies-literature

Wnt-5a-CKI{alpha} signaling promotes {beta}-catenin/E-cadherin complex formation and intercellular adhesion in human breast epithelial cells.

Project description:Wnt-5a is a non-transforming Wnt protein that is implicated in cell polarity, adhesion, and motility. We have previously shown that low expression of Wnt-5a is a predictor of shorter disease-free survival in human breast cancer. Here, we investigated whether beta-catenin/E-cadherin-mediated cell-cell adhesion was affected by loss of Wnt-5a in breast carcinomas, thereby promoting a metastatic behavior of the tumor. We show that Wnt-5a stimulation of human breast epithelial cells leads to an increased Ca(2+)-dependent cell-cell adhesion. Furthermore, Wnt-5a/casein kinase Ialpha (CKIalpha)-specific Ser-45 phosphorylation of beta-catenin is associated with an increased complex formation of beta-catenin/E-cadherin. Mutation of Ser-45 decreases the beta-catenin/E-cadherin association. Also, the inhibitory effect of Wnt-5a on breast epithelial cell invasion is reduced upon mutation of beta-catenin-Ser-45. The Wnt-5a-CKIalpha-induced Ser-45 phosphorylation does not lead to degradation of beta-catenin. Finally we show that human breast cancers lacking Wnt-5a protein have a significantly lower level of membrane-associated beta-catenin. Down-regulation of Wnt-5a expression and subsequent reduction of membrane-associated beta-catenin in invasive breast cancer, can therefore contribute to a decreased cell-cell adhesion and increased motility resulting in a higher probability for metastatic disease.

| S-EPMC2667782 | biostudies-literature

Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force.

Project description:Linkage between the adherens junction (AJ) and the actin cytoskeleton is required for tissue development and homeostasis. In vivo findings indicated that the AJ proteins E-cadherin, ?-catenin, and the filamentous (F)-actin binding protein ?E-catenin form a minimal cadherin-catenin complex that binds directly to F-actin. Biochemical studies challenged this model because the purified cadherin-catenin complex does not bind F-actin in solution. Here, we reconciled this difference. Using an optical trap-based assay, we showed that the minimal cadherin-catenin complex formed stable bonds with an actin filament under force. Bond dissociation kinetics can be explained by a catch-bond model in which force shifts the bond from a weakly to a strongly bound state. These results may explain how the cadherin-catenin complex transduces mechanical forces at cell-cell junctions.

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