Interaction of alpha-actinin with the cadherin/catenin cell-cell adhesion complex via alpha-catenin

Cadherins are Ca(2+)-dependent, cell surface glycoproteins involved in cell-cell adhesion. Extracellularly, transmembrane cadherins such as E- , P-, and N-cadherin self-associate, while intracellularly they interact indirectly with the actin-based cytoskeleton. Several intracellular proteins termed catenins, including alpha-catenin, beta- catenin, and plakoglobin, are tightly associated with these cadherins and serve to link them to the cytoskeleton. Here, we present evidence that in fibroblasts alpha-actinin, but not vinculin, colocalizes extensively with the N-cadherin/catenin complex. This is in contrast to epithelial cells where both cytoskeletal proteins colocalize extensively with E-cadherin and catenins. We further show that alpha- actinin, but not vinculin, coimmunoprecipitates specifically with alpha- and beta-catenin from N- and E-cadherin-expressing cells, but only if alpha-catenin is present. Moreover, we show that alpha-actinin coimmunoprecipitates with the N-cadherin/catenin complex in an actin- independent manner. We therefore propose that cadherin/catenin complexes are linked to the actin cytoskeleton via a direct association between alpha-actinin and alpha-catenin.     

A C-terminal dimerization motif is required for focal adhesion targeting of Talin1 and the interaction of the Talin1 I/LWEQ module with F-actin

Focal adhesion complexes are plasma membrane-associated multicomponent complexes that are essential for integrin-linked signal transduction as well as cell adhesion and cell motility. The cytoskeletal protein Talin1 links integrin adhesion receptors with the actin cytoskeleton. Talin1 and the other animal and amoebozoan talins are members of the I/LWEQ module superfamily, which also includes fungal Sla2 and animal Hip1/Hip1R. The I/LWEQ module is a conserved C-terminal structural element that is critical for I/LWEQ module protein function. The I/LWEQ module of Talin1 binds to F-actin and targets the protein to focal adhesions in vivo. The I/LWEQ modules of Sla2 and Hip1 are required for the participation of these proteins in endocytosis. In addition to these roles in I/LWEQ module protein function, we have recently shown that the I/LWEQ module also contains a determinant for protein dimerization. Taken together, these results suggest that actin binding, subcellular targeting, and dimerization are associated in I/LWEQ module proteins. In this report we have used alanine-scanning mutagenesis of a putative coiled coil at the C-terminus of the Talin1 I/LWEQ module to show that the amino acids responsible for dimerization are necessary for F-actin binding, the stabilization of actin filaments, the cross-linking of actin filaments, and focal adhesion targeting. Our results suggest that this conserved dimerization motif in the I/LWEQ module plays an essential role in the function of Talin1 as a component of focal adhesions and, by extension, the other I/LWEQ module proteins in other multicomponent assemblies involved in cell adhesion and vesicle trafficking.     

The C-terminal globular domain of the prion protein is necessary and sufficient for import into the endoplasmic reticulum

The mammalian prion protein (PrP) is composed of an unstructured flexible N-terminal region and a C-terminal globular domain. We examined the import of PrP into the endoplasmic reticulum (ER) of neuronal cells and show that information present in the C-terminal globular domain is required for ER import of the N terminus. N-terminal fragments of PrP, devoid of structural domains located in the C terminus, remained in the cytosol with an uncleaved signal peptide and were rapidly degraded by the proteasome. Conversely, the separate C-terminal domain of PrP, comprising the highly ordered helix 2-loop-helix 3 motif, was entirely imported into the ER. As a consequence, two PrP mutants linked to inherited prion disease in humans, PrP-W145Stop and PrP-Q160Stop, were partially retained in the cytosol. The cytosolic fraction was characterized by an uncleaved N-terminal signal peptide and was degraded by the proteasome. Our study identified a new regulatory element in the C-terminal globular domain of PrP necessary and sufficient to promote import of PrP into the ER.     

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.     

Calcium-dependent adhesion is necessary for the maintenance of prosomeres

Cell adhesion has been suggested to function in the establishment and maintenance of the segmental organization of the central nervous system. Here we tested the role of different classes of adhesion molecules in prosencephalic segmentation. Specifically, we examined the ability of progenitors from different prosomeres to reintegrate and differentiate within various brain regions after selective maintenance or removal of different classes of calcium-dependent versus -independent surface molecules. This analysis implicates calcium-dependent adhesion molecules as central to the maintenance of prosomeres. Only conditions that spared calcium-dependent adhesion systems but ablated more general (calcium-independent) adhesion systems resulted in prosomere-specific integration after transplantation. Among the members of this class of adhesion molecules, R-cadherin shows a striking pattern of prosomeric expression during development. To test whether expression of this molecule was sufficient to direct progenitor integration to prosomeres expressing R-cadherin, we used a retroviral-mediated gain-of-function approach. We found that progenitors originally isolated from prosomere P2 (a region which does not express R-cadherin), when forced to express this molecule, can now integrate more readily into R-cadherin-expressing regions, such as the cortex, the ventral thalamus, and the hypothalamus. Nonetheless, our analysis suggests that while calcium-dependent molecules are able to direct prosomere-specific integration, they are not sufficient to induce progenitors to change their regional identity. While diencephalic progenitors from R-cadherin-expressing regions of prosomere 5 could integrate into R-cadherin-expressing regions of the cortex, they did not express the cortex-specific gene Emx1 or the telencephalic-specific gene Bf-1. Furthermore, diencephalic progenitors that integrate heterotopically into the cortex do not persist postnatally, whereas the same progenitors survive and differentiate when they integrate homotopically into the diencephalon. Together our results implicate calcium-dependent adhesion molecules as key mediators of prosomeric organization but suggest that they are not sufficient to bestow regional identities.     

Directed actin polymerization is the driving force for epithelial cell-cell adhesion

We have found that epithelial cells engage in a process of cadherin-mediated intercellular adhesion that utilizes calcium and actin polymerization in unexpected ways. Calcium stimulates filopodia, which penetrate and embed into neighboring cells. E-cadherin complexes cluster at filopodia tips, generating a two-rowed zipper of embedded puncta. Opposing cell surfaces are clamped by desmosomes, while vinculin, zyxin, VASP, and Mena are recruited to adhesion zippers by a mechanism that requires alpha-catenin. Actin reorganizes and polymerizes to merge puncta into a single row and seal cell borders. In keratinocytes either null for alpha-catenin or blocked in VASP/Mena function, filopodia embed, but actin reorganization/polymerization is prevented, and membranes cannot seal. Taken together, a dynamic mechanism for intercellular adhesion is unveiled involving calcium-activated filopodia penetration and VASP/Mena-dependent actin reorganization/polymerization.     

An evolutionarily conserved motif in the TAB1 C-terminal region is necessary for interaction with and activation of TAK1 MAPKKK

TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor beta, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique alpha-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and its Caenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. elegans homolog of TAB1, TAP-1, was able to interact with and activate the C. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo.     

Coordinate recruitment of E-cadherin and ALCAM to cell-cell contacts by alpha-catenin

Here we report on the role of alpha-catenin in the cellular localization of activated leukocyte cell adhesion molecule, ALCAM, and cadherin-mediated cell adhesion in human prostate cancer cells. Cell lines that have a functional E-cadherin-mediated cell adhesion (DU-145 and LNCaP) show ALCAM staining at cell-cell contacts. In contrast, in cell lines that lack alpha-catenin expression (ALVA-31, PC-3, and PPC-1), E-cadherin-mediated adhesion is disturbed and ALCAM staining is cytoplasmic. A role of alpha-catenin in the recruitment of E-cadherin and ALCAM to cell-cell contacts was established by transfection of an alpha-N-catenin construct into cell lines ALVA-31 and PC-3. This resulted not only in the correct assembly of E-cadherin/alpha-catenin complexes at the cell membrane but also in localization of ALCAM to cell-cell contacts, indicating that indeed alpha-catenin affects ALCAM localization.     

The interaction of azurin and C-terminal domain of p53 is mediated by nucleic acids

Azurin is bacterial protein, which was been reported to promote cancer cell death in vitro. The interaction of azurin and p53 is important for the cytotoxic effect of azurin towards cancer cells. In this study, it was found that nucleic acids mediated the interaction of azurin and the C-terminal domain of p53 (residues 352-393). The results provide novel insight into the interaction, and raising the possibility that the allosteric regulation of C-terminus of p53 by nucleic acids play an important role in the interaction of p53 with azurin. Meanwhile an elongated expressed product of azurin was cloned and purified, which was found to have stronger interaction with C-terminal domain of p53. Cytotoxicity studies showed that the cytotoxic effect of this elongated expressed product of azurin was stronger than wild-type azurin. The difference found in the cytotoxic effect of azurin with various sequence may provide valuable insight for finding more effective anticancer peptides.     

Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation

The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.     

Inactivation of Cdc42 is necessary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation

Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.     

Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.     

The N-terminal adenosine triphosphate binding domain of Hsp90 is necessary and sufficient for interaction with estrogen receptor

To understand how the molecular chaperone Hsp90 participates in conformational maturation of the estrogen receptor (ER), we analyzed the interaction of immobilized purified avian Hsp90 with mammalian cytosolic ER. Hsp90 was either immunoadsorbed to BF4 antibody-Sepharose or GST-Hsp90 fusion protein (GST.90) was adsorbed to glutathione-Sepharose. GST.90 was able to retain specifically ER, similarly to immunoadsorbed Hsp90. When cells were treated with estradiol and the hormone treatment was maintained during cell homogenization, binding, and washing steps, GST.90 still interacted efficiently with ER, suggesting that ER may form complexes with Hsp90 even after its activation by hormone and salt extraction from nuclei. The GST.90-ER interaction was consistently reduced in the presence of increasing concentrations of potassium chloride or when cytosolic ER-Hsp90 complexes were previously stabilized by molybdate, indicating that GST.90-ER complexes behave like cytosolic Hsp90-ER complexes. A purified thioredoxin-ER fusion protein was also able to form complexes with GST.90, suggesting that the presence of other chaperones is not required. ER was retained only by GST.90 deletion mutants bearing an intact Hsp90 N-terminal region (1-224), the interaction being more efficient when the charged region A was present in the mutant (1-334). The N-terminal fragment 1-334, devoid of the dimeric GST moiety, was also able to interact with ER, pointing to the monomeric N-terminal adenosine triphosphate binding region of Hsp90 (1-224) as the region necessary and sufficient for interaction. These results contribute to understand the Hsp90-dependent process responsible for conformational competence of ER.     

Proteasome-dependent degradation of alpha-catenin is regulated by interaction with ARMc8alpha

ARMc8 (armadillo-repeat-containing protein 8) is a key component of the CTLH (C-terminal to lissencephaly type-1-like homology motif) complex in mammalian cells. This complex is well conserved in Saccharomyces cerevisiae and has been characterized as a FBPase (fructose-1, 6-bisphosphatase)-degrading complex. The yeast homologue of ARMc8, Gid (glucose-induced degradation) 5p, plays an essential role in the ubiquitin- and proteasome-dependent degradation of FBPase. To elucidate the function of ARMc8, we used a yeast two-hybrid system to screen a human skeletal muscle cDNA library. alpha-Catenin was isolated as a binding protein of ARMc8alpha. This association was confirmed by co-immunoprecipitation assay using MDCK (Madin-Darby canine kidney) cells in which exogenous alpha-catenin and ARMc8alpha were overexpressed. The association was also confirmed by co-immunoprecipitation assay using endogenous proteins in untransfected MDCK cells. We then used immunofluorescence microscopy of MDCK cells and C2C12 cells to investigate the intracellular distribution of ARMc8. Exogenously expressed ARMc8 was co-localized with alpha-catenin and beta-catenin along the cell membrane, suggesting an association between alpha-catenin and ARMc8 in the cells. To compare the binding domain of alpha-catenin with ARMc8alpha with that of beta-catenin, we performed a co-immunoprecipitation assay, again using 5'- and 3'-deletion constructs of alpha-catenin. The N-terminal sequence (amino acids 82-148) of alpha-catenin was sufficient to bind to both ARMc8alpha and beta-catenin. Next, we investigated the proteasome-dependent degradation of alpha-catenin by immunoblotting using proteasome inhibitors. Co-expression of ARMc8alpha with alpha-catenin resulted in rapid degradation of the exogenous alpha-catenin. Furthermore, ARMc8 knockdown inhibited alpha-catenin degradation and prolonged the half-life of alpha-catenin. We conclude that ARMc8alpha associates with alpha-catenin and up-regulates its degradation.     

Re-orientation of cortical F-actin is not necessary for wound-induced microtubule re-orientation and cell polarity establishment

Summary To assess the relative roles of cortical actin and microtubule re-orientation in the establishment of new cell polarity, we have examined the kinetics of cortical actin re-orientation around a wedge-shaped wound in pea roots. Cortical actin re-orients from a transverse alignment to an approximately longitudinal orientation between 5 and 24h after wounding, that is, after the re-alignment of microtubules, which is known to occur before 5h post-wounding. F-actin in root cortical cells does not appear to be necessary for the establishment of new cell polarity around wounds, since normal MT re-alignment, and new planes of cell division are still established around a wound in cytochalasin treated roots. The cytochalasin treatment appeared to totally disrupt cortical and cytoplasmic F-actin in cells of the root cortex. However, in the apparent absence of F-actin in these cells, the rate of wound-induced cell division, but not cell expansion, is slower, and we suggest that an effect on the phragmosomal actin is involved. Finally, we demonstrate that new cell polarity around a wound is not established if microtubules are disrupted by the herbicide oryzalin, but after re-establishment of these arrays following a wash-out of the drug, the typical new planes of cell expansion are observed. We conclude that microtubules play a critical role in establishing and maintaining cell polarity in this system, and that cortical F-actin has a minor and presently unclear function in these processes.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - DMSO dimethylsulphoxide - EGTA ethyleneglycol-bis-(-aminoethyleter)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MBS m-maleido-benzoyl N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - MT microtubule - PIPES 1,4-piperazine-dietha-nesulphonic acid - PPB pre-prophase band - Rh-ph rhodamine phalloidin     

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.