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Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin.

ABSTRACT: The dynamic functional linkage of cadherins with the underlying actin cytoskeleton is tightly regulated to achieve proper cell-cell adhesion. p120-catenin (p120) regulates both cadherin stability and actin dynamics, but the relationship between these two functions remains unclear. Using a novel proteomic approach called reversible cross-link immunoprecipitation, or ReCLIP, we previously identified a physical interaction between p120 and Rho-associated protein kinase 1 (ROCK1), a major effector of RhoA. In this paper, we show that a discrete fraction of cellular ROCK1 coimmunoprecipitates with p120 and precisely colocalizes to adherens junctions (AJs). Manipulation of AJs using a calcium-switch assay and cadherin-blocking antibodies indicates direct recruitment of ROCK1 to newly forming junctions. Importantly, we find that p120 links ROCK1 to the cadherin complex, as ROCK1 coimmunoprecipitates with wild-type but not p120-uncoupled E-cadherin. Moreover, depletion of ROCK1 using short-hairpin RNA results in dramatic mislocalization of the cadherin complex and junctional actin. These data are consistent with a model in which p120 dynamically regulates Rho-GTPase activity at the cadherin complex through transient interaction with several of its up- and downstream effectors, including ROCK1.

SUBMITTER: Smith AL

PROVIDER: S-EPMC3248908 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin.

Smith Andrew L AL Dohn Michael R MR Brown Meredith V MV Reynolds Albert B AB

Molecular biology of the cell 20111026 1

The dynamic functional linkage of cadherins with the underlying actin cytoskeleton is tightly regulated to achieve proper cell-cell adhesion. p120-catenin (p120) regulates both cadherin stability and actin dynamics, but the relationship between these two functions remains unclear. Using a novel proteomic approach called reversible cross-link immunoprecipitation, or ReCLIP, we previously identified a physical interaction between p120 and Rho-associated protein kinase 1 (ROCK1), a major effector o ...[more]

PMID: 22031287

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Project description:BackgroundDespite recent advances in the diagnosis and treatment of breast cancer, metastasis remains the main cause of death. Since migration of tumor cells is considered a prerequisite for tumor cell invasion and metastasis, a pressing goal in tumor biology has been to elucidate factors regulating their migratory activity. Protein kinase C alpha (PKC?) is a serine-threonine protein kinase implicated in cancer metastasis and associated with poor prognosis in breast cancer patients. In this study, we set out to define the signaling axis mediated by PKC? to promote breast cancer cell migration.MethodsOncomine™ overexpression analysis was used to probe for PRKCA (PKC?) and FOXC2 expression in mRNA datasets. The heat map of PRKCA, FOXC2, and CTNND1 were obtained from the UC Santa Cruz platform. Survival data were obtained by PROGgene and available at http://www.compbio.iupui.edu/proggene . Markers for EMT and adherens junction were assessed by Western blotting and quantitative polymerase chain reaction. Effects of PKC? and FOXC2 on migration and invasion were assessed in vitro by transwell migration and invasion assays respectively. Cellular localization of E-cadherin and p120-catenin was determined by immunofluorescent staining. Promoter activity of p120-catenin was determined by dual luciferase assay using a previously validated p120-catenin reporter construct. Interaction between FOXC2 and p120-catenin promoter was verified by chromatin immunoprecipitation assay.ResultsWe determined that PKC? expression is necessary to maintain the migratory and invasive phenotype of both endocrine resistant and triple negative breast cancer cell lines. FOXC2 acts as a transcriptional repressor downstream of PKC?, and represses p120-catenin expression. Consequently, loss of p120-catenin leads to destabilization of E-cadherin at the adherens junction. Inhibition of either PKC? or FOXC2 is sufficient to rescue p120-catenin expression and trigger relocalization of p120-catenin and E-cadherin to the cell membrane, resulting in reduced tumor cell migration and invasion.ConclusionsTaken together, these results suggest that breast cancer metastasis may partially be controlled through PKC?/FOXC2-dependent repression of p120-catenin and highlight the potential for PKC? signal transduction networks to be targeted for the treatment of endocrine resistant and triple negative breast cancer.

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Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin.

Publications

Association of Rho-associated protein kinase 1 with E-cadherin complexes is mediated by p120-catenin.

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