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Journal of Cell Science, Vol 104, Issue 3 751-762, Copyright © 1993 by Company of Biologists
JOURNAL ARTICLES |
PA Piepenhagen and WJ Nelson
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305-5426.
Ca(2+)-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include alpha-, beta-, and gamma-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either beta- or gamma-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and beta-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of gamma-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and beta-catenin co-immunoprecipitate with E-cadherin. Laser-scanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with gamma-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for < 50% of the gamma-catenin signal. Two-dimensional gel electrophoresis resolved the gamma-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI approximately 6.1). The other protein comigrated with gamma-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of approximately 4.25; we refer to this protein as gamma-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains alpha-, beta-, and gamma-catenin. Although plakoglobin shares sequence similarity with beta-catenin, and comigrates with gamma-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The association of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.
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