Selective Uncoupling of P120ctn from E-Cadherin Disrupts Strong Adhesion

Abstract

p120ctn is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell–cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin–p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin–deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike α- and β-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin–mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin–mediated transition to tight cell–cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

“…1A, and shows that each cadherin is capable of stabilizing α-catenin and β-catenin as expected. Thoreson et al developed E-cadherin mutants with a triple alanine substitution (764EED/AAA) in the p120 binding site that prevents the binding of p120 to the cadherin [8]. When E-cadherin with the AAA mutation (Ep120AAA) or N-cadherin with an analogous mutation (Np120AAA) were expressed in MiaPaCa cells they also conferred an epithelial phenotype on the cells (Fig.…”

“…A mutation in N-cadherin (Np120AAA) that prevents binding to p120 was generated by recombinant PCR. The amino-acid alterations, E780A, E781A and D782A, correspond to those reported to be effective in uncoupling Ecadherin from p120 [8]. The details of the construction are available upon request.…”

“…Mutant E-cadherin (Ep120AAA) that cannot bind to p120 has been described [8]. A mutation in N-cadherin (Np120AAA) that prevents binding to p120 was generated by recombinant PCR.…”

“…p120 has multiple biological functions including regulation of cell adhesion by controlling cadherin turnover at the cell surface [7]. Cells expressing Ecadherin with a defective p120 binding site (764EED/AAA) failed to recruit p120 into the cadherin-catenin complex, and have reduced cell-cell adhesion, highlighting the importance of p120 in cadherin function [8].…”

Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline

“…Similarly, a reduction in E-cadherin often results in b-catenin degradation (Liu et al, 2002). Another protein associated with E-cadherin, p120 (Thoreson et al, 2000), is phosphorylated on both tyrosine and serine residues in response to a variety of growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and colony stimulating factor (CSF)-1, suggesting involvement in active signalling (Downing and Reynolds, 1991;Shibamoto et al, 1995). Thus, cell -cell adhesion serves not only a structural role but dictates cellular behaviour.…”