Regulation of E-cadherin/Catenin Association by Tyrosine Phosphorylation

doi:10.1074/jbc.274.51.36734

Regulation of E-cadherin/Catenin Association by Tyrosine Phosphorylation*

From the ^‡Unitat de Biologia Cellular i Molecular, Institut Municipal d'Investigació Mèdica, Universitat Pompeu Fabra, C/. Dr. Aiguader 80, 08003 Barcelona and the ^‖Unitat de Biofı́sica, Departament de Bioquı́mica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

Abstract

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and β-catenins. We have examined the role of this modification in these proteins in the control of β-catenin/E-cadherin binding usingin vitro assays with recombinant proteins. Recombinant pp60^c-src efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for β-catenin and p120-catenin, respectively. pp60^c-src phosphorylation had opposing effects on the affinities of β-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of β-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of β-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) β-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60^c-src, only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of β-catenin to E-cadherin in vivo is controlled by phosphorylation of β-catenin Tyr-654.

Footnotes

↵* This work was supported in part by Grants SAF 97-0080 from the Comisión Interministerial de Ciencia y Tecnologı́a, PB95-069 from the Dirección General de Investigación Cientı́fica y Técnica, and 1997SGR31 from the Direcció General de Recerca.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ These authors made equivalent contributions to this work.
↵¶ Supported by a predoctoral fellowship from the CIRIT (Generalitat de Catalunya).
↵** Supported by a predoctoral fellowship from the Ministerio de Educación y Ciencia.
↵‡ To whom correspondence may be addressed. Tel.: 34-93-221-1009; Fax: 34-93-221-3237; E-mail: agarcia@imim.es.
↵§§ To whom correspondence may be addressed. Tel.: 34-93-581-1870; Fax: 34-93-581-1907; E-mail: mireia.dunach@uab.es.
↵2 J. Piedra, A. Garcı́a de Herreros, and M. Duñach, unpublished observations.
Abbreviations:

EGF

epidermal growth factor

GST

glutathione S-transferase

cytoEcad

cytosolic domain of E-cadherin

PAGE

polyacrylamide gel electrophoresis

Tyr(P)

phosphotyrosine

mAb

monoclonal antibody

PBS

phosphate-buffered saline
- Received March 26, 1999.
- Revision received September 17, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.