Regulation of β-Catenin Structure and Activity by Tyrosine Phosphorylation

José Piedra,Daniel Martı́Nez,Daniel Martinez,Julio Castaño,Julio Castano,Susana Miravet,Mireia Dunach,Mireia Duñach,Antonio Garcı́A De Herreros

doi:10.1074/jbc.m100194200

José Piedra, Daniel Martı́Nez + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m100194200

Copy DOI

Abstract

beta-Catenin plays a dual role as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Phosphorylation of Tyr-654, a residue placed in the last armadillo repeat of beta-catenin, decreases its binding to E-cadherin. We show here that phosphorylation of Tyr-654 also stimulates the association of beta-catenin to the basal transcription factor TATA-binding protein. The structural bases of these different affinities were investigated. Our results indicate that the beta-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to E-cadherin. Phosphorylation of beta-catenin Tyr-654 decreases armadillo-C-terminal tail association, uncovering the last armadillo repeats. In a C-terminal-depleted beta-catenin, the presence of a negative charge at Tyr-654 does not affect the interaction of the TATA-binding protein to the armadillo domain. However, in the case of E-cadherin, the establishment of ion pairs dominates its association with beta-catenin, and its binding is greatly dependent on the absence of a negative charge at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-beta-catenin interaction, even though the steric hindrance of the C-tail is no longer present. These results explain how phosphorylation of beta-catenin in Tyr-654 modifies the tertiary structure of this protein and the interaction with its different partners.

Highlights

Dillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to Ecadherin
In the placed in the last armadillo repeat of ␤-catenin, de- case of E-cadherin, we have recently demonstrated that phoscreases its binding to E-cadherin
Samples were separated by SDS-polyacrylamide gel electrophoresis, and the presence of bound proteins in the complex was analyzed by Western blot with specific monoclonal antibodies against ␤-catenin C terminus (Transduction Laboratories, Lexington, KY), ␤-catenin armadillo core (Alexis Biochemicals, San Diego, CA), E-cadherin cytosolic domain (Transduction Labs), or Tcf-4 N terminus (Santa Cruz Biotechnology)

Summary

EXPERIMENTAL PROCEDURES

Expression of Recombinant Proteins—Expression and purification of full-length ␤-catenin, fragments 1–106 and 696-end, and ␤-catenin point mutants Tyr-86 3 Glu, Tyr-86 3 Phe, Tyr-654 3 Glu and Tyr-654 3 Phe have been previously described [6]. Samples were separated by SDS-polyacrylamide gel electrophoresis, and the presence of bound proteins in the complex was analyzed by Western blot with specific monoclonal antibodies (mAbs) against ␤-catenin C terminus (Transduction Laboratories, Lexington, KY), ␤-catenin armadillo core (Alexis Biochemicals, San Diego, CA), E-cadherin cytosolic domain (Transduction Labs), or Tcf-4 N terminus (Santa Cruz Biotechnology). Protease Sensitivity of ␤-Catenin—1 ␮g of the different forms of ␤-catenin, phosphorylated or not by pp60c-src, were incubated in the presence of trypsin (60 ng) at 24 °C in a final volume of 100 ␮l in a buffer containing 90 mM Tris-HCl, pH 8.5, 2 mM CaCl2, and 4 mM dithiothreitol. The extent of the digestion was determined analyzing the samples by SDS-polyacrylamide gel electrophoresis and Western blot with a mAb anti-␤catenin C terminus, which recognizes an epitope situated between amino acids 696 and 781 of this protein. A quantitation of the reaction was performed scanning the autoradiograms and representing the amount of full-length ␤-catenin at the different times of incubation relative to the initial time

RESULTS

Findings

DISCUSSION