Identification of Src Phosphorylation Sites in the Catenin p120ctn

Deborah J Mariner,Panos Anastasiadis,Heike Keilhack,Frank-D Böhmer,Jue Wang,Albert B Reynolds

doi:10.1074/jbc.m102443200

Abstract

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.

Highlights

P120-catenin (p120ctn) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions
Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120 – 8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosinephosphorylated p120 in cells stimulated with epidermal growth factor
The cytoplasmic domains interact with a group of proteins called catenins (e.g. p120ctn, ␣-catenin, and ␤-catenin), which regulate cadherin interactions with the actin cytoskeleton. ␤-Catenin [1] and p120 [2, 3] are Armadillo (Arm)1 repeat domain proteins that bind to the cadherin catenin-binding [4] and juxtamembrane domains [5,6,7], respectively. ␤-Catenin is hardwired to the actin cytoskeleton through its interaction with ␣-catenin [8, 9], which binds to F-actin directly [10] or through accessory proteins such as

Summary

Introduction

P120-catenin (p120ctn) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified Changing all of these sites to phenylalanine resulted in a p120 mutant, p120 – 8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosinephosphorylated p120 in cells stimulated with epidermal growth factor. Another study showed that inhibition of the catalytic activity of the Src kinases promotes the stability of cadherin-dependent cell-cell contacts [29], suggesting that Src kinase activity is normally required for junction disassembly The latter result is consistent with data from oncogene-transformed or mitogenstimulated cells, where increased tyrosine kinase activity is associated with weak junctions (for review see Ref. 30).

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