The Noncatalytic Domain of Protein-tyrosine Phosphatase-PEST Targets Paxillin for Dephosphorylation in Vivo

Yu Shen,Patrick Lyons,Marion Cooley,Dominique Davidson,André Veillette,Ravi Salgia,James D Griffin,Michael D Schaller

doi:10.1074/jbc.275.2.1405

Yu Shen, Patrick Lyons + Show 6 more

Open Access

https://doi.org/10.1074/jbc.275.2.1405

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Abstract

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. This binding site has been narrowed to a 52-residue sequence that is composed of two nonoverlapping, weak paxillin binding sites. The PTP-PEST binding site on paxillin has been mapped to the two carboxyl-terminal LIM (lin11, isl-1, and mec-3) domains. Transient expression of PTP-PEST reduced tyrosine phosphorylation of p130(cas), as anticipated. A PTP-PEST mutant defective for binding p130(cas) does not cause a reduction in its tyrosine phosphorylation in vivo. Expression of PTP-PEST also caused a reduction of phosphotyrosine on paxillin. Expression of mutants of PTP-PEST with deletions in the paxillin-binding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin. These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation.

Highlights

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin
Using mutants of PTP-PEST, we demonstrate that the interaction of p130cas and paxillin with the noncatalytic domain of PTP-PEST is required for their dephosphorylation in vivo
Residues 297– 485 of PTP-PEST were expressed as a GAL4 DNA binding domain fusion protein and the amino-terminal domain of paxillin was expressed as a GAL4 activation domain fusion protein

Summary

Introduction

The noncatalytic domain of protein-tyrosine phosphatase (PTP)-PEST contains a binding site for the focal adhesion-associated protein paxillin. Expression of mutants of PTP-PEST with deletions in the paxillinbinding site did not associate with paxillin in vivo and failed to cause a reduction in the phosphotyrosine content of paxillin These results demonstrate that paxillin can serve as a PTP-PEST substrate in vivo and support the model that a noncatalytic domain interaction recruits paxillin to PTP-PEST to facilitate its dephosphorylation. PTPs function to dephosphorylate tyrosine-phosphorylated substrates and a number of candidate PTPs that may regulate signaling through focal adhesion-associated proteins have recently been identified. In fibroblasts lacking functional SHP2, the phosphotyrosine content of FAK and paxillin is perturbed and the cells exhibit retarded spreading on fibronectin and reduced motility [18, 19]. The PTP-PESTϪ/Ϫ cells spread more rapidly than wild type cells but show reduced motility [24]

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