Receptor-type Protein-tyrosine Phosphatase-κ Regulates Epidermal Growth Factor Receptor Function

Yiru Xu,Li-Jun Tan,Vladimir Grachtchouk,John J Voorhees,Gary J Fisher

doi:10.1074/jbc.m507722200

Abstract

Epidermal growth factor receptor (EGFR), the prototypic receptor protein tyrosine kinase, is a major regulator of growth and survival for many epithelial cell types. We report here that receptor-type protein-tyrosine phosphatase-kappa (RPTP-kappa) dephosphorylates EGFR and thereby regulates its function in human keratinocytes. Protein-tyrosine phosphatase (PTP) inhibitors induced EGFR tyrosine phosphorylation in intact primary human keratinocytes and cell-free membrane preparations. Five highly expressed RPTPs (RPTP-beta, delta, kappa, mu, and xi) were functionally analyzed in a Chinese hamster ovary (CHO) cell-based expression system. Full-length human EGFR expressed in CHO cells, which lack endogenous EGFR, displayed high basal (i.e. in the absence of ligand) tyrosine phosphorylation. Co-expression of RPTP-kappa, but not other RPTPs, specifically reduced basal EGFR tyrosine phosphorylation. RPTP-kappa also reduced epidermal growth factor-dependent EGFR tyrosine phosphorylation in CHO cells. Purified RPTP-kappa preferentially dephosphorylated EGFR tyrosines 1068 and 1173 in vitro. Overexpression of wild-type or catalytically inactive RPTP-kappa reduced or enhanced, respectively, basal and EGF-induced EGFR tyrosine phosphorylation in human keratinocytes. Furthermore, siRNA-mediated knockdown of RPTP-kappa increased basal and EGF-stimulated EGFR tyrosine phosphorylation and augmented downstream Erk activation in human keratinocytes. RPTP-kappa levels increased in keratinocytes as cells reached confluency, and overexpression of RPTP-kappa in subconfluent keratinocytes reduced keratinocyte proliferation. Taken together, the above data indicate that RPTP-kappa is a key regulator of EGFR tyrosine phosphorylation and function in human keratinocytes.

Highlights

Protein-tyrosine phosphatase (PTP) and active PTKs has recently been estimated to be very similar [3, 4]
In addition to the reduction of constitutive Epidermal growth factor receptor (EGFR) tyrosine phosphorylation, expression of receptor-type PTPs (RPTPs)-␬ reduced EGF-stimulated EGFR tyrosine phosphorylation in Chinese hamster ovary (CHO) cells (Fig. 3B). These results indicate that RPTP-␬ is capable of reducing EGFR intrinsic tyrosine kinase-catalyzed phosphorylation when co-expressed in CHO cells
Because reduction of endogenous RPTP-␬ potentiates basal and EGF-induced EGFR activation, we investigated the effect of RPTP-␬ knockdown on Erk activation. siRNA-mediated RPTP-␬ knockdown resulted in a 2-fold increase of basal phosphorylation of p44 and p42 Erk, in primary human keratinocytes (Fig. 9C)

Summary

Introduction

PTPs and active PTKs has recently been estimated to be very similar [3, 4]. Emerging evidence indicates that, depending on the particular pathway, protein tyrosine dephosphorylation can be of equal or greater importance than protein tyrosine phosphorylation for the regulation of cellular function [5]. We have further demonstrated that RPTP-␬ regulates both basal and ligand-induced EGFR tyrosine phosphorylation and function. Dephosphorylation reactions were terminated by the addition of SDS sample loading buffer, and the level of EGFR tyrosine phosphorylation was measured by Western analysis probed with phospho-EGFR antibody. Expression of RPTP-␬ in primary keratinocytes increased with cell confluency (see Fig. 10A).

Results

Conclusion