Prolactin Decreases Epidermal Growth Factor Receptor Kinase Activity via a Phosphorylation-dependent Mechanism

Victor J Quijano,Lewis G Sheffield

doi:10.1074/jbc.273.2.1200

Abstract

Previously, we have shown that prolactin inhibits epidermal growth factor (EGF)-induced mitogenesis in mouse mammary epithelial cells without altering the response to other growth promoting agents. This effect has been associated with reduced EGF-induced EGF receptor (EGFR) tyrosine phosphorylation, Grb-2 association, and Ras activation. Our current hypothesis is that prolactin induces an alteration in EGFR kinase activity via a phosphorylation-dependent mechanism. To test this hypothesis, we treated normal murine mammary gland cells with or without 100 ng/ml prolactin. EGFR isolated by wheat germ agglutinin affinity chromatography from nontreated cells exhibited substantial ligand-induced phosphorylation, and EGFR isolated from prolactin-treated cells displayed minimal EGF-induced EGFR phosphorylation, as well as decreased kinase activity toward exogenous substrates. The observed decrease in ligand-induced EGFR phosphorylation could not be attributed to either differential amounts of EGFR, decreased EGF binding affinity, or the presence of a phosphotyrosine phosphatase or ATPase. EGFR isolated from prolactin-treated cells exhibited increased phosphorylation on threonine. Removal of this phosphorylation with alkaline phosphatase restored EGFR kinase activity to levels observed in nontreated cells. Therefore, these results suggest that prolactin antagonizes EGF signaling by increasing EGFR threonine phosphorylation and decreasing EGF-induced EGFR tyrosine phosphorylation.

Highlights

EGF1 and its receptor (EGFR) play a crucial role in mammary epithelial proliferation [1, 2]
Phosphoamino acid analysis showed that epidermal growth factor (EGF)-induced EGFR phosphorylation was predominantly on tyrosines (ϳ90%) and was substantially decreased in EGFR isolated from Prl-treated cells
We have shown that receptors isolated from NMuMG cells treated with Prl exhibit a decrease in EGFinduced EGFR phosphorylation as well as a decrease in the ability of EGFR to phosphorylate angiotensin II or Src peptides, an effect that could not be attributed to differential EGFR concentrations, increasing phosphotyrosine phosphatases, ATPase activity, or varying binding affinities for EGF

Summary

Introduction

EGF1 and its receptor (EGFR) play a crucial role in mammary epithelial proliferation [1, 2] This effect is manifested in the context of a variety of other hormones including estrogen, progesterone, growth hormone, and insulin-like growth factor-1 (reviewed in Ref. 1). The receptor undergoes inter- and intramolecular autophosphorylation on tyrosines in the C-terminal cytoplasmic domain [3, 10, 11, 12] These phosphotyrosines serve as docking sites for Src homology 2-containing proteins such as Grb, phospholipase C-␥, phosphatidylinositol 3-kinase, and Shc [13, 14]. In addition to tyrosine phosphorylation sites needed for signal transduction, certain serine and threonine residues exist in the cytoplasmic portion of the EGFR that, upon phosphorylation, reduce EGF binding, induce receptor desensitization, or promote down-regulation (10, 19 –21). The best described of these events is mediated by protein kinase C [19, 20], but others have been reported [21, 22]

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