Role of Tyrosine Kinase Activity of Epidermal Growth Factor Receptor in the Lysophosphatidic Acid-stimulated Mitogen-activated Protein Kinase Pathway

Jess M Cunnick,Jay F Dorsey,Todd Standley,James Turkson,Alan J Kraker,David W Fry,Richard Jove,Jie Wu

doi:10.1074/jbc.273.23.14468

Abstract

Recent evidence indicates that the epidermal growth factor (EGF) receptor mediates a branch of lysophosphatidic acid (LPA)-induced signal transduction pathways that activate mitogen-activated protein (MAP) kinase. However, it is unclear whether the intrinsic tyrosine kinase activity of EGF receptor is involved. We previously showed that reactive oxygen species (ROS) were involved in the LPA-stimulated MAP kinase pathway. Here, we identify tyrosine phosphorylation of EGF receptor as an LPA signaling step that requires ROS. To evaluate the role of the tyrosine kinase activity of EGF receptor in the LPA-stimulated MAP kinase pathway, we examined the effects of an EGF receptor-specific tyrosine kinase inhibitor, PD158780. PD158780 potently inhibited the LPA-stimulated MAP kinase kinase 1/2 (MKK1/2) activation and EGF receptor tyrosine phosphorylation in HeLa cells, while it had no detectable effect on c-Src kinase activity. PD158780 also inhibited LPA-induced MKK1/2 activation and DNA synthesis in NIH 3T3 cells. Furthermore, we compared LPA-stimulated MKK1/2 and MAP kinase activation, transcriptional activity of the c-fos promoter, and DNA synthesis in B82L cells, which lack endogenous EGF receptor, and B82L cells expressing kinase-defective or wild-type human EGF receptor. Results obtained from analysis of these cell lines suggest that the EGF receptor tyrosine kinase contributes to the LPA-stimulated MAP kinase activation, c-fos transcription, and mitogenesis.

Highlights

Lysophosphatidic acid (LPA)1 is a bioactive phospholipid present in serum
Expression of a truncated human epidermal growth factor (EGF) receptor lacking the cytoplasmic domain in Rat1 cells abrogated LPA-stimulated mitogen-activated protein (MAP) kinase activation, suggesting that the EGF receptor mediates at least a branch of the LPA-stimulated MAP kinase activation pathway [14]
Quantification of the EGF receptor bands by a densitometer indicated that a 2.5-min stimulation of HeLa cells with 10 ␮M LPA increased tyrosine phosphorylation of the EGF receptor approximately 3-fold (3.1 Ϯ 0.5 in four experiments), which is similar to that induced by 0.17 nM EGF in HeLa cells

Summary

Introduction

Lysophosphatidic acid (LPA) is a bioactive phospholipid present in serum. LPA concentrations in serum are normally in the range of 2–20 ␮M [1], but higher concentrations have been reported [2]. The LPA-stimulated MAP kinase pathway is sensitive to certain protein tyrosine kinase inhibitors such as genistein [6].2 These observations suggest that regulation of protein tyrosine phosphorylation is an important signaling mechanism of LPA and other agonists of G protein-coupled receptors. Expression of a truncated human EGF receptor lacking the cytoplasmic domain in Rat cells abrogated LPA-stimulated MAP kinase activation, suggesting that the EGF receptor mediates at least a branch of the LPA-stimulated MAP kinase activation pathway [14] It is unclear whether intrinsic tyrosine kinase activity of EGF receptor is involved in G protein-coupled receptor-stimulated tyrosine phosphorylation of the EGF receptor, MAP kinase activation, and downstream cellular responses such as gene transcription and DNA synthesis. Role of EGF Receptor Kinase in LPA Signaling ments indicated that c-Src is not involved in LPA signaling in Rat cells [3, 17]

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