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

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. Lysophosphatidic acid (LPA)1 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). LPA induces cellular responses by binding to a specific cell surface receptor(s) that is coupled to G i , G q , and G 12/13 subfamilies of heterotrimeric G-proteins (1, 3). LPA rapidly activates the mitogen-activated protein (MAP) kinase cascade, consisting of Ras, Raf-1, MAP kinase kinase (MKK or MEK) 1 and MKK 2 (MKK1/2), and MAP kinases (also called ERKs) (4 -9). It has been observed that LPA, as well as other agonists of G protein-coupled receptors, induces tyrosine phosphorylation of several signaling proteins in diverse cell types (6, 10 -15). Activation of protein tyrosine kinases, such as Src and PYK2, by agonists of G protein-coupled receptors have been reported in certain cell types (15, 16), but not others (17). The LPA-stimulated MAP kinase pathway is sensitive to certain protein tyrosine kinase inhibitors such as genistein (6). 2These observations suggest that regulation of protein tyrosine phosphorylation is an important signaling mechanism of LPA and other agonists of G protein-coupled receptors.Recently, it was reported that LPA and some other agonists of G protein-coupled receptors stimulate tyrosine phosphorylation of the epidermal growth factor (EGF) receptor (14, 15). Expression of a truncated human EGF receptor lacking the cytoplasmic domain in Rat1 cells abrogated LPA-stimulated MAP kinase activation, suggesting that the EGF receptor mediates at ...

The platelet-derived-growth-factor receptor, not the epidermal-growth-factor receptor, is used by lysophosphatidic acid to activate p42/44 mitogen-activated protein kinase and to induce prostaglandin G/H synthase-2 in mesangial cells

“…There is, however, considerable cell specificity concerning the type of protein tyrosine kinase receptor used for trans-activation. One of the best studied systems is activation of the epidermal-growth-factor (EGF) receptor by LPA, which has been observed in fibroblasts [9][10][11], keratinocytes [10], HeLa cells [11] or transfected Cos cells [10]. These results are not without controversy, as EGF receptor trans-activation in Rat-1 cells described by Daub et al [9,12] was not detected by Kranenburg et al [13].…”

Requirement of Activated Cdc42-Associated Kinase for Survival of v-Ras-Transformed Mammalian Cells

“…4A and B). Several tyrosine kinase inhibitors, including PD158780, an inhibitor for the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (29), and PD157432, an inhibitor for EGFR (30), were studied for their ability to inhibit the kinase activity of GST-ACKD in vitro. PD158780 has the strongest inhibitory activity, whereas quercetin, genistein, wortmannin, and PD157432 exhibited very weak activity ( Fig.…”

Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes

“…Tyrosine phosphorylation of EGF receptor was analysed by immunoblotting with anti-phosphotyrosine antibody RC20 after immunoprecipitation with anti-EGF receptor antibody LA22 as described [17]. ERK activation was analysed by immunoblotting with anti-Active-MAPK antibody that reacts only with active ERK1 and ERK2 [17].…”

“…These include : MAP kinase activation, calcium mobilization and arachidonic acid release, activation of the small G protein Rho, and inhibition of adenylate cyclase. In a number of cell lines, LPA induces tyrosine phosphorylation of EGF receptor [14][15][16][17]. Preincubation of cells with specific inhibitors of the EGF receptor tyrosine kinase blocks LPA-induced EGF receptor tyrosine phosphorylation and MAP kinase activation [14,17].…”

“…Importantly, we find that 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B % (LTB % ) are required for LPA-stimulated H # O # release in HaCaT cells. [17][18][19] was generously provided by Alan Kraker (Parke-Davis Pharmaceutical Research). Zileuton [20,21] was provided by Abbott Laboratories.…”