Related Adhesion Focal Tyrosine Kinase and the Epidermal Growth Factor Receptor Mediate the Stimulation of Mitogen-activated Protein Kinase by the G-protein-coupled P2Y2 Receptor: PHORBOL ESTER OR [Ca2+]iELEVATION CAN SUBSTITUTE FOR RECEPTOR ACTIVATION

Stephen P Soltoff

doi:10.1074/jbc.273.36.23110

Abstract

The activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells. Previously, we demonstrated that the activation of G-protein-coupled P2Y2 receptors by extracellular ATP and UTP stimulated MAP (p42 ERK2) kinase by a mechanism that was dependent on the elevation of [Ca2+]i and the activation of related adhesion focal tyrosine kinase (RAFTK) (also called PYK2, CAKbeta, and CADTK) and protein kinase C (PKC). Here, we examine further the signaling cascade between the P2Y2 receptor and MAP kinase. MAP kinase was transiently activated by exposure of PC12 cells to UTP. UTP, ionomycin, and phorbol ester (phorbol 12-myristate 13-acetate) increased MAP kinase activity and also promoted the tyrosine phosphorylation of RAFTK, the epidermal growth factor (EGF) receptor, SHC, and p120(cbl). Down-regulation of PKC and inhibition of the elevation of [Ca2+]i, conditions that block the activation of MAP kinase, also blocked the increases in the tyrosine phosphorylation of RAFTK and the EGF receptor. AG1478, a tyrphostin selective for the EGF receptor, reduced the activation of MAP kinase, the tyrosine phosphorylation of SHC, the association of Grb2 with SHC, and the tyrosine phosphorylation of the EGF receptor and p120(cbl) but did not block the tyrosine phosphorylation of RAFTK. The similar effects of UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA) on these signaling proteins demonstrate that the two signaling molecules from phosphatidylinositol 4,5-bisphosphate hydrolysis ([Ca2+]i, from inositol 1,4,5-trisphosphate production, and diacylglycerol) can individually initiate the activation of MAP kinase in an EGF receptor-dependent manner. These results demonstrate that the P2Y2 receptor-mediated transactivation of the EGF receptor occurs at a point downstream of RAFTK and indicate that the EGF receptor is required for P2Y2 receptor-mediated MAP kinase activation. Although P2Y2 and EGF receptors may both activate a similar multiprotein signaling cascade immediately upstream of MAP kinase, the P2Y2 receptor appears to uniquely utilize [Ca2+]i, PKC, and, subsequently, RAFTK.

Highlights

The activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells
Time Dependence of UTP on MAP Kinase Activity—An established paradigm in PC12 cells is that agents that cause proliferation (e.g. epidermal growth factor (EGF)) produce short lived increases in MAP kinase, while agents that cause differentiation (e.g. NGF) produce much longer lived increases that are sustained for hours [17, 18]
Along with the inhibitory effects of AG1478 on MAP kinase (Fig. 4), these results suggest that the activation of the EGF receptor is between related adhesion focal tyrosine kinase (RAFTK) and MAP kinase in the signaling cascade promoted by UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA)

Summary

Introduction

The activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells. The similar effects of UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA) on these signaling proteins demonstrate that the two signaling molecules from phosphatidylinositol 4,5-bisphosphate hydrolysis ([Ca2؉]i, from inositol 1,4,5-trisphosphate production, and diacylglycerol) can individually initiate the activation of MAP kinase in an EGF receptordependent manner. The effects of a number of GPCRs have been reported to involve increases in tyrosine phosphorylation Signaling proteins such as SHC [4, 5] and members of the Src kinase family may contribute to GPCR-mediated activation of signaling pathways, including MAP kinase (4 –11). There are similarities in the signaling cascades involved in MAP kinase activation initiated by EGF and UTP, notably EGF receptor and SHC tyrosine phosphorylation, RAFTK and PKC are uniquely involved in the P2Y2 receptormediated cascade

Methods

Results

Conclusion