Rap2B-Dependent Stimulation of Phospholipase C-ε by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

Abstract

Receptor tyrosine kinase regulation of phospholipase C-ε (PLC-ε), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca²⁺ signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ε, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ε, and Rap2B-dependent translocation of PLC-ε to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca²⁺ and expression of lipase-inactive PLC-γ1 but not of PLC-ε. Expression of RasGRP3, a Ca²⁺/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca²⁺ signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ε.

We thank K. Baden, M. Hagedorn, H. Geldermann, D. Petermeyer, and A. Kötting-Dorsch for expert technical assistance, C. Rimmbach for assistance in the PCR experiments, C. Heneweer for advice on the microscopic analysis, and A. Blaukat, J. L. Bos, C. von Eichel-Streiber, J. de Gunzburg, D. Illenberger, J. W. Lomasney, J. T. Parsons, H. Rehmann, J. de Rooij, P.-G. Suh, and A. Ullrich for providing toxins and cDNA constructs.

This work was supported by the Deutsche Forschungsgemeinschaft, the Interne Forschungsförderung Essen, and the Fonds der Chemischen Industrie.