RAPV12 ANTAGONIZES RAS-DEPENDENT ACTIVATION OF ERK1 AND ERK2 BY LPA AND EGF IN RAT-1 FIBROBLASTS

Rap1 is a small Ras-related GTPase which when over-expressed is able t o revert transformation by Ki-Ras. We have investigated the role of Ra p1 in regulating 'normal' Ras function by studying the activation of t he mitogen-activated protein (MAP) kinases ERK1 and ERK2 by two fundam entally different growth factors, epidermal growth factor (EGF) and 1- oleoyl-lyso-phosphatidic acid (LPA). Conditional expression of RasN17 (a dominant-negative mutant) in Rat-1 cells inhibited activation of MA P kinases by EGF and also LPA, the first time a defined G-protein-coup led receptor mitogen has been shown to require Ras to exert its effect s. Conditional or constitutive expression of even low levels of RapV12 (a mutant insensitive to Rap-GAP) attenuated activation of MAP kinase s by EGF and LPA, but did not interfere with growth factor-stimulated increases in Ras-GTP, indicating that signalling from receptors to Ras was not impaired. Inhibition of Ras-mediated signalling with either R asN17 or RapV12 attenuated DNA synthesis by EGF and LPA. We conclude t hat receptor tyrosine kinases and G-protein-coupled receptors use Ras as a common step in signalling to MAP kinases and that Rap-GTP (RapV12 ) at physiological levels interferes with downstream signalling from R as to MAP kinases in vivo.