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 to revert transformation by Ki-Ras. We have investigated the role of Rap1 in regulating 'normal' Ras function by studying the activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 by two fundamentally 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 MAP kinases by EGF and also LPA, the first time a defined G-protein-coupled receptor mitogen has been shown to require Ras to exert its effects. Conditional or constitutive expression of even low levels of RapV12 (a mutant insensitive to Rap-GAP) attenuated activation of MAP kinases 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 RasN17 or RapV12 attenuated DNA synthesis by EGF and LPA. We conclude that 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 Ras to MAP kinases in vivo.
Cook
S J
SJ
Onyx Pharmaceuticals Inc., Richmond, CA 94806.
Rubinfeld
B
B
Albert
I
I
McCormick
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D011905:Genes, ras; D007700:Kinetics; D008246:Lysophospholipids / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D020668:rap GTP-Binding Proteins
D008246:Lysophospholipids; D010455:Peptides; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins; C032881:lysophosphatidic acid