Activation of the mitogen-activated protein kinase pathway in Triton X- 100 disrupted NIH-3T3 cells by p21 ras and in vitro by plasma membranes from NIH 3T3 cells [published erratum appears in Mol Biol Cell 1995 Sep;6(9):1261]

P Dent, J Wu, G Romero, LA Vincent, D Castle and TW Sturgill

Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.

We describe a novel Triton-disrupted mammalian cell system wherein the pathways for activation of mitogen-activated protein (MAP) kinases (MAPKs) are capable of direct biochemical manipulation in vitro. MAPKs p42mapk and p44mapk are activated in signal transduction cascade(s) initiated by occupancy of plasma membrane receptors for peptide growth factors, hormones, and neurotransmitters. One likely activation pathway for MAPKs consists of sequential activations of c-ras, c-raf-1, and a protein-tyrosine/threonine kinase, MAP kinase kinase. Triton-disrupted cells retained capacity for activation of the pathway by both peptide growth factors and by addition of GTP-loaded p21 rasVal12. Incubation of disrupted cells with an antibody that neutralized the function of c- ras (Y13-259) abolished receptor-mediated stimulation of MAPK as did acute addition of 200 microM azatyrosine. Activation of the pathway was reconstituted in a cell-free system using high-speed supernatants generated from Triton-disrupted cells together with purified plasma membranes from parental cells and as a heterogeneous system using purified plasma membranes from v-ras-transformed cells. These systems will allow biochemical dissection in vitro of the interaction(s) between c-ras and the MAPK pathway in mammalian cells.

Volume 4, Issue 5, pp. 483-493, 05/01/1993
Copyright © 1993 by The American Society for Cell Biology

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