ACTIVATION OF THE MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY IN TRITON-X-100 DISRUPTED NIH-3T3 CELLS BY P21 RAS AND INVITRO BY PLASMA-MEMBRANES FROM NIH-3T3 CELLS

We describe a novel Triton-disrupted mammalian cell system wherein the pathways for activation of mitogen-activated protein (MAP) kinases (M APKs) are capable of direct biochemical manipulation in vitro. MAPKs p 42mapk and p44mapk are activated in signal transduction cascade(s) ini tiated by occupancy of plasma membrane receptors for peptide growth fa ctors, 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 ras(Val12). Incubati on of disrupted cells with an antibody that neutralized the function o f c-ras (Y13-259) abolished receptor-mediated stimulation of MAPK as d id acute addition of 200 muM azatyrosine. Activation of the pathway wa s reconstituted in a cell-free system using high-speed supernatants ge nerated from Triton-disrupted cells together with purified plasma memb ranes from parental cells and as a heterogeneous system using purified plasma membranes from v-ras-transformed cells. These systems will all ow biochemical dissection in vitro of the interaction(s) between c-ras and the MAPK pathway in mammalian cells.