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.
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.
Dent
P
P
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Wu
J
J
Romero
G
G
Vincent
L A
LA
Castle
D
D
Sturgill
T W
TW
D016475:3T3 Cells; D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000502:physiology*; D002474:Cell-Free System; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D008854:Microscopy, Electron; D019950:Mitogen-Activated Protein Kinase 1; D017830:Octoxynol; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011092:Polyethylene Glycols; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000494:pharmacology*; D015398:Signal Transduction
D007328:Insulin; D010982:Platelet-Derived Growth Factor; C011870:beta-(5-hydroxy-2-pyridyl)alanine; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; D017830:Octoxynol; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D016283:Proto-Oncogene Proteins p21(ras); D000409:Alanine