Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.
The serine-threonine protein kinase Raf-1 is an important signal transducer in mitogenesis, phosphorylating and activating mitogen-activated protein (MAP) kinase kinase. Raf-1 activation in vivo is dependent on Ras, but the mechanism of Raf activation is unknown. The ability of preparations of plasma membranes to activate exogenous (His)6-Raf-1 was studied. Plasma membranes of v-Ras-transformed NIH 3T3 cells, but not parental cells, enhanced MAP kinase kinase kinase (MAPKKK) activity dependent on addition of (His)6-Raf-1 and ATP/Mg. Treatment of membranes with concentrations of Bacillus cereus phosphatidylcholine-specific phospholipase C that activated Raf-1 in vivo failed to enhance MAPKKK activity in vitro. Activation of (His)6-Raf-1 in vitro by membranes was dependent on binding to Ras. Membranes from v-Src-transformed cells also activated (His)6-Raf-1 and synergized with v-Ras membranes. Serum-treatment of NIH 3T3 cells stimulated the ability of membranes to activate (His)6-Raf-1. Activated (His)6-Raf-1 could be recovered on Ni(2+)-agarose, and this methodology was used to demonstrate that activation by membranes was ATP dependent. These findings demonstrate Ras- and ATP-dependent step(s) for Raf-1 activation by plasma membranes in vitro.
Dent
P
P
Howard Hughes Medical Institute, University of Virginia, Charlottesville 22908.
Sturgill
T W
TW
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D003470:Culture Media; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D011905:Genes, ras; D020930:MAP Kinase Kinase Kinases; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera; D010738:Type C Phospholipases / Q000378:metabolism
D003470:Culture Media; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D020930:MAP Kinase Kinase Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010738:Type C Phospholipases