NDLI: Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.

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Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.

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Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Dent, P. Sturgill, T. W.
Description	Author Affiliation: Dent P ( Howard Hughes Medical Institute, University of Virginia, Charlottesville 22908.);
Abstract	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.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	20
Volume Number	91
Language	English
Publisher	National Academy of Sciences
Publisher Date	1994-10-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Genes, Ras Protein-Serine-Threonine Kinases Metabolism Proto-Oncogene Proteins 3T3 Cells Amino Acid Sequence Animals Cell Line Cell Line, Transformed Cell Membrane Culture Media Enzyme Activation Fibroblasts Enzymology MAP Kinase Kinase Kinases Mice Mitogen-Activated Protein Kinase Kinases Molecular Sequence Data Protein Kinases Isolation & Purification Proto-Oncogene Proteins C-raf Recombinant Proteins Spodoptera Type C Phospholipases Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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