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Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 1994 Sep 02; Vol. 269 (35), pp. 22340-6. - Publication Year :
- 1994
-
Abstract
- Four overlapping peptide fragments of human c-Raf-1 (residues 55-132, 55-117, 77-132, and 77-117) were expressed in Escherichia coli as carboxyl-terminal extensions of maltose binding protein (MBP). The MBP-Raf fusions were purified by affinity chromatography on amylose resin and tested for binding to Ras.GTP indirectly by measuring their ability to inhibit the stimulation of Ras GTPase activity by GTPase activating protein (GAP120) in vitro. MBP-Raf(55-132) was a potent inhibitor in this assay (50% inhibition at 100 nM concentration), but the other fusion proteins had no measurable effect. The fusion partners were cleaved with Factor Xa protease and separated by gel filtration. The 8960-dalton Raf(55-132) fragment retained full activity as a competitive inhibitor of GAP120. It also blocked Ras-stimulated germinal vesicle breakdown in frog oocytes. Raf(55-132) was further characterized by circular dichroism and nuclear magnetic resonance spectroscopy. The results indicate that this fragment of c-Raf-1 adopts a highly structured, monomeric conformation in solution.
- Subjects :
- Amino Acid Sequence
Animals
Baculoviridae
Base Sequence
Binding Sites
Carrier Proteins metabolism
Cell Line
Circular Dichroism
Humans
Magnetic Resonance Spectroscopy
Maltose metabolism
Maltose-Binding Proteins
Molecular Sequence Data
Moths
Oligodeoxyribonucleotides
Oocytes
Protein Structure, Secondary
Proto-Oncogene Proteins c-raf
Recombinant Fusion Proteins metabolism
Xenopus laevis
ATP-Binding Cassette Transporters
Escherichia coli Proteins
Guanosine Triphosphate metabolism
Monosaccharide Transport Proteins
Oncogene Protein p21(ras) metabolism
Protein Serine-Threonine Kinases metabolism
Proto-Oncogene Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9258
- Volume :
- 269
- Issue :
- 35
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 8071362