Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP.
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.
Scheffler
J E
JE
Department of Oncology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Waugh
D S
DS
Bekesi
E
E
Kiefer
S E
SE
LoSardo
J E
JE
Neri
A
A
Prinzo
K M
KM
Tsao
K L
KL
Wegrzynski
B
B
Emerson
S D
SD
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002942:Circular Dichroism; D029968:Escherichia coli Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009036:Moths; D009838:Oligodeoxyribonucleotides; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D009865:Oocytes; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014982:Xenopus laevis
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D015689:Oncogene Protein p21(ras)