Difference in the mechanism of interaction of Raf-1 and B-Raf with H-Ras.
Ras is known to possess multiple cellular targets including Raf-1. Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms. B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays. Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1. On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK. These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf.
Shinkai
M
M
Department of Physiology II, Kobe University School of Medicine, Japan.
Masuda
T
T
Kariya
K
K
Tamada
M
M
Shirouzu
M
M
Yokoyama
S
S
Kataoka
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D011905:Genes, ras; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D051381:Rats; D011994:Recombinant Proteins; D018411:Spodoptera; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins