A novel GTPase-activating protein for R-Ras.
R-Ras, belonging to the Ras small GTP-binding protein superfamily, has been implicated in regulation of various cell functions such as gene expression, cell proliferation, and apoptotic cell death. In the present study, we purified an R-Ras-interacting protein with molecular mass of about 98 kDa (p98) from bovine brain cytosol by glutathione S-transferase (GST)-R-Ras affinity column chromatography. This protein bound to GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).R-Ras but not to GDP.R-Ras, GTP gamma S.R-Ras with a mutation in the effector domain (R-RasA64), GTP gamma S.Ha-Ras, or GTP gamma S.RalA. We obtained a cDNA encoding p98 on the basis of its partial amino acid sequences. The predicted protein consists of 834 amino acids whose calculated mass, 95,384 Da, is close to the apparent molecular mass of p98. The amino acid sequence shows a high degree of sequence similarity to the entire sequence of Gap1m, one of the GTPase-activating proteins (GAP) for Ha-Ras. A recombinant protein consisting of the GAP-related domain of p98 fused to maltose-binding protein stimulated GTPase activity of R-Ras, and showed a weak effect on that of Ha-Ras but not that of Rap1 or Rho. These results clearly indicate that p98 is a novel GAP for R-Ras. Thus, we designated this protein as R-Ras GAP.
Yamamoto
T
T
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Japan.
Matsui
T
T
Nakafuku
M
M
Iwamatsu
A
A
Kaibuchi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D005982:Glutathione Transferase; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D005982:Glutathione Transferase; D018631:ras Proteins