Biochemical characterization of yeast RAS2 mutants reveals a new region of ras protein involved in the interaction with GTPase-activating proteins.
We report biochemical characterization of two recently identified mutants of yeast RAS2, RAS2-E99K and RAS2-E130K. These mutants exhibit dominant activating phenotypes in yeast. Characterization of their intrinsic GTPase and GDP dissociation as well as their ability to stimulate adenylate cyclase showed that these activities of RAS2-E99K mutant protein were similar to those of the wild type protein. RAS2-E130K protein, on the other hand, differed from the wild type protein with a fast GDP dissociation rate and 2-fold higher activation of adenylate cyclase. When the sensitivity to GTPase-activating protein (GAP) was examined, we found that the RAS2-E99K protein was approximately 1200-fold less sensitive to NF1-GAP activity. In addition, the affinity for NF1 as revealed by competition binding experiments was reduced more than 150-fold with RAS2-E99K protein. Thus, the RAS2-E99K mutation affects interaction with GAP proteins. This mutation is particularly interesting because it is the first mutation identified in the alpha 3 region of ras protein that affects GAP interaction. The alpha 3 region appears to be directly involved in interaction with NF1, since peptides containing the sequence encompassing residue 99 of RAS2 inhibit NF1-GAP activity. These results suggest that the interaction between ras and GAP involves a larger region within ras than previously recognized.
Wood
D R
DR
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Poullet
P
P
Wilson
B A
BA
Khalil
M
M
Tanaka
K
K
Cannon
J F
JF
Tamanoi
F
F
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006153:Guanosine Diphosphate / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins; D006153:Guanosine Diphosphate; D020558:GTP Phosphohydrolases; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases