Ras interaction with the GTPase-activating protein (GAP).
Biologically active forms of Ras complexed to GTP can bind to the GTPase-activating protein (GAP), which has been implicated as possible target of Ras in mammalian cells. In order to study the structural features of Ras required for this interaction, we have evaluated a series of mutant ras proteins for the ability to bind GAP and a series of Ras peptides for the ability to interfere with this interaction. Point mutations in the putative effector region of Ras (residues 32-40) that inhibit biological activity also impair Ras binding to GAP. An apparent exception is the Thr to Ser substitution at residue 35; [Ser-35]Ras binds to GAP as effectively as wild-type Ras even though this mutant is biologically weak in both mammalian and S. cerevisiae cells. In vitro, [Ser-35]Ras can also efficiently stimulate the S. cerevisiae target of Ras, adenylyl cyclase, indicating that other factors may influence Ras/protein interactions in vivo. Peptides having Ras residues 17-44 and 17-32 competed with the binding of Ras to E. coli-expressed GAP with IC50 values of 2.4 and 0.9 microM, respectively, whereas Ras peptide 17-26 was without effect up to 400 microM. A related peptide from the yeast GTP-binding protein YPT1 analogous to Ras peptide 17-32 competed with an IC50 value of 19 microM even though the YPT1 protein itself is unable to bind to GAP. These results suggest that determinants within Ras peptide 17-32 may be important for Ras binding to GAP.
Schaber
M D
MD
Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Garsky
V M
VM
Boylan
D
D
Hill
W S
WS
Scolnick
E M
EM
Marshall
M S
MS
Sigal
I S
IS
Gibbs
J B
JB
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005656:Fungal Proteins / Q000235:genetics; D019204:GTP-Binding Proteins; D020690:GTPase-Activating Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship; D020703:ras GTPase-Activating Proteins; D018631:ras Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019204:GTP-Binding Proteins; D018631:ras Proteins; D000262:Adenylyl Cyclases