A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity.
The cDNA for bovine ras p21 GTPase activating protein (GAP) has been cloned and the 1044 amino acid polypeptide encoded by the clone has been shown to bind the GTP complexes of both normal and oncogenic Harvey (Ha) ras p21. To identify the regions of GAP critical for the catalytic stimulation of ras p21 GTPase activity, a series of truncated forms of GAP protein were expressed in Escherichia coli. The C-terminal 343 amino acids of GAP (residues 702-1044) were observed to bind Ha ras p21-GTP and stimulate Ha ras p21 GTPase activity with the same efficiency (kcat/KM congruent to 1 x 10(6) M-1 s-1 at 24 degrees C) as GAP purified from bovine brain or full-length GAP expressed in E. coli. Deletion of the final 61 amino acid residues of GAP (residues 986-1044) rendered the protein insoluble upon expression in E. coli. These results define a distinct catalytic domain at the C terminus of GAP. In addition, GAP contains amino acid similarity with the B and C box domains conserved among phospholipase C-II, the crk oncogene product, and the non-receptor tyrosine kinase oncogene products. This homologous region is located in the N-terminal half of GAP outside of the catalytic domain that stimulates ras p21 GTPase activity and may constitute a distinct structural or functional domain within the GAP protein.
Marshall
M S
MS
Department of Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486.
Hill
W S
WS
Ng
A S
AS
Vogel
U S
US
Schaber
M D
MD
Scolnick
E M
EM
Dixon
R A
RA
Sigal
I S
IS
Gibbs
J B
JB
D000818:Animals; D002417:Cattle; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)