Cloning of bovine GAP and its interaction with oncogenic ras p21.
The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
Vogel
U S
US
Department of Molecular Biology, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Dixon
R A
RA
Schaber
M D
MD
Diehl
R E
RE
Marshall
M S
MS
Scolnick
E M
EM
Sigal
I S
IS
Gibbs
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011506:Proteins; D011518:Proto-Oncogene Proteins; 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