Showing papers by "Jackson B. Gibbs published in 1988"

Cloning of bovine GAP and its interaction with oncogenic ras p21

Abstract: 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)1–3. 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 (ref. 4). GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21 (refs 3, 5 and 6). We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity7. 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 cerevisiae8–10 and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases26,27.

Purification of ras GTPase activating protein from bovine brain

Abstract: In cytosolic extracts of bovine brain, we detected ras GTPase activating protein (GAP) activity that stimulated the GTP hydrolytic activity of normal c-Ha-ras p21 but not that of the oncogenic [Val12]p21 variant. GAP was purified 19,500-fold by a five-column procedure involving DEAE-Sephacel, Sepharose 6B, orange dye and green dye matrices, and Mono Q resins. A single major protein band of 125 kDa was observed on NaDodSO4/polyacrylamide gels that correlated with the elution of GAP activity on Mono Q. Purified GAP was devoid of inherent GTP hydrolytic activity, suggesting that it was a regulator of ras intrinsic GTPase activity. Under submaximal velocity conditions, the second-order rate constant of GTP hydrolysis at 24 degrees C for p21-GTP + GAP (4.5 X 10(6) M-1.sec-1) was at least 1000-fold greater than that for [Val12]p21-GTP + GAP (less than 3 X 10(3) M-1.sec-1).

An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations.

Abstract: Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.

Inhibition of yeast adenylate cyclase by antibodies to ras p21.

Abstract: Monoclonal antibody Y13-259 to ras p21 was shown to bind to the highly conserved residues in the region 63-73 and to neutralize ras action in the Saccharomyces cerevisiae adenylate cyclase system. Inhibition of adenylate cyclase activity in isolated membranes by antibody Y13-259 occurred after a lag period of 6 min. This lag corresponded to the time necessary for binding of antibody Y13-259 to the membranes in a ras-dependent manner. The mechanism of inhibition appeared to be steric in nature because antibody Y13-259 neutralized ras p21 bound to a stable GTP analogue. Monoclonal antibodies Y13-4 and Y13-128 also inhibited yeast adenylate cyclase activity, and the epitopes for both the these antibodies were localized to ras region 65-75. However, the ras residues essential for binding of antibodies Y13-4 and Y13-128 to ras p21 (positions 65, 66, 68 and 75) were different from those essential for binding of antibody Y13-259 (positions 63, 65, 66, 67, 70 and 73). These results indicate that residues 63-75 constitute a major neutralizing epitope on ras p21.