Mutational and kinetic analyses of the GTPase-activating protein (GAP)-p21 interaction: the C-terminal domain of GAP is not sufficient for full activity.

Abstract

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-'. Mutations of residues in loop Li (Gly-12 and Gly-13), in loop L2 , and in loop LA (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.p21 proteins are the products of the N-, K-, and H-ras genes. They are small guanine nucleotide-binding proteins that are believed to function as signal switch molecules: in the active GTP-bound state, they are switched on; in the GDP-bound state, they are switched off. p21 has an intrinsic GTPase activity, the rate of which determines the lifetime of the active state (5, 10).Mutated forms of the ras genes have been identified in ca. 30% of human tumors. These ras oncogenes code for p21 proteins with point mutations in only three residues: Gly-12, Gly-13, and Gln-61. The mutated proteins have a reduced GTPase activity (5, 8, 9). The GTPase-activating protein (GAP) has been identified by its property of stimulating the conversion of normal p21-GTP to p21-GDP in Xenopus oocytes (32, 41). It has been cloned and sequenced and has a molecular mass of 120 kDa. The fact that it does not stimulate the GTPase activity of oncogenic mutants can explain the transforming properties of those mutants (18,41,42,44).The p21-GAP interaction has not been investigated in detail, and the stimulation of the GTPase by GAP was estimated to be 200-fold (41). By mutational analysis, it has been established that loop L2 of p21 is involved in the interaction with GAP (1,13,18,32). From the threedimensional structure, it would appear that loop L4 could also be involved in this interaction (33,36,37,40). It has also been assumed that the C-terminal domain of GAP is sufficient for the GTPase stimulatory activity (44). We have reevaluated the properties of GAP and show that the maximal stimulation of the GTPase activity by GAP is 105-fold and that mutations in loops Ll, L2, and LA affect the p21-GAP interaction in different ways. We also show that the C-terminal domain of GAP is not sufficient for full activating activity and is apparently modulated by the SH2 (src homologous) domains of GAP.

Structure of ERA in complex with the 3′ end of 16S rRNA: Implications for ribosome biogenesis

“…The intrinsic GTP-hydrolyzing activity of Ras is low (15), but can be accelerated Ͼ10 5 -fold by GAP (16,17). The intrinsic GTP-hydrolyzing activity of ERA is also low (18), ranging from 0.01 to 0.02 mmol min Ϫ1 mmol Ϫ1 , but is stimulated 3-to 12-fold in the presence of 16S rRNA (6,7).…”

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function

“…The likely explanation for this observation is that the SRJ ligands deplete the pool of GTP-Ras over time by allowing intrinsic hydrolysis of bound GTP while preventing reactivation via GEF-catalyzed GDP-GTP exchange. This mechanism can operate because oncogenic mutations prevent the ability of GAP to accelerate the intrinsic GTPase activity of Ras and only modestly reduce the actual intrinsic GTPase activity (41)(42)(43)(44). In showing that GEF-mediated nucleotide exchange is critical for oncogenic Ras signaling, our findings represent an important proof of concept for anti-Ras drug discovery.…”

Trapping the transition state of an ATP-binding cassette transporter: Evidence for a concerted mechanism of maltose transport

“…The basal rate of GTP hydrolysis by Ras is very slow, and GAP stimulates the activity by several orders of magnitude (42). Aluminum f luoride, like vanadate, stabilizes a transition state complex of GDP-Ras and GAP.…”