Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

Abstract

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a >1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (-100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.The cellular ras genes encode plasma membrane-associated proteins (p2ls) of 189 amino acids which bind GDP and GTP with high affinity and display a low level of GTPase activity (see reference 1 for a review). These properties of p21 are analogous to the G proteins, which transduce signals from a variety of cell surface receptors to enzymes which affect metabolism of second messengers (see reference 11 for a review). It is therefore an attractive hypothesis that p2is function analogously to the G proteins to affect second messenger pathway(s) which can result in abnormal cell proliferation.One approach to understanding the function of p21 has been comparative analysis of the biological and biochemical activities of the products of mutated ras genes. Activation of the transforming potential of ras genes in human tumors has been found to result from single amino acid substitutions at positions 12, 13, and 61 (1). The viral rasH and rasK genes also contain activating mutations at position 59 (1). Activating mutations at positions 12 and 61 decrease the GTPase activity of p21 approximately 10-fold (6, 10, 14, 15, 18). Since GTP hydrolysis mediates physiological deactivation of the G proteins, these results are consistent with the Gprotein analogy. However, reduced GTPase does not correlate quantitatively with the transforming ...

ras activation in human tumors and in animal model systems.

“…This difference was statistically significant. In agreement with our findings, a recent report showed that an in vitro mutagenized H-ras gene with a mutation in codon 146 had less transforming activity than did a ras gene with a codon 12 mutation (27).…”

Activation of NF-κB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa -induced mucin overproduction in epithelial cells

“…All the cells were then exposed to P. aeruginosa for 6 hr. Forty hours after being transfected, HM3 cells were either exposed or not exposed to PAO1 culture supernatant (CS)with a dominant-negative mutant form of Ras (RasN17) (25) reduced the response by 80% (Fig. 2C), and cotransfection with a dominant-negative mutant form of c-Src (SrcRF) inhibited the response by 45% (Fig.…”

HRAS mutants identified in Costello syndrome patients can induce cellular senescence: possible implications for the pathogenesis of Costello syndrome

“…It has been reported that the nucleotide exchange rate of both p.K117R and p.A146V HRAS is increased, relative to wild type. 13,27,28 However, the transformational potential of p.A146V HRAS is partially activated, 27 whereas that of p.K117R-HRAS is not; its transformational activity is instead similar to that of GTPase impaired mutants. 28 Our results and those of other reports suggest that p.K117R and p.A146T have milder effects on downstream effectors than do mutations in codon 12/13.…”

“…Patients with p.K117R or p.A146V had a milder and more unusual CS phenotype, compared with patients with mutations in codon 12 or 13. Though detailed analyses of some mutants have been performed, 13,[25][26][27][28] a comprehensive comparison of the substitutions identified in patients with CS has not been conducted.…”