Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.

Clanton, David J.; Lü, Youyong; Blair, Donald G.; Shih, Thomas Y.

doi:10.1128/mcb.7.9.3092

Cited by 53 publications

(44 citation statements)

References 30 publications

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“…In contrast to Asn-17 p21, the protein encoded by the Asn-16 mutant ras gene displayed a comparable reduction (-100-fold) in affinity for both GDP and GTP. Additional ras mutants with reduced affinities for both GTP and GDP also lack growth inhibitory properties in mammalian cells (7,8,12,15,52). In fact, in some cases decreased nucleotide-binding affinities and increased rates of nucleo-VOL.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Feig¹,

Cooper²

1988

Mol. Cell. Biol.

729

606

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Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20-to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.The mammalian rais gene family contains three members, ras , ras and rasN, which encode similar 21,000-dalton proteins referred to as p21's (for a review, see reference 1). These proteins reside on the inner face of the plasma membrane, bind GTP and GDP with an equally high affinity. and display a weak GTP hydrolysis activity (1). These properties are similar to those of G proteins that transduce signals from a wide variety of cell surface receptors to enzymes which affect the metabolism of second messengers, including adenylate cyclase, cyclic GMP phosphodiesterase, and phospholipases C and A2 and may also directly regulate ion channels (for a review, see reference 21). Based on this analogy, it has been proposed that r-as proteins also act as signal-transducing molecules. The fact that r-as genes activated by point mutations confer upon cultured cells some aspects of an oncogenic phenotype implies that their encoded proteins function in the transduction of signals that regulate cell proliferation. Other pathways are also probably affected, since p21 is expressed in nondividing cells (6. 19) and activated rcas genes can induce neuronal differentiation of PC12 cells (2, 22), concomitant with a cessation of cell division.G proteins characterized to date have a common structural design. They exist as oligomers made up of ox, ,B, and y subunits (21). In the inactive form. the a subunit is bound to GDP as well as to the a and -y subunits (21). An excited membrane receptor activates the G protein by enhancing the rate of exchange of bound GDP for GTP. Go then dissociates from 13 and y and alters the activity of its target enzyme or ion channel. The ot chain then hydrolyzes its bound GTP to GDP, thereby terminating activation (21). Although no 1 a...

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Section: Methodsmentioning

confidence: 99%

“…In fact, in some cases decreased nucleotide-binding affinities and increased rates of nucleo-VOL. 8,1988 on May 12, 2018 by guest http://mcb.asm.org/ Downloaded from tide exchange activate ras-transforming potential (13. 41, 50).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Feig¹,

Cooper²

1988

Mol. Cell. Biol.

729

606

View full text Add to dashboard Cite

show abstract

“…Mutations which alter the affinity of p21 for GTP have also been identified by in vitro mutagenesis (3,4,7,8,17,21). These mutations decrease the equilibrium binding affinity of p21 for GTP primarily by increasing the dissociation rate of bound nucleotide, thereby resulting in an increased rate of nucleotide exchange (8).…”

Section: * Corresponding Authormentioning

confidence: 99%

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

Feig¹,

Cooper²

1988

Mol. Cell. Biol.

186

140

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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 ...

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“…G10E mutation, detected in NRAS, is highly transforming and tumourigenic since the protein behaves as the wild-type v-ras p21, and belongs to a class of mutations whose biological activities do not require GTP-binding (29). The highly conserved glycine at position 10, changed to glutamic acid, is the first residue of a major site of activation (nucleotide phosphate-binding, 10-17) (27) of the p21 protein transforming properties.…”

Section: Ink4amentioning

confidence: 99%

Three cases of rare salivary gland tumours: a molecular study of TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes

Falbo

Floridia²,

Censi³

et al. 2011

Oncol Rep

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Abstract. Salivary gland tumours are rare tumours characterized by histopathologic complexity and a wide variety of morphologic features. Studies on genetic changes in different histological subtypes of salivary gland tumours are important to better understand molecular pathogenetic mechanisms and to identify diagnostic and prognostic markers. Data are even more scanty dealing with unusual subtypes of these tumours. The aim of the present study was to analyse two high grade transformation adenoid cystic carcinomas (hgACC) and one hybrid tumour in order to identify, by mutational and microsatellite analysis, genetic alterations in TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes. The two hgACCs showed snps missense in RAS genes and alterations with allelic instability in CDKN2A/ARF; moreover, a double mutation in TP53 was detected in one case. The hybrid tumour showed alterations in CDKN2A/ARF gene and snps missense in NRAS genes. Our data suggest that CDKN2A/ARF pathway might be involved in pathogenesis of the salivary gland tumours analysed. Further molecular analyses of these very rare tumours are necessary to better understand the role of other genetic alterations detected in our study.

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Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis.

Cited by 53 publications

References 30 publications

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

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

Three cases of rare salivary gland tumours: a molecular study of TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes

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