Mutations of the Ras Gene Product P21 That Abolish Guanine Nucleotide Binding

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1986 Jul 1

PMID 3014531

Citations 28

Authors

D J Clanton

S Hattori

T Y Shih

Affiliations

Soon will be listed here.

Abstract

We have constructed several point mutations affecting the GTP-binding site of p21, the ras-encoded protein. Both lysine (116K) and tyrosine (116Y) mutations of asparagine-116, which, by analogy with the crystal structure of elongation factor Tu (EF-Tu), has critical interactions with the guanine base, abolish GTP binding and transforming activities of p21. These activities are retained by proteins with a mutation at position 117 or 118. Both 116K and 116Y mutant p21s, when overproduced in Escherichia coli, are apparently devoid of GTP-binding and autokinase activities. Similarly, the mutant DNAs do not transform NIH 3T3 cells in a focus-forming assay. By cotransfection with pSV-neo, cell clones resistant to the neomycin analog G418 have been isolated. Cells transfected with 116K or 116Y mutant DNA are contact inhibited. In contrast to competent clones, the defective mutants have no detectable phosphorylated p21. The present results suggest that the basic structure of the GTP-binding site is conserved between p21 and EF-Tu and that this binding site is crucial for ras gene function.

Citing Articles

Novel variants in GNAI3 associated with auriculocondylar syndrome strengthen a common dominant negative effect.

Romanelli Tavares V, Gordon C, Zechi-Ceide R, Kokitsu-Nakata N, Voisin N, Tan T Eur J Hum Genet. 2014; 23(4):481-5.

PMID: 25026904 PMC: 4666574. DOI: 10.1038/ejhg.2014.132.

The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae.

Sengottaiyan P, Spetea C, Lagerstedt J, Samyn D, Andersson M, Ruiz-Pavon L BMC Biochem. 2012; 13:11.

PMID: 22726655 PMC: 3477016. DOI: 10.1186/1471-2091-13-11.

Effects of site-directed mutagenesis of mglA on motility and swarming of Myxococcus xanthus.

Fremgen S, Burke N, Hartzell P BMC Microbiol. 2010; 10:295.

PMID: 21083931 PMC: 3000849. DOI: 10.1186/1471-2180-10-295.

Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.

Tartaglia M, Gelb B Ann N Y Acad Sci. 2010; 1214:99-121.

PMID: 20958325 PMC: 3010252. DOI: 10.1111/j.1749-6632.2010.05790.x.

Noonan syndrome: clinical aspects and molecular pathogenesis.

Tartaglia M, Zampino G, Gelb B Mol Syndromol. 2010; 1(1):2-26.

PMID: 20648242 PMC: 2858523. DOI: 10.1159/000276766.

References

McCormick F, Clark B, la Cour T, Kjeldgaard M, Nyborg J . A model for the tertiary structure of p21, the product of the ras oncogene. Science. 1985; 230(4721):78-82. DOI: 10.1126/science.3898366. View

Jurnak F . Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985; 230(4721):32-6. DOI: 10.1126/science.3898365. View

la Cour T, Nyborg J, Thirup S, Clark B . Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography. EMBO J. 1985; 4(9):2385-8. PMC: 554514. DOI: 10.1002/j.1460-2075.1985.tb03943.x. View

SANGER F, Nicklen S, Coulson A . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74(12):5463-7. PMC: 431765. DOI: 10.1073/pnas.74.12.5463. View

Lowy D, Rands E, Scolnick E . Helper-independent transformation by unintegrated Harvey sarcoma virus DNA. J Virol. 1978; 26(2):291-8. PMC: 354067. DOI: 10.1128/JVI.26.2.291-298.1978. View

Chang E, Maryak J, Wei C, Shih T, Shober R, Cheung H . Functional organization of the Harvey murine sarcoma virus genome. J Virol. 1980; 35(1):76-92. PMC: 288784. DOI: 10.1128/JVI.35.1.76-92.1980. View

Southern P, Berg P . Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982; 1(4):327-41. View

Furth M, Davis L, Fleurdelys B, Scolnick E . Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982; 43(1):294-304. PMC: 256120. DOI: 10.1128/JVI.43.1.294-304.1982. View

Dhar R, Ellis R, Shih T, Oroszlan S, Shapiro B, Maizel J . Nucleotide sequence of the p21 transforming protein of Harvey murine sarcoma virus. Science. 1982; 217(4563):934-6. DOI: 10.1126/science.6287572. View

10.

Shih T, Stokes P, Smythers G, Dhar R, Oroszlan S . Characterization of the phosphorylation sites and the surrounding amino acid sequences of the p21 transforming proteins coded for by the Harvey and Kirsten strains of murine sarcoma viruses. J Biol Chem. 1982; 257(19):11767-73. View

11.

Lautenberger J, Ulsh L, Shih T, Papas T . High-level expression in Escherichia coli of enzymatically active Harvey murine sarcoma virus p21ras protein. Science. 1983; 221(4613):858-60. DOI: 10.1126/science.6308763. View

12.

Leberman R, Egner U . Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21. EMBO J. 1984; 3(2):339-41. PMC: 557346. DOI: 10.1002/j.1460-2075.1984.tb01808.x. View

13.

Finkel T, Der C, Cooper G . Activation of ras genes in human tumors does not affect localization, modification, or nucleotide binding properties of p21. Cell. 1984; 37(1):151-8. DOI: 10.1016/0092-8674(84)90310-6. View

14.

McGrath J, Capon D, Goeddel D, Levinson A . Comparative biochemical properties of normal and activated human ras p21 protein. Nature. 1984; 310(5979):644-9. DOI: 10.1038/310644a0. View

15.

Lacal J, Santos E, Notario V, Barbacid M, Yamazaki S, Kung H . Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins. Proc Natl Acad Sci U S A. 1984; 81(17):5305-9. PMC: 391692. DOI: 10.1073/pnas.81.17.5305. View

16.

Sweet R, Yokoyama S, Kamata T, Feramisco J, Rosenberg M, Gross M . The product of ras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature. 1984; 311(5983):273-5. DOI: 10.1038/311273a0. View

17.

Gibbs J, Sigal I, Poe M, Scolnick E . Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984; 81(18):5704-8. PMC: 391779. DOI: 10.1073/pnas.81.18.5704. View

18.

Hurley J, Simon M, Teplow D, Robishaw J, GILMAN A . Homologies between signal transducing G proteins and ras gene products. Science. 1984; 226(4676):860-2. DOI: 10.1126/science.6436980. View

19.

Zoller M, Smith M . Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984; 3(6):479-88. DOI: 10.1089/dna.1.1984.3.479. View

20.

Willumsen B, Norris K, Papageorge A, Hubbert N, Lowy D . Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J. 1984; 3(11):2581-5. PMC: 557733. DOI: 10.1002/j.1460-2075.1984.tb02177.x. View