Biochemical and biological properties of the human N-ras p21 protein.

Trahey, Meg; Milley, Robert; Cole, G; Innis, Michael A.; Paterson, Hugh; Cj, Marshall; Hall, Alan; McCormick, Frank

doi:10.1128/mcb.7.1.541

Cited by 104 publications

(58 citation statements)

References 38 publications

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“…Recombinant mutant (Val-12) and wild-type H-ras proteins were expressed from pGEX-2T vectors in E. Coli strain BL21DE3 (LysE) and puri®ed by a nity chromatography on glutathione-sepharose (Pharmacia, Piscataway, NJ, USA) as described (Trahey et al, 1987). The GST-fusion protein was eluted by cleavage with thrombin and dialysed against 10 mM Tris-HCl pH 7.5, 114 mM KCl, 15 mM NaCl, 5 mM MgCl 2 .…”

Section: Bacterial Expression and Puri®cation Of Recombinant Proteinsmentioning

confidence: 99%

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras

1999

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Given the high frequency of ras oncogene activation in several common human cancers, its signal pathways are an important target for novel therapy. For practical reasons, however, these have been studied mainly in the context of transformation of established ®broblast cell lines, whereas ras acts at an earlier stage in human tumorigenesis and predominantly on epithelial cells. Here we have developed a more directly relevant model ± human primary thyroid epithelial cells ± which are a major target of naturally-occurring Ras mutation, and in which expression of mutant Ras in culture induces clonal expansion without morphological transformation, closely reproducing the phenotype of the corresponding tumour in vivo. Transient or stable expression of mutant H-ras (by scrapeloading or retroviral infection) at levels which stimulated proliferation induced sustained activation and translocation of MAP kinase (MAPK) in these cells. Inhibition of the MAPK pathway at the level of MAPKK, by expression of a dominant-negative mutant or by the pharmacological inhibitor PD98059, e ciently blocked the proliferative response. Conversely, selective activation of MAPK by a constitutively-active MAPKK1 mutant failed to mimic the action of Ras and, although this was achievable with activated Raf, micro-injection of anti-ras antibodies showed that this still required endogenous wild-type Ras function. In contrast to recent results obtained with a rodent thyroid cell line (WRT), therefore, activation of the MAPK pathway is necessary, but not su cient, for the proliferogenic action of mutant Ras on primary human thyroid cells. These data emphasize the unreliability of extrapolation from cell lines and establish the feasibility of using a more representative human epithelial model for Ras signalling studies.

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Section: Bacterial Expression and Puri®cation Of Recombinant Proteinsmentioning

confidence: 99%

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras

1999

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“…MEK then phosphorylates MAPK on threonine and tyrosine, and the activated MAPK which is a serine/threonine kinase is able to phosphorylate several nuclear transcription factors inluding Myc, Elk and Rsk (Siegfried and Zi , 1990;Kolch et al, 1991;Alexandropoulos et al, 1992;Blenis, 1993;Thomas, 1992;Crews et al, 1992). It is now known that not only can the activated Ras oncogene cause cellular transformation (Barbacid, 1987;Trahey et al, 1987), but that activated v-Raf or a constitutively activated MAPKK can also result in transformation (Heidecker et al, 1990;Cleveland et al, 1994;Samuels et al, 1993;Mansour et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis

et al. 1997

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“…The N-RAS p21 expression plasmid was constructed as described (10) The effect of the valine 13C carbonyl label is to broaden and possibly split three peaks, A, F, and K (Fig. 2a), relative to the singly labeled protein (Fig.…”

Section: Methodsmentioning

confidence: 99%

Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.

Campbell

Papastavros²,

McCormick³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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A sample ofEscherichia coil-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with '5N at each of the 14 glycine amide positions. Twodimensional proton-observe '5N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three ofthese resonances were asigned to residues 10, 15, and 115 from the spectrum ofa sample that was also labeled with ['3C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13Cdecoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several sitespecific probes of critical domains in p21. In this report we describe methods used to identify important glycine residues in N-RAS-encoded p21 (p21), a guanine nucleotide-binding protein that plays a crucial role in control of cellular growth (6). N-RAS is one of three distinct mammalian genes (N-RAS, K-RAS, and H-RAS) that encode highly related 21-kDa proteins. These proteins are related to several other families of guanine nucleotide-binding proteins (mammalian signal-transducing proteins, elongation factor Tu). Certain mutations in the coding sequence of RAS genes give rise to p21 proteins with transforming properties, and these altered proteins have been found in many human and animal tumor cells. The structure of N-RAS p21 has been inferred by analogy with elongation factor Tu (7, 8), and a preliminary x-ray study of H-RAS-encoded p21 has appeared (9). Six glycines common to both H-RAS and N-RAS p21 are near the guanine nucleotide-binding site . Several oncogenic activating positions near this site have been genetically identified (residues 12, 13, 15, 16, 59, 116, and 119). In particular, Gly-12 is in the phosphatebinding domain. This position appears to be crucial since substitution ofany other amino acid (except proline) generates mutant p21 proteins with transformation-inducing properties.In addition to the normal N-RAS gene product, we have also studied a p21 transforming mutant that contains aspartate instead of glycine at position 12. We have used isotopeedited NMR methods to identify active site glycine resonances in ['-N]glycine p21. These resonances have been used as site-specific probes to investigate spectral variations associated with p21 point mutations or ligand changes in critical domains of p21 (S.C.B., unpublished data).The NMR methods used to obtain resolved signals will not be detailed here. As already indicated, through a combination of proton and '5N pulsed excitation we can obtain a 2D NMR spectral map that shows peaks only from the few "5NH 817The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance wi...

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Biochemical and biological properties of the human N-ras p21 protein.

Cited by 104 publications

References 38 publications

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras

Activation of mitogen-activated protein kinase is necessary but not sufficient for proliferation of human thyroid epithelial cells induced by mutant Ras

Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis

Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.

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