[10] Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements

Lenzen, Christian; Cool, Robbert H.; Wittinghofer, Alfred

doi:10.1016/s0076-6879(95)55012-7

Cited by 127 publications

(142 citation statements)

References 18 publications

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“…[17,24,25] Recombinant H-p21ras (2 μg) was loaded with a fluorescent nucleotide analogue N-methylanthraniloyl GDP (Mant-GDP), which exhibits fluorescence only when bound to the protein. The p21ras was incubated 1 h at 37°C with an excess of Mant-GDP in a binding buffer (potassium phosphate buffer (50 mM), NaCl (50 mM), MgCl 2 (5 mM) and EDTA (5 mM)) and the protein-Mant-GDP complex was purified on a PL-6 spin column.…”

Section: P21ras Activity Assaymentioning

confidence: 99%

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

Mahadevan

Clavreul

Huang

et al. 2007

Free Radical Biology and Medicine

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Abstractp21ras GTPase is the protein product of the most commonly mutated human oncogene and has been identified as a target for reactive oxygen and nitrogen species (ROS/RNS). Post-translational modification of reactive thiols, by reversible S-glutathiolation and S-nitrosation, and potentially also by irreversible oxidation, may have significant effects on p21ras activity. Here we used an isotopecoded affinity tag (ICAT) and mass spectrometry to quantitate the reversible and irreversible oxidative post-translational thiol modifications of p21ras caused by peroxynitrite (ONOO − ) or glutathione disulfide (GSSG). The activity of p21ras was significantly increased following exposure to GSSG, but not to ONOO − . The results of LC-MS/MS analysis of tryptic peptides of p21ras treated with ONOO − showed that ICAT labeling of Cys 118 was decreased by 47%, whereas Cys 80 was not significantly affected and was thereby shown to be less reactive. The extent of S-glutathiolation of Cys 118 by GSSG was 53%, and that of the terminal cysteines was 85%, as estimated by the decrease in ICAT labeling. The changes in ICAT labeling caused by GSSG were reversible by chemical reduction, but those caused by peroxynitrite were irreversible. The quantitative changes in thiol modification caused by GSSG associated with increased activity demonstrate the potential importance of redox modulation of p21ras.

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Section: P21ras Activity Assaymentioning

confidence: 99%

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

Mahadevan

Clavreul

Huang

et al. 2007

Free Radical Biology and Medicine

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“…2',3'-bis(O)-(Nmethylanthranoloyl)-guanosine-diphosphate, by incubating them in the presence of 10 mM EDTA and a 20-fold excess of mGDP for 4 ± 10 h at room temperature (Lenzen et al, 1995). Protein-nucleotide complex was separated from unbound nucleotides by a small NAP5 prepacked gel ®ltration column (Pharmacia).…”

Section: Expression and Puri®cation Of A Catalytic Fragment Of C3gmentioning

confidence: 99%

“…Protein-nucleotide complex was separated from unbound nucleotides by a small NAP5 prepacked gel ®ltration column (Pharmacia). Nucleotide content was assayed by HPLC (Lenzen et al, 1995) and related to the protein concentration determined by the Bradford assay (Bradford, 1976). mGDP loading e ciency usually was between 70 and 95%, except for Rap2A (30%).…”

Section: Expression and Puri®cation Of A Catalytic Fragment Of C3gmentioning

confidence: 99%

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

Berghe¹,

Cool²,

Horn³

et al. 1997

Oncogene

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A catalytically active fragment of the Rap-speci®c guanine-nucleotide exchange factor C3G was expressed in E coli. It was puri®ed and its interaction with GTPbinding proteins was investigated using¯uorescence spectroscopy. C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily. Like the corresponding fragment from CDC25 Mm , the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A . GDP up to 100 mM, indicating an apparent K M higher than 100 mM. Unlike the Ras-CDC25 Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro. These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative e ects.

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“…The activation of Ras by receptor tyrosine kinases proceeds through the recruitment of the nucleotide-exchange factor Son of sevenless (Sos) to the plasma membrane, where it encounters Ras and stimulates release of GDP, allowing its replacement by GTP (2)(3)(4)(5)(6). In some cells, G protein-coupled receptors rely on relatives of Sos, such as Ras guanine nucleotide-releasing factor 1 (RasGRF1), also known as p140 Ras-GRF or Cdc25, for initiating Ras signaling (7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

“…In contrast to Sos, which requires Ras binding to the allosteric site for activity, the Cdc25 domain of RasGRF1 is active on its own (Fig. 1b) (11,17).…”

mentioning

confidence: 99%

A Ras-induced conformational switch in the Ras activator Son of sevenless

Freedman

Sondermann

Friedland

et al. 2006

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

129

152

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The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-Å resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Å resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity.Cdc25 ͉ nucleotide-exchange factor ͉ crystal structure ͉ Ras exchanger motif (Rem) domain ͉ Ras guanine nucleotide-releasing factor 1

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[10] Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements

Cited by 127 publications

References 18 publications

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

A Ras-induced conformational switch in the Ras activator Son of sevenless

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