Enhancement of the GDP-GTP Exchange of RAS Proteins by the Carboxyl-Terminal Domain of SCD25

Créchet, Jean‐Bernard; Poullet, Patrick; Mistou, Michel‐Yves; Parmeggiani, Andrea; Camonis, Jacques; Boy‐Marcotte, Emmanuelle; Damak, Faten; Jacquet, Michel

doi:10.1126/science.2188363

Cited by 148 publications

(106 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…GDP and p21 . GTP complexes, as already reported (Crechet et al, 1990a), but its stimulation of the k', value of the GTP and GDP complex only differed by a factor of two (Fig. 4A).…”

Section: Kinetic and Equilibrium Constants Of The Ras -Gdp(gtp)supporting

confidence: 62%

Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins

Poullet

Créchet

Bernardi

et al. 1995

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia culi recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95 %) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc2Sp-C was stronger for Ras2p . GDP than Ras2p . GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p. GDP (GTP) complex was also enhanced by Sdc2Sp-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc2Sp-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc2Sp-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc2Sp-C (truncated Sdc2Sp-C) showing the same specific activity as the 64-kDa Sdc2Sp-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1 : 1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S 17NIp21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc2Sp-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81DIRas2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc2Sp.

show abstract

“…GDP and p21 . GTP complexes, as already reported (Crechet et al, 1990a), but its stimulation of the k', value of the GTP and GDP complex only differed by a factor of two (Fig. 4A).…”

Section: Kinetic and Equilibrium Constants Of The Ras -Gdp(gtp)supporting

confidence: 62%

Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins

Poullet

Créchet

Bernardi

et al. 1995

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Specific exchange factors have been also described for the ras gene family in bovine brain [27], rat brain [28] and in yeast [29]. In the latter organism, a cdc25 gene product from Succharomyces cerevisiue has been shown to increase the adenylate cyclase activity [29 -311, probably by enhancing the GDP-exchange rate from p21 [32]. However, any structural homology between cdc25 and translational exchange factors is absent [31].…”

Section: Discussionmentioning

confidence: 99%

Mapping the functional domains of the eukaryotic elongation factor 1βγ

Damme¹,

Amons²,

Janssen³

et al. 1991

European Journal of Biochemistry

View full text Add to dashboard Cite

The functional domains of the eukaryotic elongation factor (EF) 1Py have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1P and EF-ly. By means of limited proteolysis, it was possible to obtain large fragments of EF-ID. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-ID were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-la. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 By containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1P and two monoclonal antibodies against EFl y were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-I y. The antiserum against EF-1P and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-ly, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-IB and EF-ly are firmly attached to each other, and that the carboxy-terminal part of EF-lP interacts with EF-la.Protein synthesis requires factors for proper translation [I]. In prokaryotes, correct binding of aminoacyl tRNA to translating ribosomes is mediated by elongation factor EFTu, a protein which is able to form a ternary complex with aminoacyl tRNA and GTP. Under simultaneous hydrolysis of GTP into GDP and Pi, the cognate aminoacyl tRNA becomes bound to the ribosome mRNA complex [2]. Replacement of GDP, in the EF-Tu . GDP complex, by GTP is enhanced by a separate factor, EF-Ts, the nucleotide-exchange factor [2]. The situation in eukaryotes is similar; here, however, EF-I a ( Z 50 kDa) replaces EF-Tu, whereas a complex between two polypeptide chains, EF-1fi and EF-ly, functions as the nucleotide-exchange factor [2]. The actual exchange activity, which resides on EF-lP (~2 6 kDa), is modulated by phosphorylation of its residue Ser89 [3]. The role of EF-ly (~4 6 kDa), a protein with strong hydrophobic properties, is not yet clear, although it possesses a stimulating effect on the exchange activity of EF-1P [4]. High-molecular-mass complexes between EF-la and EF-1Py have been described in many eukaryotic tissues [5 -111.In an attempt to unravel the requirements for nucleotideexchange activity and the involvement of the protein stretch around the phosphorylated residue Ser89 in EF-IP, we have isolated large, well-defined proteolytic fragments of this pro- Abbreviations. EF-la, EF-Ib, EF-Iy and EF-16, the a, b, y and 6 subunits of the eukaryotic protein synthesis elongation factor 1 ; EFTu, prokaryotic equivalent of the eukaryotic protein synthesis elongation factor 13; EF-Ts, prokaryotic equivalent of the eukaryotic protein synthesis elongation factor 1 f l y ; mAb, monoclonal antibody.Enzymes. Trypsin (EC 3.4.21.4); clo...

show abstract

“…In signal transduction processes across membranes, an activated membrane receptor acts as the exchange factor (Bourne, Sanders & McCormick, 1991). In oncogenesis, soluble cytoplasmic factors serve as the guanine-nucleotide exchange factor (Crechet et al, 1990). Neither the binding site of the exchange factor on the guanine nucleotide-binding protein nor the mechanism of exchange is well understood.…”

Section: Introductionmentioning

confidence: 99%

Extension of the diffraction resolution of crystals

Schick

Jurnak²

1994

Acta Cryst D

View full text Add to dashboard Cite

The diffraction resolution of crystals of the guanine nucleotide-exchange factor complex, EF-Tu-Ts, has been extended from 5.0 to 2.5 A by lowering the solvent content in the crystals as well as the temperature of data collection. The common form of EF-Tu-Ts crystal belongs to space group P212121 with a = 81.1, b = 109.9, c = 207.5 A and has a solvent content of 61%. The crystals diffract to a resolution of 5.0 A, at 293 K and 4.0 A at 273 K. When cryoprotective agents are slowly diffused into the crystals, the cell constants slu'ink to a = 74.4, b = 109.9, c = 198.7A and the solvent content falls to 55%.

show abstract

Enhancement of the GDP-GTP Exchange of RAS Proteins by the Carboxyl-Terminal Domain of SCD25

Cited by 148 publications

References 16 publications

Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins

Properties of the Catalytic Domain of Sdc25p, a Yeast GDP/GTP Exchange Factor of Ras Proteins

Mapping the functional domains of the eukaryotic elongation factor 1βγ

Extension of the diffraction resolution of crystals

Contact Info

Product

Resources

About