Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells

Chevallier-Multon, M C; Schweighoffer, Fabien; Barlat, Isabelle; Baudouy, Nicole; Fath, I; Duchesne, Marc; Tocqué, Bruno

doi:10.1016/s0021-9258(18)82099-3

Cited by 17 publications

(2 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A region of 200-300 amino acids, the RasGEF domain, is shared by all GEFs which act on members of the Ras subfamily, and their activity is specific toward either Ras, Ral, or Rap. The fact that truncated versions of various lengths, containing this RasGEF domain, have been shown to be active RasGEFs in vivo and in vitro (4,(13)(14)(15)(16) confirms that this region indeed represents the Ras-specific guanine nucleotide exchange domain.…”

mentioning

confidence: 83%

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25^Mm

et al. 1998

View full text Add to dashboard Cite

Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543-12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.

show abstract

mentioning

confidence: 83%

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25^Mm

et al. 1998

View full text Add to dashboard Cite

show abstract

“…In S. cerevisiae, two GDP/GTP exchange factors were identified, Cdc25p and Sdc25p, which are members of the conserved CDC25-like family [for references, see Parmeggiani et al (1993)]. Their catalytic domains were shown to have a GEF activity in vitro (Crechet et al, 1990;Jones et al, 1991;Lai et al, 1993;Chevallier-Multon et al, 1993;Jacquet et al, 1994).…”

mentioning

confidence: 99%

Properties and regulation of the catalytic domain of Ira2p, Saccharomyces cerevisiae GTPase-activating protein of Ras2p

Parrini

Jacquet²,

Bernardi³

et al. 1995

Biochemistry

View full text Add to dashboard Cite

This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography. The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times. The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable. Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414. The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts. The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S. cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle. The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383. A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

et al. 1995

View full text Add to dashboard Cite

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.

show abstract

Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells

Cited by 17 publications

References 0 publications

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25^Mm

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25^Mm

Properties and regulation of the catalytic domain of Ira2p, Saccharomyces cerevisiae GTPase-activating protein of Ras2p

Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

Contact Info

Product

Resources

About

Saccharomyces cerevisiae CDC25 (1028-1589) is a guanine nucleotide releasing factor for mammalian ras proteins and is oncogenic in NIH3T3 cells

Cited by 17 publications

References 0 publications

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25Mm

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25Mm

Properties and regulation of the catalytic domain of Ira2p, Saccharomyces cerevisiae GTPase-activating protein of Ras2p

Guanine nucleotide exchange factors: Activators of the Ras superfamily of proteins

Contact Info

Product

Resources

About

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25^Mm

Kinetic Analysis by Fluorescence of the Interaction between Ras and the Catalytic Domain of the Guanine Nucleotide Exchange Factor Cdc25^Mm