Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator.

Martegani, Enzo; Vanoni, Marco; Zippel, Renata; Coccetti, Paola; Brambilla, Riccardo; Ferrari, Cristina; Sturani, E.; Alberghina, Lilia

doi:10.1002/j.1460-2075.1992.tb05274.x

Cited by 237 publications

(145 citation statements)

References 58 publications

Supporting

Mentioning

139

Contrasting

Unclassified

Order By: Relevance

“…Many aspects of the yeast Ras pathway are similar to the mammalian Ha-Ras pathway (2,40,62). To determine whether mammalian Ha-Ras affects expression of mammalian hsp genes, we measured induction of HSP68 in rat fibroblasts (FR3T3), untransformed or transformed with Ha-ras.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Engelberg¹,

Zandi²,

Parker³

et al. 1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-rastransformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.The ras proto-oncogenes are highly conserved from yeasts to humans (reviewed in reference 12). These genes encode small membrane-associated GTP binding proteins that transduce signals generated at the cell surface to intracellular effectors (3,7,24,59). The mammalian Ha-Ras protein seems to interact with the serine or threonine kinase [77][78][79]85) and somehow leads to its activation. This results in activation of a protein kinase cascade (18,73,83) that induces gene expression through phosphorylation of transcription factors (reviewed in reference 25). One transcription factor affected by Ha-Ras is c-Jun (6), and another is NF-KB (17). In addition to positive modulation of the transcription factors' activity, HaRas may also negatively regulate certain transcription factors.In the yeast Saccharomyces cerevisiae, It was shown that expression of heat shock proteins (hsps) under nonshock conditions confers hyperresistance to heat shock (27,42,56). Therefore, it seems reasonable that resistance and sensitivity to heat shock are mediated at the level of hsp gene transcription. Indeed, it was shown that cyri cells express three proteins with a molecular mass of 72 kDa (65). These proteins, which were probably hsps, were not induced in the bcyl strain (65). Other studies showed that one of the heat shock genes, SSA3, was activated in the cyril strain in the absence of heat shock (9, 80). Also, the UBI4 gene, coding for polyubiquitin, was found to be regulated both by heat shock and cAMP (72). It is still not known, however, whether the Ras-cAMP pathway is a general regulator of hsp gene expression. It is also not known whether mammalian Ha-Ras activation also affects hsp gene expression. Furthermore, both in yeasts and in mammals, the relationship between the Ras pathway and the heat shock transcription factor (HSF [described bel...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Many aspects of Ras signalling are similar in yeasts and mammals (2,20,40,62). It is not yet known, however, whether the effects of Ras activity on gene expression are similar in yeasts and in mammals.…”

mentioning

confidence: 99%

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Engelberg¹,

Zandi²,

Parker³

et al. 1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…: 33-1-6933-4180; Fax: 33-1-6933-4840. 1 The abbreviations used are: CDC25 Mm , full-length murine CDC25 Mm gene product of 1262 amino acid residues; C-CDC25 Mm285 and C-CDC25 Mm631 , C-terminal fragments of CDC25 Mm comprising the last 285 and 631 amino acid residues, respectively; MBP, maltosebinding protein; GEF, guanine nucleotide exchange factor; GRF, guanine nucleotide releasing factor; ME, 2-mercaptoethanol; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase. been replaced by a SalI site.…”

Section: Production and Purification Of Cdc25mentioning

confidence: 99%

The N-terminal Moiety of CDC25Mm, a GDP/GTP Exchange Factor of Ras Proteins, Controls the Activity of the Catalytic Domain

Baouz

Jacquet

Bernardi

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

This work describes the in vitro properties of fulllength CDC25 Mm (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25 Mm , isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21⅐GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25 Mm285 ) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25 Mm was increased severalfold by the Ca 2؉ -dependent protease calpain that cleaves around a PEST-like region (residues 798 -853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25 Mm (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25 Mm -induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca 2؉ ions. Calmodulin also inhibited C-CDC25 Mm285 but with a ϳ100 times higher IC 50 than in the case of CDC25 Mm (ϳ10 M versus 0.1 M, respectively). Together, these results emphasize the role of the other domains of CDC25 Mm in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25 Mm activity.The mouse CDC25 Mm 1 protein is a guanine nucleotide exchange factor (GEF) regenerating the active form of H-ras p21, the complex with GTP (1-3). Homologous products were found in rat (p140-rasGRF) (4) and human (H-GRF) (5, 6). These rasGEFs have been described to be specific for the central nervous system (4 -9). Some evidence has also been reported for the existence of full-length and truncated forms of these exchange factors in other tissues (10,11). Experiments in vivo suggest that the upstream connection of this GEF involves G-protein-coupled receptors (9, 12, 13) and not hormone-receptor-bound tyrosine kinases via the adaptor protein GRB2, as has been found for SOS, a ubiquitous rasGEF (14 -16). CDC25 Mm contains in the N-terminal moiety two domains of pleckstrin homology (PH1 and PH2), one of DBL homology (DH) and a coiled-coil region that follows the PH1 domain (cf. (21) and the DH is a domain sharing similarity with a GDP/GTP exchange factor of members of the Rho family (2,4,22,23). Farnsworth et al. (24) reported that in vivo the activity of the homologous p140-rasGRF from rat brain is enhanced by raising the calcium concentration, an effect associated with the binding of calmodulin, and that p140-rasGRF and calmodulin form a stable complex. A direct action of calmodulin was supported by the presence in the N-terminal region of CDC25 Mm of an IQ domain, a sequence frequently found in proteins interacting with calmodulin (25, 26). Very recent experiments in vivo have indicated that PH1, ...

show abstract

“…In mammalian cells two classes of ras-speci®c GEF have been identi®ed. One class is mostly brain-speci®c and has been called CDC25 Mm , Ras Grf, hGRF55 by various groups (Martegani et al, 1992;Shou et al, 1992;Schweigho er et al, 1993;Wei et al, 1993Wei et al, , 1994Sturani et al, 1997). The other class is expressed ubiquitously and is called Sos Buday and Downward, 1993;Guerrero et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts

et al. 2000

View full text Add to dashboard Cite

Ras proteins are small GTPases playing a pivotal role in cell proliferation and di erentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25 Mm -GEF), conserved in all rasspeci®c GEFs identi®ed so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to e ciently displace wild-type GEF from p21 ras and to originate a stable ras/GEF binary complex due to the reduced a nity of the nucleotide-free ras/ GEF complex for the incoming nucleotide. We show here that this`ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse ®broblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEF W1056E in stable transfected cells strongly reduces intracellular ras´GTP levels in k-ras transformed ®broblasts. Accordingly, the transfected ®broblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-speci®c GEFs as a tool to down-regulate tumor growth is discussed.

show abstract

Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator.

Cited by 237 publications

References 58 publications

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

The N-terminal Moiety of CDC25Mm, a GDP/GTP Exchange Factor of Ras Proteins, Controls the Activity of the Catalytic Domain

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts

Contact Info

Product

Resources

About