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John S Lazo - One of the best experts on this subject based on the ideXlab platform.
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Cdc25b dual specificity phosphatase inhibitors identified in a high throughput screen of the nih compound library
Assay and Drug Development Technologies, 2009Co-Authors: Paul A Johnston, Peter Wipf, Caleb Foster, Marni Brisson Tierno, Tong Ying Shun, Sunita N Shinde, William D Paquette, Kay M Brummond, John S LazoAbstract:Abstract The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 μM met the active criterion of ≥50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC50) values <50 μM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kin...
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independent mechanistic inhibition of Cdc25 phosphatases by a natural product caulibugulone
Molecular Pharmacology, 2007Co-Authors: Marni Brisson, Peter Wipf, Theresa Nguyen, Caleb Foster, Robert J Tomko, John S LazoAbstract:Caulibugulones are novel but poorly characterized cytotoxic isoquinoline quinones and iminoquinones identified in extracts from the marine bryozoan Caulibugula intermis . We now report that the caulibugulones are selective in vitro inhibitors of the Cdc25 family of cell cycle-controlling protein phosphatases compared with either human vaccinia H1-related phosphatase (VHR) or tyrosine phosphatase 1B (PTP1B). The in vitro inhibition of Cdc25B by caulibugulone A was irreversible and attenuated by reducing agents or catalase, consistent with direct oxidation of the enzyme by reactive oxygen species. Mechanistically, caulibugulone A directly inhibited cellular Cdc25B activity, generated intracellular reactive oxygen species and arrested cells in both G1 and G2/M phases of the cell cycle. Caulibugulone A also caused the selective degradation of Cdc25A protein by a process that was independent of reactive oxygen species production, proteasome activity, and the Chk1 signaling pathway. Instead, caulibugulone A stimulated the phosphorylation and subsequent activation of p38 stress kinase, leading to Cdc25A degradation. Thus, caulibugulone inhibition of cellular Cdc25A and B phosphatases occurred through at least two different mechanisms, leading to pronounced cell cycle arrest.
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discovery and characterization of novel small molecule inhibitors of human Cdc25b dual specificity phosphatase
Molecular Pharmacology, 2004Co-Authors: Marni Brisson, Peter Wipf, Theresa Nguyen, Andreas Vogt, Jack C Yalowich, Angela Giorgianni, Dror Tobi, Ivet Bahar, Corey R J Stephenson, John S LazoAbstract:Cdc25A and Cdc25B dual-specificity phosphatases are key regulators of cell cycle transition and proliferation. They have oncogenic properties and are overexpressed in many human tumors. Because selective Cdc25 phosphatase inhibitors would be valuable biological tools and possible therapeutic agents, we have assayed a small molecule library for in vitro inhibition of Cdc25. We now report the identification of two new structurally distinct classes of Cdc25 inhibitors with cellular activity. The cyclopentaquinoline 3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4,8-dicarboxylic acid (5661118) and the naphthofurandione 3-benzoyl-naphtho[1,2-b]furan-4,5-dione (5169131) had in vitro IC50 values of 2.5 to 11 M against recombinant Cdc25 and were less potent inhibitors of other phosphatases. Unlike 5661118, 5169131 caused reversible inhibition of Cdc25B and displayed competitive inhibitor kinetics. No growth inhibitory activity was seen with 5661118, whereas 10 to 30 M 5169131 caused G1/S and G2/M arrest. We also found that 5169131 inhibited human PC-3 prostate and MDAMB-435 breast cancer cell proliferation. Concentration-dependent Tyr15 hyperphosphorylation was seen on cyclin-dependent kinase with a 1-h 5169131 treatment, consistent with Cdc25 inhibition. Cells resistant to DNA toposiomerase II inhibitors were as sensitive to 5169131 as parental cells, indicating that this quinone compound does not inhibit topoisomerase II in vivo. Molecular modeling was used to predict a potential interaction site between the inhibitor and Cdc25B and to provide insights as to the molecular origins of the experimental observations. Based on its kinetic profile and cellular activity, we suggest that 5169131 could be an excellent tool for further studies on the cellular roles of Cdc25.
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binding and inhibition of Cdc25 phosphatases by vitamin k analogues
Biochemistry, 2003Co-Authors: Iliya Lefterov, John S Lazo, Meifang Wang, Colleen N Scott, Craig S Wilcox, Brian I CarrAbstract:A synthetic K vitamin analogue, 2-(2-mercaptothenol)-3-methyl-1,4-naphthoquinone or Cpd 5, was previously found to be a potent inhibitor of cell growth [Nishikawa et al., (1995) J. Biol. Chem. 270, 28304−28310]. The mechanisms of cell growth were hypothesized to include the inactivation of cellular protein tyrosine phosphatases, especially the Cdc25 family [Tamura et al. (2000) Cancer Res. 60, 1317−1325]. In this study, we synthesized PD 49, a new biotin containing Cpd 5 derivative, to search for evidence of direct interaction of these arylating analogues with Cdc25A, Cdc25B, and Cdc25C phosphatases. PD 49 was shown to directly bind to GST-Cdc25A, GST-Cdc25B, their catalytic fragments, and GST-Cdc25C. The binding could be competed with excess glutathione or Cpd 5, and a cysteine-to-serine mutation of the catalytic cysteine abolished binding. This was consistent with an involvement in binding of cysteine in the catalytic domain. This interaction between PD 49 and Cdc25 also occurred in lysates of treated c...
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dual g1 and g2 phase inhibition by a novel selective Cdc25 inhibitor 7 chloro 6 2 morpholin 4 ylethylamino quinoline 5 8 dione
Journal of Biological Chemistry, 2002Co-Authors: Lixia Pu, Andrew A Amoscato, Mark E Bier, John S LazoAbstract:Abstract The Cdc25 dual specificity phosphatases coordinate cell cycle progression, but potent and selective inhibitors have generally been unavailable. In the present study, we have examined one potential inhibitor, 7-chloro-6-(2-morpholin-4-ylethylamino)-quinoline-5,8-dione (NSC 663284), that was identified in the compound library of the National Cancer Institute. We found that NSC 663284 arrested synchronized cells at both G1 and G2/M phase, and blocked dephosphorylation and activation of Cdk2 and Cdk1 in vivo, as predicted for a Cdc25 inhibitor. Using the natural Cdc25A substrate, Tyr15-phosphorylated Cdk2/cyclin A, we demonstrated that NSC 663284 blocked reactivation of Cdk2/cyclin A kinase by Cdc25A catalytic domain in vitro. In-gel trypsin digestion followed by capillary liquid chromatography-electrospray ionization mass spectrometry and tandem mass spectrometry revealed the direct binding of NSC 663284 to one of the two serine residues in the active site loop HCEFSSER of the Cdc25A catalytic domain. Cdc25 binding and inhibition could contribute to the anti-proliferative activity of NSC 663284 and its ability to arrest cell cycle progression. Moreover, NSC 663284 should be a valuable reagent to probe the actions of Cdc25 phosphatases within cells and may also be useful structure for the design of more potent and selective antiproliferative agents.
Andrea Parmeggiani - One of the best experts on this subject based on the ideXlab platform.
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properties of the catalytic domain of Cdc25 a saccharomyces cerevisiae gdp gtp exchange factor comparison of its activity on full length and c terminal truncated ras2 proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: Eric Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of Cdc25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two Cdc25 fragments containing the conserved catalytic domain, only Cdc25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by Cdc25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. Cdc25-509 displayed a higher specific activity than the catalytic domains of the two Cdc25-like proteins: S. cerevisiae SDC25 and mouse Cdc25 Mm .
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Properties of the Catalytic Domain of Cdc25, a Saccharomyces cerevisiae GDP/GTP Exchange Factor: Comparison of Its Activity on Full-Length and C-Terminal Truncated RAS2 Proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: Eric Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of Cdc25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two Cdc25 fragments containing the conserved catalytic domain, only Cdc25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by Cdc25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. Cdc25-509 displayed a higher specific activity than the catalytic domains of the two Cdc25-like proteins: S. cerevisiae SDC25 and mouse Cdc25 Mm .
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The C-terminal part of the Cdc25 gene product has Ras-nucleotide exchange activity when present in a chimeric SDC25-Cdc25 protein
Current Genetics, 1993Co-Authors: Emmanuelle Boy-marcotte, Andrea Parmeggiani, Christine Soustelle, Patrick Poullet, Michel JacquetAbstract:The Cdc25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of Cdc25 gives no detectable exchange activity. A chimera between the 3′ regions of these two genes was constructed by homeologous recombination. This chimeric gene suppresses Cdc25 mutations. When expressed in E. coli, the chimeric product is detectable by antibodies directed against the carboxy-terminal Cdc25 peptide and has an exchange-factor activity on the Ras2 protein. Therefore, the carboxy-terminal parts of both the Cdc25 and the SDC25 gene products are structurally and functionally similar. The Cdc25 part of the chimeric protein contains an intrinsic guanine exchange factor which does not require an additional cofactor.
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a mouse Cdc25 like product enhances the formation of the active gtp complex of human ras p21 and saccharomyces cerevisiae ras2 proteins
Journal of Biological Chemistry, 1992Co-Authors: Eric Jacquet, Enzo Martegani, Maria A Vanoni, Cristina Ferrari, Lilia Alberghina, Andrea ParmeggianiAbstract:Abstract GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals. A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae Cdc25 gene, whose product acts as a GDS of RAS proteins. However, except for Cdc25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these Cdc25-like proteins has yet been available. We show that the product of a recently isolated mouse Cdc25-like gene (Cdc25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro. As a consequence, the Cdc25Mm induces a rapid formation of the biologically active Ras.GTP complex. This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins. The mouse GDS can efficiently substitute for yeast Cdc25 in an in vitro adenylylcyclase assay on RAS2 Cdc25 yeast membranes. Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of Cdc25-like proteins.
Bernard Ducommun - One of the best experts on this subject based on the ideXlab platform.
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cell cycle control by the Cdc25 phosphatases
Anti-cancer Agents in Medicinal Chemistry, 2008Co-Authors: Bernadette Aressy, Bernard DucommunAbstract:: Cell division cycle 25 (Cdc25) phosphatases are key actors in eukaryotic cell cycle control. They are responsible for the dephosphorylations that activate the cyclin-dependent kinases (CDK) at specific stages of the cell cycle. Human Cdc25A, Cdc25B and Cdc25C are also central targets and regulators of the G2/M checkpoint mechanisms activated in response to DNA injury. The expression and activity of these enzymes is finely regulated by multiple mechanisms including post-translational modifications, interactions with regulatory partners, control of their intracellular localization, and cell cycle-regulated degradation. Altered expression of these phosphatases is associated with checkpoint bypass and genetic instability. Accordingly, increased expression of Cdc25A and Cdc25B is found in many high-grade tumors and is correlated with poor prognosis in human cancers. This review summarizes our current knowledge within this domain and discusses the data that support therapeutic strategies targeting Cdc25 activity in the treatment of cancer.
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receptor based virtual ligand screening for the identification of novel Cdc25 phosphatase inhibitors
Journal of Chemical Information and Modeling, 2008Co-Authors: Matthieu Montes, Bernard Ducommun, Mariepriscille Brun, Emmanuelle Braud, Odile Mondesert, Maria A Miteva, Christiane Garbay, Marylorene Goddard, Stephanie Kolb, Bruno O VilloutreixAbstract:Cdc25 phosphatases play critical roles in cell cycle regulation and are attractive targets for anticancer therapies. Several small non-peptide molecules are known to inhibit Cdc25, but many of them appear to form a covalent bond with the enzyme or act through oxidation of the thiolate group of the catalytic cysteine. Structure-based virtual ligand screening computations were performed with FRED, Surflex, and LigandFit, a compound collection of over 310 000 druglike molecules and the crystal structure of Cdc25B in order to identify novel classes of ligands. In vitro experiments carried out on a selected list of 1500 molecules led to the discovery of 99 compounds able to inhibit Cdc25B activity at 100 μM. Further docking computations were applied, allowing us to propose a binding mode for the most potent molecule (IC50 = 13 μM). Our best compounds represent promising new classes of Cdc25 inhibitors that also exhibit antiproliferative properties.
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The when and wheres of Cdc25 phosphatases.
Current Opinion in Cell Biology, 2006Co-Authors: Rose Boutros, Christine Dozier, Bernard DucommunAbstract:The Cdc25 phosphatases are key regulators of normal cell division and the cell's response to DNA damage. Earlier studies suggested non-overlapping roles for each isoform during a specific cell cycle phase. However, recent data suggest that multiple Cdc25 isoforms cooperate to regulate each cell cycle transition. For instance, although Cdc25A was initially thought to exclusively regulate the G(1)-S transition, recent data demonstrate a significant role for Cdc25A in the G(2)-M transition. Further evidence demonstrates that in addition to the ATM/ATR-CHK pathway, a p38-MAPKAP pathway is also involved in controlling Cdc25 activity during G(2)/M checkpoint activation. Together with the fact that Cdc25 overexpression is reported in many cancers, these data highlight the significance of developing specific Cdc25 inhibitors for cancer therapy.
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inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of Cdc25 phosphatases
Molecular Cancer Therapeutics, 2005Co-Authors: Mariechristine Brezak, Odile Mondesert, Muriel Quaranta, Marieodile Contourgalcera, Olivier Lavergne, Pierrick Auvray, Philip G Kasprzyk, Gregoire Prevost, Bernard DucommunAbstract:Cell cycle regulators, such as the Cdc25 phosphatases, are potential targets for the development of new anticancer drugs. Here we report the identification and the characterization of BN82685, a quinone-based Cdc25 inhibitor that is active in vitro and in vivo . BN82685 inhibits recombinant Cdc25A, B, and C phosphatases in vitro . It inhibits the growth of human tumor cell lines with an IC50 in the submicromolar range, independently of their resistance to chemotherapeutic agents. This inhibitory effect is irreversible on both the purified Cdc25 enzyme in vitro and on tumor cell proliferation. The specificity of BN82685 towards the Cdc25 phosphatases is shown by an increase in cyclin-dependent kinase 1 tyrosine 15 phosphorylation, by the reversion of the mitosis-inducing effect of Cdc25B overexpression in HeLa cells, and by the lack of a growth inhibitory effect in an assay based on the use of a Cdc25-independent fission yeast model. Finally, when administered p.o., BN82685 is shown to inhibit the growth of the human pancreatic tumor Mia PaCa-2 xenografted in athymic nude mice. BN82685 is therefore a promising new compound targeting Cdc25, which confirms the interest of the inhibition of these enzymes as an anticancer therapeutic strategy.
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design synthesis and biological evaluation of novel naphthoquinone derivatives with Cdc25 phosphatase inhibitory activity
Bioorganic & Medicinal Chemistry, 2005Co-Authors: Mariepriscille Brun, Bernard Ducommun, Emmanuelle Braud, Delphine Angotti, Odile Mondesert, Muriel Quaranta, Matthieu Montes, Maria A Miteva, Nohad Gresh, Christiane GarbayAbstract:Cdc25 dual-specificity phosphatases are essential key regulators of eukaryotic cell cycle progression and the Cdc25A and B isoforms are over-expressed in different tumors and related cancer cell lines. Cdc25s are now considered to be interesting targets in the search for novel anticancer agents. We describe new compounds derived from vitamin K3 that inhibit Cdc25B activity with IC50 values in the low micromolar range. These naphthoquinone derivatives also display antiproliferative activity on HeLa cells as expected for Cdc25 inhibitors and inhibit cell growth in a clonogenic assay at submicromolar concentrations. They increase inhibitory tyrosine 15 phosphorylation of CDK and induce the cleavage of PARP, a hallmark of apoptosis.
Eric Jacquet - One of the best experts on this subject based on the ideXlab platform.
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properties of the catalytic domain of Cdc25 a saccharomyces cerevisiae gdp gtp exchange factor comparison of its activity on full length and c terminal truncated ras2 proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: Eric Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of Cdc25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two Cdc25 fragments containing the conserved catalytic domain, only Cdc25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by Cdc25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. Cdc25-509 displayed a higher specific activity than the catalytic domains of the two Cdc25-like proteins: S. cerevisiae SDC25 and mouse Cdc25 Mm .
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Properties of the Catalytic Domain of Cdc25, a Saccharomyces cerevisiae GDP/GTP Exchange Factor: Comparison of Its Activity on Full-Length and C-Terminal Truncated RAS2 Proteins
Biochemical and Biophysical Research Communications, 1994Co-Authors: Eric Jacquet, M C Parrini, Alberto Bernardi, Enzo Martegani, Andrea ParmeggianiAbstract:Abstract Two C-terminal fragments (334 and 509 amino acid residues) of Cdc25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E. coli , using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two Cdc25 fragments containing the conserved catalytic domain, only Cdc25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by Cdc25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. Cdc25-509 displayed a higher specific activity than the catalytic domains of the two Cdc25-like proteins: S. cerevisiae SDC25 and mouse Cdc25 Mm .
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a mouse Cdc25 like product enhances the formation of the active gtp complex of human ras p21 and saccharomyces cerevisiae ras2 proteins
Journal of Biological Chemistry, 1992Co-Authors: Eric Jacquet, Enzo Martegani, Maria A Vanoni, Cristina Ferrari, Lilia Alberghina, Andrea ParmeggianiAbstract:Abstract GDP-dissociation stimulators (GDSs) are the key element for the regeneration of the active state of ras proteins, but despite intensive investigations, little is so far known about their functional and structural properties, particularly in mammals. A growing number of genes from various organisms have been postulated to encode GDSs on the basis of sequence similarity with the Saccharomyces cerevisiae Cdc25 gene, whose product acts as a GDS of RAS proteins. However, except for Cdc25 and the related SDC25 C-domain, no biochemical evidence of ras GDS activity for these Cdc25-like proteins has yet been available. We show that the product of a recently isolated mouse Cdc25-like gene (Cdc25Mm) can strongly enhance (more than 1000 times) the GDP release from both human c-Ha-ras p21 and yeast RAS2 in vitro. As a consequence, the Cdc25Mm induces a rapid formation of the biologically active Ras.GTP complex. This GDS is much more active on the GDP than on the GTP complex and has a narrow substrate specificity, since it was found to be inactive on several ras-like proteins. The mouse GDS can efficiently substitute for yeast Cdc25 in an in vitro adenylylcyclase assay on RAS2 Cdc25 yeast membranes. Our results show that a cloned GDP to GTP exchange factor of mammalian ras belongs to the novel family of Cdc25-like proteins.
William G. Dunphy - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Xenopus Cdc25 protein.
Methods in Enzymology, 2004Co-Authors: Akiko Kumagai, William G. DunphyAbstract:Publisher Summary This chapter discusses the regulation of xenopus Cdc25 protein. The Cdc25 protein was first identified on the basis of a mutation in Schizosaccharomyces pombe that blocks the cell cycle at the G2 phase. The biochemical function of Cdc25C as a phosphatase that dephosphorylates both Thr-14 and Tyr-15 of Cdc2, thereby activating the Cdc2 protein kinase at the onset of mitosis, is well established. Cdc25 has a highly conserved catalytic domain in its C-terminal half that contains a critical cysteine residue required for catalytic activity. The N-terminal half is less conserved between different species and contains a number of phosphorylation sites. During mitosis, the N-terminal half becomes extensively phosphorylated. Cdc25 has a highly conserved catalytic domain in its C-terminal half that contains a critical cysteine residue required for catalytic activity. The N-terminal half is less conserved between different species and contains a number of phosphorylation sites.
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14 3 3 proteins act as negative regulators of the mitotic inducer Cdc25 in xenopus egg extracts
Molecular Biology of the Cell, 1998Co-Authors: Akiko Kumagai, Peter S Yakowec, William G. DunphyAbstract:Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2-cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3epsilon and to a lesser extent with 14-3-3zeta . The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2-M transition.
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purification and molecular cloning of plx1 a Cdc25 regulatory kinase from xenopus egg extracts
Science, 1996Co-Authors: Akiko Kumagai, William G. DunphyAbstract:Cdc2, the cyclin-dependent kinase that controls mitosis, is negatively regulated by phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25, the phosphatase that dephosphorylates both of these residues, undergoes activation and phosphorylation by multiple kinases at mitosis. Plx1, a kinase that associates with and phosphorylates the amino-terminal domain of Cdc25, was purified extensively from Xenopus egg extracts. Cloning of its complementary DNA revealed that Plx1 is related to the Polo family of protein kinases. Recombinant Plx1 phosphorylated Cdc25 and stimulated its activity in a purified system. Cdc25 phosphorylated by Plx1 reacted strongly with MPM-2, a monoclonal antibody to mitotic phosphoproteins. These studies indicate that Plx1 may participate in control of mitotic progression.
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regulation of the Cdc25 protein during the cell cycle in xenopus extracts
Cell, 1992Co-Authors: Akiko Kumagai, William G. DunphyAbstract:Abstract The Cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the Cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the Cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of Cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of Cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a Cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the Cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.
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the Cdc25 protein contains an intrinsic phosphatase activity
Cell, 1991Co-Authors: William G. Dunphy, Akiko KumagaiAbstract:Abstract Genetic and biochemical studies have indicated that the Cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase. We show that the isolated Cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides. The Cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators. Moreover, the phosphatase activity of the Cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis. These observations indicate that the Cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.