The Experts below are selected from a list of 13949574 Experts worldwide ranked by ideXlab platform
John S. Lazo - One of the best experts on this subject based on the ideXlab platform.
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hypoxia mediated regulation of cdc25a phosphatase by p21 and mir 21
Cell Cycle, 2009Co-Authors: Pierre Queiroz E De Oliveira, Lin Zhang, Zhenghe Wang, John S. LazoAbstract:Hypoxia is a common feature of solid tumors and represents a critical factor in their progression and responsiveness to chemotherapy and radiotherapy. We now report that hypoxic exposure of colon cancer cells decreased the protein levels of the cell cycle-controlling phosphatase Cdc25A. Hypoxia decreased the mitotic population and caused S-phase arrest in these cells. Suppression of Cdc25A was phosphatase family member-specific, as a similar decrease was not observed with closely related CDC25B or Cdc25C phosphatases. Pharmacological and genetic blockade of Chk1 and Chk2 failed to inhibit the hypoxia-mediated loss of Cdc25A, indicating this process was not regulated by a traditional ATM/ATR checkpoint response. In addition, hypoxia did not affect ectopically expressed Cdc25A levels suggesting independence from an increase in proteasomal degradation. Cdc25A mRNA levels also decreased in human colon cancer cells 24 hr after hypoxia supporting a mechanistic role for decreased Cdc25A expression or mRNA stability. The reduction in Cdc25A mRNA and protein was dependent on the cyclin-dependent kinase inhibitor p21 and miR-21, which were upregulated in HCT116 colon cancer cells during hypoxia. These results reveal previously unknown mechanisms for the transient suppression of Cdc25A, providing a coordinated and fundamental adaptive change that may be exploited by cancer cells conferring proliferative and survival advantages.
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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, Marni Brisson Tierno, Tong Ying Shun, Sunita N Shinde, William D Paquette, Kay M Brummond, Caleb Foster, Peter Wipf, 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, Robert J Tomko, Caleb Foster, Theresa Nguyen, Peter Wipf, 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, Jack C Yalowich, Angela Giorgianni, Dror Tobi, Theresa Nguyen, Andreas Vogt, Peter Wipf, 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, Colleen N Scott, Meifang Wang, John S. Lazo, 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...
Peter Wipf - 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, Marni Brisson Tierno, Tong Ying Shun, Sunita N Shinde, William D Paquette, Kay M Brummond, Caleb Foster, Peter Wipf, 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, Robert J Tomko, Caleb Foster, Theresa Nguyen, Peter Wipf, 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, Jack C Yalowich, Angela Giorgianni, Dror Tobi, Theresa Nguyen, Andreas Vogt, Peter Wipf, 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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identification of a potent and selective pharmacophore for cdc25 dual specificity phosphatase inhibitors
Molecular Pharmacology, 2002Co-Authors: John S. Lazo, Katharine E Pestell, Kathleen A Cooley, Eileen C Southwick, Daniel W Zaharevitz, William Furey, Douglas A Mitchell, Kaoru Nemoto, Rick Gussio, Peter WipfAbstract:Small molecules provide powerful tools to interrogate biological pathways but many important pathway participants remain refractory to inhibitors. For example, Cdc25 dual-specificity phosphatases regulate mammalian cell cycle progression and are implicated in oncogenesis, but potent and selective inhibitors are lacking for this enzyme class. Thus, we evaluated 10,070 compounds in a publicly available chemical repository of the National Cancer Institute for in vitro inhibitory activity against oncogenic, full-length, recombinant human CDC25B. Twenty-one compounds had mean inhibitory concentrations of 75% were quinones and >40% were of the para -naphthoquinone structural type. Most notable was NSC 95397 (2,3-bis-[2-hydroxyethylsulfanyl]-[1,4]naphthoquinone), which displayed mixed inhibition kinetics with in vitro K i values for Cdc25A, -B, and -C of 32, 96, and 40 nM, respectively. NSC 95397 was more potent than any inhibitor of dual specificity phosphatases described previously and 125- to 180-fold more selective for Cdc25A than VH1-related dual-specificity phosphatase or protein tyrosine phosphatase 1b, respectively. Modification of the bis -thioethanol moiety markedly decreased enzyme inhibitory activity, indicating its importance for bioactivity. NSC 95397 showed significant growth inhibition against human and murine carcinoma cells and blocked G 2 /M phase transition. A potential Cdc25 site of interaction was postulated based on molecular modeling with these quinones. We propose that inhibitors based on this chemical structure could serve as useful tools to probe the biological function of Cdc25.
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discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase cdc25
Journal of Medicinal Chemistry, 2001Co-Authors: John S. Lazo, Kathleen A Cooley, Eileen C Southwick, Diana C Aslan, Alexander P Ducruet, And Andreas Vogt, Peter WipfAbstract:The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations <1 μM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against CDC25B2 as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, CDC25B2, and Cdc25C with Ki values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against CDC25B2 in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential ma...
Bernard Ducommun - One of the best experts on this subject based on the ideXlab platform.
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development of novel thiazolopyrimidines as CDC25B phosphatase inhibitors
ChemMedChem, 2009Co-Authors: Odile Mondesert, Marylorene Goddard, Stephanie Kolb, Bruno O Villoutreix, Denis Jullien, Bernard DucommunAbstract:The development of CDC25 phosphatase inhibitors is an interesting approach toward new antitumor agents, as CDC25 play key roles in cell-cycle regulation and are overexpressed in numerous cancers. We previously reported a novel compound belonging to the thiazolopyrimidine family that inhibits CDC25 activity with an IC(50) value of 13 microM and displays cytotoxic properties against HeLa cells. Structural modifications were subsequently conducted on this new pharmacophore which led to a library of 45 thiazolopyrimidines. Regarding the in vitro effects, 14 compounds inhibit CDC25B with IC(50)<20 microM, with the most efficient inhibitor 44 improving the potency to 4.5 microM. Steady-state kinetics were performed and showed a mixed inhibition pattern for all tested compounds. Furthermore, 44 was able to revert the bypass of genotoxicity-induced G(2) arrest upon CDC25B overexpression, indicating that this compound targets the dual-specificity phosphatase in cultured cells. Finally, the cytotoxic activities of the compounds were determined against two human cancer cell lines. The results indicate that the prostatic LNCaP cell line is more sensitive to these derivatives than the pancreatic adenocarcinoma MiaPaCa-2 line. With its interesting enzymatic and cellular properties, compound 44 appears to be a promising CDC25B inhibitor for further development.
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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, Mariepriscille Brun, Odile Mondesert, Marylorene Goddard, Maria A Miteva, Stephanie Kolb, Emmanuelle Braud, Christiane Garbay, Bernard Ducommun, 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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CDC25B involvement in the centrosome duplication cycle and in microtubule nucleation.
Cancer Research, 2007Co-Authors: Rose Boutros, Valérie Lobjois, Bernard DucommunAbstract:Centrosome amplification is frequently reported in human cancers, although the molecular mechanisms that are responsible for this remain unclear. There is significant evidence to support a role for cyclin-dependent kinase (CDK)-cyclin complexes in centrosome duplication. The activities of CDK-cyclin complexes are, in turn, regulated by the CDC25 family of phosphatases in a strict spatiotemporal manner, and we have recently reported that CDC25B localizes to the centrosomes from early S phase. In the present study, we have investigated the role of centrosomally localized CDC25B in centrosome duplication. We first observed that overexpression of CDC25B under an inducible promoter in S phase results in centrosome overduplication. We found that forced expression of wild-type but not phosphatase-inactive CDC25B at the centrosomes results in centrosome amplification, aberrant microtubule organization, and abnormal accumulation of gamma-tubulin. In contrast, inhibition of CDC25B phosphatase activity inhibits the assembly of interphase microtubules and the centrosomal localization of gamma-tubulin. We propose that CDC25B is part of the pathway that controls the localization of gamma-tubulin to the centrosomes, thereby regulating centrosome duplication during S phase and the nucleation of microtubules. We speculate that abnormal expression of CDC25B in numerous human tumors might therefore have a critical role in centrosome amplification and genomic instability.
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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, Philip G. Kasprzyk, P. Auvray, 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.
Brian Gabrielli - One of the best experts on this subject based on the ideXlab platform.
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cdc25 dependent activation of cyclin a cdk2 is blocked in g2 phase arrested cells independently of atm atr
Oncogene, 2001Co-Authors: Sherilyn Goldstone, J Sinnamon, Sandra Pavey, Alistair R. R. Forrest, Brian GabrielliAbstract:Cyclin A/cdk2 is active during S and G2 phases of the cell cycle, but its regulation and function during G2 phase is poorly understood. In this study we have examined the regulation of cyclin A/cdk2 activity during normal G2 phase progression and in genotoxin-induced G2 arrest. We show that cyclin A/cdk2 is activated in early G2 phase by a cdc25 activity. In the G2 phase checkpoint arrest initiated in response to various forms of DNA damage, the cdc25-dependent activation of both cyclin A/cdk2 and cyclin B1/cdc2 is blocked. Ectopic expression of CDC25B, but not cdc25C, in G2 phase arrested cells efficiently activated both cyclin A/cdk2 and cyclin B1/cdc2. Finally, we demonstrate that the block in cyclin A/cdk2 activation in the G2 checkpoint arrest is independent of ATM/ATR. We speculate that the ATM/ ATR-independent block in G2 phase cyclin A/cdk2 activation may act as a further layer of checkpoint control, and that blocking G2 phase cyclin A/cdk2 activation contributes to the G2 phase checkpoint arrest.
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cdc25 dependent activation of cyclin a cdk2 is blocked in g2 phase arrested cells independently of atm atr
Oncogene, 2001Co-Authors: Sherilyn Goldstone, J Sinnamon, Sandra Pavey, Alistair R. R. Forrest, Brian GabrielliAbstract:Cyclin A/cdk2 is active during S and G2 phases of the cell cycle, but its regulation and function during G2 phase is poorly understood. In this study we have examined the regulation of cyclin A/cdk2 activity during normal G2 phase progression and in genotoxin-induced G2 arrest. We show that cyclin A/cdk2 is activated in early G2 phase by a cdc25 activity. In the G2 phase checkpoint arrest initiated in response to various forms of DNA damage, the cdc25-dependent activation of both cyclin A/cdk2 and cyclin B1/cdc2 is blocked. Ectopic expression of CDC25B, but not cdc25C, in G2 phase arrested cells efficiently activated both cyclin A/cdk2 and cyclin B1/cdc2. Finally, we demonstrate that the block in cyclin A/cdk2 activation in the G2 checkpoint arrest is independent of ATM/ATR. We speculate that the ATM/ ATR-independent block in G2 phase cyclin A/cdk2 activation may act as a further layer of checkpoint control, and that blocking G2 phase cyclin A/cdk2 activation contributes to the G2 phase checkpoint arrest.
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hyperphosphorylation of the n terminal domain of cdc25 regulates activity toward cyclin b1 cdc2 but not cyclin a cdk2
Journal of Biological Chemistry, 1997Co-Authors: Brian Gabrielli, Joanne M Clark, Andrew K Mccormack, Kay A. O. EllemAbstract:Abstract Cdc25 regulates entry into mitosis by regulating the activation of cyclin B/cdc2. In humans, at least two cdc25 isoforms have roles in controlling the G2/M transition. Here we show, using bacterially expressed recombinant proteins, that two CDC25B splice variants, CDC25B2 and CDC25B3, are capable of activating cyclin A/cdk2 and cyclin B/cdc2, but that mitotic hyperphosphorylation of these proteins increases their activity toward only cyclin B1/cdc2. Cdc25C has only very low activity in its unphosphorylated form, and following hyperphosphorylation it will efficiently catalyze the activation of only cyclin B/cdc2. This was reflected by the in vivo activity of the immunoprecipitated CDC25B and cdc25C from interphase and mitotic HeLa cells. The increased activity of the hyperphosphorylated cdc25s toward cyclin B1/cdc2 was in large part due to increased binding of this substrate. The substrate specificity, activities, and timing of the hyperphosphorylation of CDC25B and cdc25C during G2 and M suggest that these two mitotic cdc25 isoforms are activated by different kinases and perform different functions during progression through G2 into mitosis.
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cytoplasmic accumulation of CDC25B phosphatase in mitosis triggers centrosomal microtubule nucleation in hela cells
Journal of Cell Science, 1996Co-Authors: Brian Gabrielli, C P C De Souza, Ian D Tonks, J M Clark, Nicholas K Hayward, Kay A. O. EllemAbstract:The formation of the mitotic spindle is an essential prerequisite for successful mitosis. The dramatic changes in the level of microtubule (Mt) nucleation at the centrosomes and Mt dynamics that occur in prophase are presumed to be initiated through the activity of cdc2/cyclin B. Here we present data that the CDC25B isoform functions to activate the cytoplasmic pool of cdc2/cyclin B responsible for these events. In contrast to cdc25C, CDC25B is present at low levels in HeLa cells during interphase, but sharply increases in prophase, when CDC25B accumulation in the cytoplasm correlates with prophase spindle formation. Overexpression of wild type and dominant negative mutants of CDC25B and cdc25C shows that prophase Mt nucleation is a consequence of cytoplasmic CDC25B activity, and that cdc25C regulates nuclear G2/M events. Our data also suggest that the functional status of the centrosome can regulate nuclear mitotic events.
Alexander P Ducruet - One of the best experts on this subject based on the ideXlab platform.
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discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase cdc25
Journal of Medicinal Chemistry, 2001Co-Authors: John S. Lazo, Kathleen A Cooley, Eileen C Southwick, Diana C Aslan, Alexander P Ducruet, And Andreas Vogt, Peter WipfAbstract:The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations <1 μM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against CDC25B2 as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, CDC25B2, and Cdc25C with Ki values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against CDC25B2 in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential ma...
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discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase cdc25
Journal of Medicinal Chemistry, 2001Co-Authors: John S. Lazo, Kathleen A Cooley, Eileen C Southwick, Diana C Aslan, Alexander P Ducruet, And Andreas Vogt, Beomjun Joo, Peter WipfAbstract:The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations <1 microM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against CDC25B(2) as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, CDC25B(2), and Cdc25C with K(i) values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against CDC25B(2) in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential mapping suggested the need for an electron-deficient 7-position for maximal inhibitor activity. Using a chemical complementation assay, we found that NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression.
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spatial analysis of key signaling proteins by high content solid phase cytometry in hep3b cells treated with an inhibitor of cdc25 dual specificity phosphatases
Journal of Biological Chemistry, 2001Co-Authors: Andreas Vogt, Alexander P Ducruet, Takahito Adachi, Jon Chesebrough, Brian I. Carr, Kaoru Nemoto, John S. LazoAbstract:Abstract Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for Cdc25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor κB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, andin vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of Cdc25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that Cdc25A regulates Erk phosphorylation status.
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spatial analysis of key signaling proteins by high content solid phase cytometry in hep3b cells treated with an inhibitor of cdc25 dual specificity phosphatases
Journal of Biological Chemistry, 2001Co-Authors: Andreas Vogt, Alexander P Ducruet, Takahito Adachi, Jon Chesebrough, Brian I. Carr, Kaoru Nemoto, John S. LazoAbstract:Abstract Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for Cdc25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor κB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, andin vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of Cdc25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that Cdc25A regulates Erk phosphorylation status.