Biomedical subjects

K Galaktionov

Publications and source records attributed to K Galaktionov.

9 recordsLinked to original sources

PubMed2000

E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression.

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.

Binding Sites↗

PubMed1996

Cdc25 cell-cycle phosphatase as a target of c-myc.

The product of the proto-oncogene c-myc, in partnership with Max, forms a transcription factor that can promote either oncogenic transformation or apoptosis. The Myc/Max heterodimer binds to elements in the cdc25A gene and activates transcription. Like myc, cdc25A, itself a proto-oncogene, can induce apoptosis in cells depleted of growth factor, and Myc-induced apoptosis also requires cdc25A. These findings indicate that cdc25A is a physiologically relevant transcriptional target of c-myc.

3T3 Cells↗

PubMed1995

p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase.

In normal human fibroblasts, cyclin A-CDK2 exists in a quaternary complex that contains p21 and PCNA. In many transformed cells, p21 disappears, and a substantial fraction of cyclin A-CDK2 complexes with p9CKS1/CKS2, p19, and p45. To investigate the significance of these rearrangements, we have isolated cDNAs encoding p19 and p45. In vitro reconstitution demonstrated that binding of p19 to cyclin A-CDK2 requires p45. Addition of these proteins to the kinase had no substantial effect on the kinase activity in vitro. Interference with p45 function in vivo by microinjection of antibodies or antisense oligonucleotides prevented entry into S phase in both normal and transformed cells. Cyclin A-CDK2 has previously been identified as a kinase whose activity is essential for S phase. Our results identify p45 as an essential element of this activity. The abundance of p45 is greatly increased in many transformed cells. This could result in changes in cell cycle control that contribute to the process of cellular transformation.

Amino Acid Sequence↗

PubMed1995

CDC25 phosphatases as potential human oncogenes.

Cyclin-dependent kinases (CDKs) are activated by CDC25 phosphatases, which remove inhibitory phosphate from tyrosine and threonine residues. In human cells, CDC25 proteins are encoded by a multigene family, consisting of CDC25A, CDC25B, and CDC25C. In rodent cells, human CDC25A or CDC25B but not CDC25C phosphatases cooperate with either Ha-RASG12V or loss of RB1 in oncogenic focus formation. Such transformants were highly aneuploid, grew in soft agar, and formed high-grade tumors in nude mice. Overexpression of CDC25B was detected in 32 percent of human primary breast cancers tested. The CDC25 phosphatases may contribute to the development of human cancer.

Animals↗

PubMed1995

14-3-3 proteins associate with cdc25 phosphatases.

The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening. 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery.

14-3-3 Proteins↗

PubMed1995

Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation.

The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth. Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases. We show here that Cdc25 phosphatase associates with raf1 in somatic mammalian cells and in meiotic frog oocytes. Furthermore, Cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner. We suggest that activation of the cell cycle by the Ras/Raf1 pathways might be mediated in part by Cdc25.

Amino Acid Sequence↗

PubMed1994

Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I.

Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.

Animals↗

PubMed1991

Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins.

Two previously unidentified human cdc25 genes have been isolated, cdc25A and cdc25B. Both genes rescue a cdc25ts mutant of fission yeast. Microinjection of anti-cdc25A antibodies into HeLa cells causes their arrest in mitosis. cdc25A and cdc25B display endogenous tyrosine phosphatase activity that is stimulated several-fold, in the absence of cdc2, by stoichiometric addition of either cyclin B1 or B2 but not A or D1. Association between cdc25A and cyclin B1/cdc2 was detected in the HeLa cells. These findings indicate that B-type cyclins are multifunctional proteins that not only act as M phase regulatory subunits of the cdc2 protein kinase, but also activate the cdc25 tyrosine phosphatase, of which cdc2 is the physiological substrate. A region of amino acid similarity between cyclins and tyrosine PTPases has been detected. This region is absent in cdc25 phosphatases. The motif may represent an activating domain that has to be provided to cdc25 by intermolecular interaction with cyclin B.

Amino Acid Sequence↗

PubMedDate not supplied

Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase.

A universal intracellular factor, the "M phase-promoting factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25. We describe here a colorimetric assay of recombinant human cdc25A tyrosine phosphatase used as a cell cycle-specific target to screen for antimitotic compounds. The glutathione-S-transferase/cdc25A tyrosine phosphatase fusion protein is produced in large amounts of Escherichia coli and easily purified by affinity chromatography on glutathione-agarose. Optimal purification, storage and assay conditions (concentrations of enzyme, p-nitrophenylphosphate and dithiothreitol; duration of assay) have been determined. Using this system we tested 15 compounds currently used in cancer treatment; none of them displayed any inhibitory activity. However, the assay detected the inhibitory activity of vanadate, a reported tyrosine phosphatase inhibitor. The simplicity, speed and possible extensive automation of this assay using an essential cell cycle-regulating component provide a highly specific mechanism-based screen for antimitotic drugs discovery.

Amino Acid Sequence↗