Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Entry into mitosis in Schizosaccharomyces pombe is negatively regulated by the wee1+ gene, which encodes a protein kinase with serine-, theonine-, and tyrosine-phosphorylating activities. The wee1+ kinase negatively regulates mitosis by phosphorylating p34cdc2 on tyrosine 15, thereby inactivating the p34cdc2-cyclin B complex. The human homolog of the wee1+ gene (WEE1Hu) was overproduced in bacteria and assayed in an in vitro system. Unlike its fission yeast homolog, the product of the WEE1Hu gene encoded a tyrosine-specific protein kinase. The human WEE1 kinase phosphorylated the p34cdc2-cyclin B complex on tyrosine 15 but not on threonine 14 in vitro and inactivated the p34cdc2-cyclin B kinase. This inhibition was reversed by the human Cdc25C protein, which catalyzed the dephosphorylation of p34cdc2. These results indicate that the product of the WEE1Hu gene directly regulates the p34cdc2-cyclin B complex in human cells and that a kinase other than that encoded by WEE1Hu phosphorylates p34cdc2 on threonine 14.
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PMID:Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. 138 26

Human WEE1 (WEE1Hu) was cloned on the basis of its ability to rescue wee1+ mutants in fission yeast [Igarashi, M., Nagata, A., Jinno, S., Suto, K. & Okayama, H. (1991) Nature (London) 353, 80-83]. Biochemical studies carried out in vitro with recombinant protein demonstrated that WEE1Hu encodes a tyrosine kinase of approximately 49 kDa that phosphorylates p34cdc2 on Tyr-15 [Parker, L. L. & Piwnica-Worms, H. (1992) Science 257, 1955-1957]. To study the regulation of WEE1Hu in human cells, two polyclonal antibodies to bacterially produced p49WEE1Hu were generated. In addition, a peptide antibody generated against amino acids 361-388 of p49WEE1Hu was also used. Unexpectantly, these antibodies recognized a protein with an apparent molecular mass of 95 kDa in HeLa cells, rather than one of 49 kDa. Immunoprecipitates of p95 phosphorylated p34cdc2 on Tyr-15, indicating that p95 is functionally related to p49WEEIHu, and mapping studies demonstrated that p95 is structurally related to p49WEE1Hu. In addition, the substrate specificity of p95 was more similar to that of fission yeast p107wee1 than to that of human p49WEE1. Finally, the kinase activity of p95 toward p34cdc2/cyclin B was severely impaired during mitosis. Taken together, these results indicate that the original WEE1Hu clone isolated in genetic screens encodes only the catalytic domain of human WEE1 and that the authentic human WEE1 protein has an apparent molecular mass of approximately 95 kDa.
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PMID:Identification of a 95-kDa WEE1-like tyrosine kinase in HeLa cells. 756 88

Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.
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PMID:Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints. 1291 80

The dual-specificity CDC25 phosphatases are critical positive regulators of cyclin-dependent kinases (CDKs). Even though an antagonistic Arabidopsis thaliana WEE1 kinase has been cloned and tyrosine phosphorylation of its CDKs has been demonstrated, no valid candidate for a CDC25 protein has been reported in higher plants. We identify a CDC25-related protein (Arath;CDC25) of A. thaliana, constituted by a sole catalytic domain. The protein has a tyrosine-phosphatase activity and stimulates the kinase activity of Arabidopsis CDKs. Its tertiary structure was obtained by NMR spectroscopy and confirms that Arath;CDC25 belongs structurally to the classical CDC25 superfamily with a central five-stranded beta-sheet surrounded by helices. A particular feature of the protein, however, is the presence of an additional zinc-binding loop in the C-terminal part. NMR mapping studies revealed the interaction with phosphorylated peptidic models derived from the conserved CDK loop containing the phosphothreonine-14 and phosphotyrosine-15. We conclude that despite sequence divergence, Arath;CDC25 is structurally and functionally an isoform of the CDC25 superfamily, which is conserved in yeast and in plants, including Arabidopsis and rice.
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PMID:A small CDC25 dual-specificity tyrosine-phosphatase isoform in Arabidopsis thaliana. 1532 14

To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy. In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells, which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.
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PMID:Construction and co-expression of bicistronic plasmid encoding human WEE1 and stem cell factor. 1568 67

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.
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PMID:Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis. 1685 85

Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.
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PMID:The cell cycle-associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit. 1758 6

Myelosuppression is one of the major side-effects of most anticancer drugs. To achieve myeloprotection, one bicistronic vector encoding anti-apoptotic protein human WEE1 (WEE1Hu) and proliferation-stimulating stem cell factor (SCF) was generated. In this study, we selected human umbilical cord blood CD34+ cells as the in vitro model in an attempt to investigate whether WEE1Hu, rather than conventional drug-resistant genes, can be introduced to rescue cells from the damage by chemotherapeutic agents such as cisplatin, adriamycin, mitomycin-c and 5-fluorouracil. Cell viability and cytotoxicity assay, colony-forming units in culture assay and externalization of phospholipid phosphatidylserine analysis showed that the expression of WEE1Hu and SCF in CD34+ cells provided the cells with some protection. These findings suggest that the expression of WEE1Hu and SCF might rescue CD34+ cells from chemotherapy-induced myelosuppression.
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PMID:Effect of human WEE1 and stem cell factor on human CD34+ umbilical cord blood cell damage induced by chemotherapeutic agents. 1768 95

The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G(2)/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1 alpha. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials.
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PMID:The cyclin-dependent kinase inhibitor seliciclib (R-roscovitine; CYC202) decreases the expression of mitotic control genes and prevents entry into mitosis. 1807 15

Large-scale comparison of sequence polymorphism and divergence at numerous genomic loci within and between closely related species can reveal signatures of natural selection. Here, we present a population genomics study based on direct sequencing of 61 mitotic cell cycle genes from 30 Arabidopsis thaliana accessions and comparison of the resulting data to the close relative Arabidopsis lyrata. We found that the Arabidopsis core cell cycle (CCC) machinery is not highly constrained but is subject to different modes of selection. We found patterns of purifying selection for the cyclin-dependent kinase (CDK), CDK subunit, retinoblastoma, and WEE1 gene families. Other CCC gene families often showed a mix of one or two constrained genes and relaxed purifying selection on the other genes. We found several large effect mutations in CDKB1;2 that segregate in the species. We found a strong signature of adaptive protein evolution in the Kip-related protein KRP6 and departures from equilibrium at CDKD;1 and CYCA3;3 consistent with the operation of selection in these gene regions. Our data suggest that within Arabidopsis, the genetic robustness of cell cycle-related processes is more due to functional redundancy than high selective constraint.
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PMID:A population genomics study of the Arabidopsis core cell cycle genes shows the signature of natural selection. 1988 Jul 99


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