Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

DNA topoisomerase II is an essential nuclear enzyme required for the proper condensation and segregation of chromosomes during mitotic and meiotic cell division. The enzyme exists in the cell as a phosphoprotein, and it is most highly phosphorylated in G2 and M-phases of the cell cycle. We have shown that topoisomerase II is the target of casein kinase II (CKII) in yeast by comparison of in vivo and in vitro phosphotryptic peptide maps. Limited proteolysis and probing with domain specific antibodies show that with the exception of a weakly modified residue between aa 660 and aa 1250, all residues modified by CKII are in the last 200 amino acids of yeast topoisomerase II. This C-terminal domain is the least conserved region of the enzyme and truncation of the enzyme shows that it is nonessential for activity in vitro. However, the fully dephosphorylated full-size protein is nearly inactive in decatenation assays, and activity can be restored by phosphorylation by CKII. To reconcile these observations, we propose that the C-terminal region is a negative regulatory domain, counteracted by phosphorylation within the domain itself. To test this hypothesis we have mutagenised 12 potential CKII phosphoacceptor sites in the C-terminus of topoisomerase II and introduced the mutant genes into a yeast strain which has a temperature sensitive top2 gene. The growth of the transformed strains is monitored at nonpermissive temperature to determine whether C-terminal phosphorylation is important for mitotic growth. In addition, we have purified the mutant enzymes to homogeneity for in vitro assays.
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PMID:The regulation of DNA topoisomerase II by casein kinase II. 773 31

Eukaryotic DNA topoisomerase II is an abundant nuclear enzyme that is essential for cell proliferation. This homodimeric enzyme catalyzes the cleavage and re-ligation of double-stranded DNA required to separate replicated sister chromatids. Both biochemical and genetic studies show that its catalytic activity is required for chromosome condensation and segregation, and that its decatenation activity can be stimulated by a variety of protein kinases in vitro. In budding yeast, topoisomerase II is most highly phosphorylated in metaphase, and casein kinase II (CKII) was shown to be the major kinase modifying topoisomerase II. We have investigated the effects of phosphorylation of yeast topoisomerase II by CKII in vitro, by means of gel-retardation and filter binding assays. The phosphorylation of the C terminus of topoisomerase II by CKII appears to increase the stability of the complex formed with linear DNA fragments, while dephosphorylation has the opposite effect. Rephosphorylation of phosphatase-treated topoisomerase II by chicken casein kinase II restores a stable protein-DNA complex using a linear DNA fragment. The enhanced stability of the topoisomerase II-DNA complex is also observed with relaxed circular DNA, but not with supercoiled minicircles, in agreement with published results using topoisomerase II from Drosophila. Limited proteolysis and probing with domain-specific antibodies shows that, with the exception of a weakly modified residue between amino acid residues 660 and 1250, all residues modified by casein kinase II are in the last 180 amino acid residues of yeast topoisomerase II.
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PMID:Phosphorylation of the C-terminal domain of yeast topoisomerase II by casein kinase II affects DNA-protein interaction. 793 31

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.
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PMID:Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain. 793 13

Immunoprecipitation of DNA topoisomerase II from yeast results in a preparation that contains casein kinase II; this suggests that the two proteins may associate in the intact cell. Purified recombinant topoisomerase II and casein kinase II associate to form a complex in vitro which is stable after topoisomerase II becomes phosphorylated by the kinase. Studies with isolated recombinant casein kinase II subunits disclosed that although the alpha (catalytic) subunit alone can efficiently phosphorylate topoisomerase II, the formation of a stable topoisomerase II-casein kinase II association requires the presence of the beta subunit of the kinase. Both proteins engaged in this complex retain their catalytic activities. Naturally occurring polyamines and polyanionic compounds appear to be crucial factors governing the interaction between the two proteins. Although the biological significance of a stable catalytically active topoisomerase II-casein kinase II molecular complex remains to be defined, these observations suggest the possibility of a novel mechanism regulating topoisomerase II and casein kinase II activities.
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PMID:DNA topoisomerase II and casein kinase II associate in a molecular complex that is catalytically active. 822 2

Drug resistance to anti-tumour agents often coincides with mutations in the gene encoding DNA topoisomerase II alpha. To examine how inactive forms of topoisomerase II can influence resistance to the chemotherapeutic agent VP-16 (etoposide) in the presence of a wild-type allele, we have expressed point mutations and carboxy-terminal truncations of yeast topoisomerase II from a plasmid in budding yeast. Truncations that terminate the coding region of topoisomerase II at amino acid (aa) 750, aa 951 and aa 1044 are localised to both the cytosol and the nucleus and fail to complement a temperature-sensitive top2-1 allele at non-permissive temperature. In contrast, the plasmid-borne wild-type TOP2 allele and a truncation at aa 1236 are nuclear localised and complement the top2-1 mutation. At low levels of expression, truncated forms of topoisomerase II render yeast resistant to levels of etoposide 2- and 3-fold above that tolerated by cells expressing the full-length enzyme. Maximal resistance is conferred by the full-length enzyme carrying a mutated active site (Y783F) or a truncation at aa 1044. The level of phosphorylation of topoisomerase II was previously shown to correlate with drug resistance in cultured cells, hence we tested mutants in the major casein kinase II acceptor sites in the C-terminal domain of yeast topoisomerase II for changes in drug sensitivity. Neither ectopic expression of the C-terminal domain alone nor phosphoacceptor site mutants significantly alter the host cell's sensitivity to etoposide.
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PMID:Ectopic expression of inactive forms of yeast DNA topoisomerase II confers resistance to the anti-tumour drug, etoposide. 863 Feb 79

It has been suggested that casein kinase II phosphorylates DNA topoisomerase II alpha (topo II alpha) in mouse FM3A cells, by comparison of phosphopeptide maps of topo II alpha labeled in intact cells and of topo II alpha phosphorylated by various kinases in vitro. The phosphorylation of purified topo II alpha by casein kinase II, which attached a maximum of two phosphate groups per topo II alpha molecule, had no effect on the activity of topo II alpha. Dephosphorylation of purified topo II alpha by potato acid phosphatase, which almost completely dephosphorylated the topo II alpha, did not reduce the activity of topo II alpha. The incubation itself, regardless of phosphorylation or dephosphorylation status, stimulated the enzyme activity in both reactions. Topo II alpha activity was stimulated by incubation in a medium containing low concentrations of glycerol but not in that containing high concentrations of glycerol, such as the 50% in which purified topo II alpha is stored. The stimulation of topo II alpha activity by incubation was dependent on the concentration of topo II alpha, requiring a relatively high concentration of topo II alpha.
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PMID:Phosphorylation-independent stimulation of DNA topoisomerase II alpha activity. 863 19

The MPM-2 antibody labels mitosis-specific and cell cycle-regulated phosphoproteins. The major phosphoproteins of mitotic chromosomes recognized by the MPM-2 antibody are DNA topoisomerase II (topoII) alpha and beta. In immunofluorescence studies of PtK1 cytoskeletons, prepared by detergent lysis in the presence of potent phosphatase inhibitors, the MPM-2 antibody labels phosphoproteins found at kinetochores, chromosome arms, midbody and spindle poles of mitotic cells. In cells extracted without phosphatase inhibitors, labeling of the MPM-2 antibodies at kinetochores is greatly diminished. However, in cytoskeletons this epitope can be regenerated through the action of kinases stably bound at the kinetochore. Various kinase inhibitors were tested in order to characterize the endogenous kinase responsible for these phosphorylations. We found that the MPM-2 epitope will not rephosphorylate in the presence of the broad specificity kinase inhibitors K-252a, staurosporine and 2-aminopurine. Several other inhibitors had no effect on the rephosphorylation indicating that the endogenous MPM-2 kinase at kinetochores is not p34cdc2, casein kinase II, MAP kinase, protein kinase A or protein kinase C. The addition of N-ethylmaleimide inactivated the endogenous kinetochore kinase; this allowed testing of several purified kinases in the kinetochore rephosphorylation assay. Active p34cdc2-cyclin B, casein kinase II and MAP kinase could not generate the MPM-2 phosphoepitope. However, bacterially expressed NIMA from Aspergillus and ultracentrifuged mitotic HeLa cell extract were able to catalyze the rephosphorylation of the MPM-2 epitope at kinetochores. Furthermore, fractionation of mitotic HeLa cell extract showed that kinases that create the MPM-2 epitope at kinetochores and chromosome arms are distinct. Our results suggest that multiple kinases (either soluble or kinetochore-bound), including a homolog of mammalian NIMA, can create the MPM-2 phosphoepitope. The kinetochore-bound kinase that catalyzes the formation of the MPM-2 phosphoepitope may play an important role in key events such as mitotic kinetochore assembly and sister chromatid separation at anaphase.
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PMID:MPM-2 antibody-reactive phosphorylations can be created in detergent-extracted cells by kinetochore-bound and soluble kinases. 937 53

DNA topoisomerase II (topo II) is an essential nuclear enzyme required for chromatin condensation and chromosome segregation during mitosis. Forced overexpression of topo IIalpha was found to cause morphological changes in recipient cells associated with apoptosis. This induction of apoptosis required nuclear localization of topo IIalpha, yet was independent of the DNA cleavage-religation activity of the enzyme. Apoptosis mediated by topo IIalpha deregulation was blocked by overexpression of crmA, a specific inhibitor of certain caspases, but not by bcl-2. topo IIalpha-induced apoptosis was also blocked by overexpression of a dominant-acting mutant of stress-activated protein kinase kinase (SEK1/MKK4) but not by the overexpression of its normal counterpart. Furthermore, apoptosis was blocked by coexpression of a dominant-negative form of the cyclin-dependent kinase cdk2 but not by dominant-negative cdc2. These results provide a rationale for the tight regulation of topo IIalpha levels through the cell cycle in that deregulation of topo IIalpha expression results in apoptotic cell death.
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PMID:Induction of apoptosis by deregulated expression of DNA topoisomerase IIalpha. 978 93

The 3F3/2 antibody recognizes a phosphoepitope that is implicated in the mitotic checkpoint regulating the metaphase-to-anaphase transition. Immunoprecipitation and Western blotting revealed that the 3F3/2 antibody binds to human DNA topoisomerase II alpha (HsTIIalpha) from mitotic but not interphase HeLa cells. Extracts from mitotic cells efficiently catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIalpha expressed in bacteria. Expression and site-directed mutagenesis of various HsTIIalpha protein fragments mapped the 3F3/2 phosphoepitope to the region of HsTIIalpha containing phosphorylated threonine 1342. This threonine lies within a consensus sequence for phosphorylation by casein kinase II (CKII). CKII is present in cellular extracts and is associated with isolated mitotic chromosomes. The 3F3/2 phosphoepitope kinase present in mitotic cell extracts was able to create the epitope using GTP and was inhibited by heparin. A kinase associated with the isolated chromosomes also generated the 3F3/2 phosphoepitope on HsTIIalpha. Recombinant CKII catalyzed the formation of the 3F3/2 phosphoepitope on fragments of HsTIIalpha containing threonine 1342. These results indicate that the mitotic 3F3/2 phosphoepitope kinase activity is attributable to CKII. We suggest that the 3F3/2 phosphoepitope reflects a CKII-catalyzed phosphorylation of threonine 1342 that may regulate mitotic functions of HsTIIalpha.
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PMID:Casein kinase II catalyzes a mitotic phosphorylation on threonine 1342 of human DNA topoisomerase IIalpha, which is recognized by the 3F3/2 phosphoepitope antibody. 980 34

Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology. The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region. The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.
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PMID:NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ. 981 6


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