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)

Cell cycle arrest in G2 phase is a common response to a variety of DNA-damaging agents. The coupling between DNA damage and G2 arrest was studied in synchronized HeLa cells using camptothecin, a highly specific inhibitor of topoisomerase I that damages DNA through the formation of reversible topoisomerase I-DNA cleavable complexes. Brief camptothecin treatment of early S-phase HeLa cells caused arrest at G2 phase and abolished the activation of p34cdc2 protein kinase. Both tyrosine dephosphorylation of p34cdc2 and cyclin B accumulation were altered. These cell cycle-dependent changes were not observed when DNA replication was inhibited by aphidicolin during the brief camptothecin treatment. Our results suggest that to produce G2 arrest, active DNA synthesis is required at the time of camptothecin treatment, as was previously shown for camptothecin-induced cytotoxicity. Furthermore, our results suggest that the interaction of the replication fork with DNA damage may ultimately trigger altered regulation of p34cdc2/cyclin B, leading to cell cycle arrest at the G2 phase.
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PMID:The involvement of active DNA synthesis in camptothecin-induced G2 arrest: altered regulation of p34cdc2/cyclin B. 131

A complementary DNA coding for a second type of activin receptor (ActRIIB) has been cloned from Xenopus laevis that fulfills the structural criteria of a transmembrane protein serine kinase. Ectodermal explants from embryos injected with activin receptor RNA show increased sensitivity to activin, as measured by the induction of muscle actin RNA. In addition, injected embryos display developmental defects characterized by inappropriate formation of dorsal mesodermal tissue. These results demonstrate that this receptor is involved in signal transduction and are consistent with the proposed role of activin in the induction and patterning of mesoderm in Xenopus embryos.
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PMID:Cloning of a second type of activin receptor and functional characterization in Xenopus embryos. 131 88

The effects of serine phosphorylation on the DNA cleavage/religation equilibrium of topoisomerase II and the sensitivity of the enzyme to antineoplastic drugs were characterized. Both casein kinase II and protein kinase C were used for these studies. Each kinase incorporated a maximum of approximately 1.4 phosphate molecules per homodimer of topoisomerase II. When the enzyme was incubated with both kinases simultaneously, phosphate incorporation increased to approximately 2.6 molecules/homodimer. In the absence of antineoplastic drugs, phosphorylation had only a slight effect on the DNA cleavage/religation equilibrium of topoisomerase II. However, in the presence of etoposide or 4'-(9-acridinylamino)methane-sulfon-m-anisidide, phosphorylation attenuated the ability of drugs to stabilize enzyme-DNA cleavage complexes. Levels of drug-induced DNA cleavage products decreased approximately 33% following phosphorylation of topoisomerase II by casein kinase II, approximately 17% following modification by protein kinase C, and approximately 50% following simultaneous phosphorylation of the enzyme by both kinases. This latter 50% reduction in DNA cleavage products correlated with an approximately 2-fold increase in the apparent first order rate constant for DNA religation mediated by simultaneously modified topoisomerase II. These results strongly suggest that the sensitivity of topoisomerase II toward antineoplastic drugs can be modulated by altering the phosphorylation state of the enzyme.
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PMID:Phosphorylation of topoisomerase II by casein kinase II and protein kinase C: effects on enzyme-mediated DNA cleavage/religation and sensitivity to the antineoplastic drugs etoposide and 4'-(9-acridinylamino)methane-sulfon-m-anisidide. 131 38

The early events of signal transduction associated with interleukin-2 (IL-2) binding to its receptor were examined using a human IL-2 dependent T-cell line, Kit225. Cell cycle analysis showed that 90% of Kit225 cells were in the G0/G1 phase after a 72-hr incubation in the absence of exogenous IL-2. At this point, stimulation of the cells with IL-2 resulted in the rapid initiation of RNA and DNA synthesis by 9 and 20 hr, respectively. Within 5 min after addition of IL-2, rapid activation of tyrosine and ribosomal S6 kinases was detected. Addition of IL-2 also increased mRNA levels for c-fos, c-myc, IL-2 receptor alpha, and IL-2 receptor beta chain. These events increased in the absence of detectable changes in free cytosolic [Ca2+]i, inositol phosphate metabolism, or the activity of several kinases including cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase, or protein kinase C. These findings demonstrate that the signals triggered by IL-2 binding to its receptors are quickly transduced into the nucleus with increased mRNA transcription of activation-associated genes. Furthermore, the data indicate that tyrosine and ribosomal S6 kinases may be important for IL-2-induced cell growth.
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PMID:Signal transduction by interleukin 2 in human T cells: activation of tyrosine and ribosomal S6 kinases and cell-cycle regulatory genes. 131 23

Cyclins are proteins which are synthesized and degraded in a cell cycle-dependent fashion and form integral regulatory subunits of protein kinase complexes involved in the regulation of the cell cycle. The best known catalytic subunit of a cyclin-dependent protein kinase complex is p34cdc2. In the cell, cyclins A and B are synthesized at different stages of the cell cycle and induce protein kinase activation with different kinetics. The kinetics of activation can be reproduced and studied in extracts of Xenopus eggs to which bacterially produced cyclins are added. In this paper we report that in egg extracts, both cyclin A and cyclin B associate with and activate the same catalytic subunit, p34cdc2. In addition, cyclin A binds a less abundant p33 protein kinase related to p34cdc2, the product of the cdk2/Eg1 gene. When complexed to cyclin B, p34cdc2 is subject to transient inhibition by tyrosine phosphorylation, producing a lag between the addition of cyclin and kinase activation. In contrast, p34cdc2 is only weakly tyrosine phosphorylated when bound to cyclin A and activates rapidly. This finding shows that a given kinase catalytic subunit can be regulated in a different manner depending on the nature of the regulatory subunit to which it binds. Tyrosine phosphorylation of p34cdc2 when complexed to cyclin B provides an inhibitory check on the activation of the M phase inducing protein kinase, allowing the coupling of processes such as DNA replication to the onset of metaphase. Our results suggest that, at least in the early Xenopus embryo, cyclin A-dependent protein kinases may not be subject to this checkpoint and are regulated primarily at the level of cyclin translation.
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PMID:Cyclin A- and cyclin B-dependent protein kinases are regulated by different mechanisms in Xenopus egg extracts. 131 71

A rat genomic clone containing 4.5 kilobases of 5'-flanking DNA and the first exon of the type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase was isolated, restriction mapped, and sequenced. The proximal 400-basepair promoter region was GC rich, lacked TATA/CAAT box motifs, and initiated transcription at multiple sites. Bandshifting and DNase-I footprinting experiments using this region of the RII beta promoter detected several related specific DNA-protein complexes formed using crude and fractionated nuclear extracts from rat ovary, brain, adrenal gland, and liver. All binding in these experiments mapped to a domain within the same region found to confer cAMP inducibility to a chloramphenicol acetyltransferase (CAT) reporter gene when transfected into primary cultures of rat granulosa cells. Although GC boxes (putative SP1-binding sites) and activator protein-2 (AP-2) elements were present in this functional region, and although expression vectors containing AP-2 sites conferred high levels of cAMP regulation of the CAT gene in cultured ovarian cells, neither the GC boxes nor the AP-2 sites were protected by footprint analyses or required for band shift activity of nuclear extract protein. These known regulatory elements, therefore, may be involved in functional activity of the RII beta promoter, but additional cis-acting DNA and trans-acting factors (yet to be characterized) also appear to interact with the functional promoter of the RII beta gene and regulate the hormone-specific expression of the A-kinase subunit in ovarian and neuronal cells.
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PMID:Identification and characterization of the GC-rich and cyclic adenosine 3',5'-monophosphate (cAMP)-inducible promoter of the type II beta cAMP-dependent protein kinase regulatory subunit gene. 131 46

Human progesterone receptors (PR) in T47D breast cancer cells are synthesized as two different sized proteins, PR-A [94 kilodaltons (kDa)] and PR-B (120 kDa). Progestin addition to cells (in vivo) causes a 2-fold increase in total phosphorylation of PR and an increase in the apparent mol wt of both PR-A and PR-B on sodium dodecyl sulfate (SDS)-gels. Time-course experiments showed that increased PR phosphorylation that results from hormone addition is a multistep process and involves a rapid increase into total 32P labeling that takes place before the more slowly occurring phosphorylation(s) responsible for the change in electrophoretic mobility of PR on SDS-gels. As an approach to test whether phosphorylation is involved in regulating PR activity, we have examined the effects of cellular modulators of protein phosphorylation on PR-mediated target gene transcription in vivo using a T47D cloned cell line containing a stably transfected mouse mammary tumor virus-chloramphenicol acetyltransferase construct. Treatment with 8-bromo-cAMP (activator of cAMP-dependent protein kinases) or okadaic acid (protein phosphatase-1 and -2A inhibitor) did not stimulate target gene expression in the absence of progestin. When added together with progestin, either compound augmented PR-mediated target gene transcription by 3- to 4-fold. The cyclic nucleotide-dependent protein kinase inhibitor H8 completely blocked target gene responsiveness to hormone. Neither 8-bromo-cAMP, okadaic acid, nor H8 altered the hormone- or DNA-binding activities of PR, as measured in vitro or affected cellular concentrations of PR. These agents, therefore, appeared to selectively modulate PR transcriptional activity. Moreover, none of these compounds altered expression from a control reporter gene, pSV2CAT, indicating that these agents affect PR-mediated processes directly and are not acting through a general effect on transcription. Effects on PR phosphorylation were assessed by measuring 32P labeling of PR in vivo. None of these treatments had a substantial effect on the extent of total 32P labeling of immune isolated PR or on the phosphorylation(s) responsible for PR up-shifts on SDS-gels. This suggests that these agents modulate PR transcriptional activity either through phosphorylation of another protein intimately involved in PR-mediated transcription or through modification of a key site(s) not measurable as a change in total PR phosphorylation or electrophoretic mobility on SDS gels.
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PMID:Effects of hormone and cellular modulators of protein phosphorylation on transcriptional activity, DNA binding, and phosphorylation of human progesterone receptors. 131 49

Mammalian RNA polymerase II contains at the C terminus of its largest subunit an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The phosphorylation of this domain is thought to play an important role in the transition of RNA polymerase II from a preinitiation complex to an elongating complex. The unphosphorylated form of RNA polymerase II is designated IIA, whereas the phosphorylated form is designated IIO. In an effort to determine the consequence of C-terminal domain phosphorylation on complex formation, 32P-labeled RNA polymerases IIA and IIO were prepared and examined for their ability to form a stable preinitiation complex on the adenovirus-2 major late promoter in the presence of a reconstituted HeLa cell transcription extract. Preinitiation complexes were formed in the absence of ATP and purified from free RNA polymerase II by chromatography on Sepharose CL-4B. The state of phosphorylation of the largest subunit was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the transcriptional activity was determined by assaying specific transcript formation upon the addition of nucleotides and a competing DNA template. RNA polymerase IIA was recovered in transcriptionally active complexes in reactions in which the input enzyme was RNA polymerase IIA. In reactions with RNA polymerase IIO as the input enzyme, no IIO was recovered in excluded fractions that normally contain preinitiation complex. In reactions with equimolar amounts of RNA polymerases IIO and IIA, purified preinitiation complexes contained almost exclusively RNA polymerase HA. These results support the idea that RNA polymerase II containing an unphosphorylated C-terminal domain preferentially associates with the adenovirus-2 major late promoter. The state of phosphorylation of the C-terminal domain can, therefore, directly influence preinitiation complex formation. We also report here the presence of an activity in HeLa cell extracts that catalyzes dephosphorylation of the C-terminal domain, thereby converting RNA polymerase IIO to IIA. This C-terminal domain phosphatase is specific in that it does not catalyze the dephosphorylation of a serine residue phosphorylated by casein kinase II. The presence of a C-terminal domain phosphatase in in vitro transcription reactions containing RNA polymerase IIO results in the formation of RNA polymerase IIA. This RNA polymerase IIA associates preferentially with preinitiation complexes.
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PMID:The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa. 131 3

Two regulatory mutants of Pseudomonas aeruginosa, R1 and RA, that affect transcription of the pilin gene were isolated. This was done by introducing a plasmid carrying a fusion of the pilin gene's promoter with the lacZ gene into a bank of P. aeruginosa DNA mutagenized with the transposon Tn5G. The block in pilin expression in these mutants was shown to be at the level of transcription, since these mutants did not synthesize either pilin mRNA or pilin antigen. A restriction fragment derived from the R1 mutant that contains the entire transposon plus flanking chromosomal DNA was cloned and used as a probe to screen a cosmid library of P. aeruginosa DNA. Cosmids that could complement the pilin expression defect in both R1 and RA were isolated. The gene inactivated in R1 was sequenced. This gene, designated pilR, encodes an approximately 50-kDa polypeptide which exhibits significant similarity to the NtrC family of response regulators of the two-component regulatory system. PilR contains the amino-terminal aspartic acid residues which are conserved among the response regulators, suggesting that pilin gene transcription is regulated via a phosphotransfer mechanism in which PilR is phosphorylated by an as yet unidentified protein kinase.
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PMID:Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene. 131 79

The transition from G1 to S phase of the cell cycle may be regulated by modification of proteins which are essential for initiating DNA replication. One of the first events during initiation is to unwind the origin DNA and this requires a single-stranded DNA binding protein. RPA, a highly conserved multi-subunit single-stranded DNA binding protein, was first identified as a cellular protein necessary for the initiation of SV40 DNA replication. The 32 kDa subunit of RPA has been shown to be phosphorylated at the start of S phase. Using SV40 replication as a model, we have reproduced in vitro the S phase-dependent phosphorylation of RPA-32 and show that it occurs specifically within the replication initiation complex. Phosphorylated RPA-32 is predominantly associated with DNA. Phosphorylation is not a pre-requisite for association with DNA, but occurs after RPA binds to single-stranded DNA formed at the origin during the initiation phase. The protein kinase(s) which phosphorylates RPA-32 is present at all stages of the cell cycle but RPA-32 does not bind to the SV40 origin or become phosphorylated in extracts from G1 cells. Therefore, the cell cycle-dependent phosphorylation of RPA-32 may be regulated by its binding to single-stranded origin DNA during replication initiation.
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PMID:Cell cycle regulated phosphorylation of RPA-32 occurs within the replication initiation complex. 131 94


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