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)

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.
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PMID:Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells. 814 55

A previous study showing that ethanol (ETOH) blocked [3H]choline incorporation into phosphatidylcholine (PC) suggested an inhibition of PC biosynthesis in human leukemic monocyte-like U937 cells. The mechanism of the inhibitory action of ETOH was investigated. Cells were pulsed with [3H]choline for 30 min and chased in the presence or absence of ETOH for up to 6 h. PC biosynthesis was inhibited drastically within 1 h after exposure to ETOH which increased intracellular cAMP appreciably. After a 3-h treatment, ETOH significantly inhibited both choline kinase (CK) and the cytosolic CTP: cholinephosphate cytidylyltransferase (CT). The inactivated CT was no longer stimulated by exogenous phosphatidylglycerol (PG). There was no evidence for redistribution of CT activity between cytosol and microsomes. When cells were exposed to 8-Bromo-cAMP ranging from 100 to 300 microM, PC biosynthesis remained unaffected despite the drastically elevated cAMP. These results seem to suggest that the raised cAMP is not a prerequisite for the inhibition of PC biosynthesis in U937 cells. Following pretreatment with protein kinase inhibitors (H-89 and K-252a), PC biosynthesis was decreased significantly and the inhibitory effect of ETOH was potentiated. Taken together, our results suggest that the inhibition of PC biosynthesis and the inhibitory effect of ETOH are independent of the activation of cAMP-dependent protein kinase. Unlike protein kinase inhibitors, pretreatment with tyrosine kinase inhibitors (erbstatin, genistein and tyrphostin 25) resulted in differential effects on PC biosynthesis and on the inhibitory action of ETOH. Genistein stimulated PC biosynthesis by 30 per cent as well as partially preventing/reversing the ETOH action, while tyrphostin 25 produced a synergistic inhibition. The relevance of tyrosine phosphorylation/dephosphorylation to the regulation of PC biosynthesis and ETOH action remains to be established.
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PMID:Mechanism by which ethanol inhibits phosphatidylcholine biosynthesis in human leukemic monocyte-like U937 cells. 816 30

A dominant mutation of Saccharomyces cerevisiae, CSE1, caused a decrease in the expression of the INO1 gene product, inositol-1-phosphate synthase. The residual activity was completely repressed by the addition of choline to the medium. A mutant carrying this mutation could not grow in the presence of choline unless inositol was added to the medium. Here we report a suppressor gene of the CSE1 mutation, SCS1 (suppressor of CSE1), which was cloned by complementation of CSE1 with a wild-type multicopy yeast genomic library. The cloned SCS1 gene contained an open reading frame which encoded 304 amino acid residues with a calculated molecular mass of 34,234 Da, and the sequence coincided with residues with a calculated molecular mass of 34,234 Da, and the sequence coincided with that of the INO2 gene. An scs1/ino2 null mutant constructed by gene replacement was viable, but auxotrophic for inositol and choline, and used for determination of the mRNA levels of various phospholipid-synthesizing enzymes. In agreement with the reported data for ino2 mutants the disruptant showed decreased expression of the INO1 and PSS genes, which are known to be regulated by inositol and choline. In addition, we newly found that the disruption of SCS1/INO2 also caused a decrease in the expression of the CKI, PEM1, and PEM2 genes, which we previously showed to belong to the inositol-choline-regulated gene family. These results confirm and strengthen the conclusion that the SCS1/INO2 gene is required for expression of inositol-choline-regulated genes in phospholipid synthesis.
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PMID:Cloning and characterization of the SCS1 gene required for the expression of genes in yeast phospholipid synthesis. 818 19

We have shown previously that N-[2-bromocinnamyl(amino)-ethyl]-5-isoquinolinesulphonamide (H-89), a selective inhibitor of cyclic-AMP-dependent protein kinase (PKA), inhibits phosphatidylcholine biosynthesis in HeLa cells. In the present study, we elucidated the mechanism underlying the described inhibition. Treatment of cells with 10 microM H-89 had no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. However, H-89 slightly affected the distribution of cytidylyltransferase between cytosol and membranes, but the cellular 1,2-diacylglycerol content was not influenced. Furthermore, pulse-chase experiments revealed that H-89 did not affect cytidylyltransferase activity. Instead, H-89 inhibited choline kinase, the enzyme catalysing the first step in the CDP-choline pathway. In the presence of 10 microM H-89, choline kinase activity was inhibited by 36 +/- 7.6% in vitro. Additionally, the phosphorylation of choline to phosphocholine was inhibited by 30 +/- 3% in cell-culture experiments. This inhibitory effect could be partly prevented by simultaneous addition of 10 microM forskolin, indicating that choline kinase is regulated in part by PKA activity.
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PMID:N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulphonamide (H-89) inhibits incorporation of choline into phosphatidylcholine via inhibition of choline kinase and has no effect on the phosphorylation of CTP:phosphocholine cytidylyltransferase. 828 Jan 5

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.
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PMID:Purification and characterization of casein kinase I from broccoli. 832 68

Okadaic acid, penetrating the human erythrocytes, almost completely inhibits P-Ser-protein phosphatase activity, whereas it unaffects Ser/Thr-protein kinase activity (casein kinases CKI and CKII), thus promoting a marked increase of the endogenous Ser-phosphorylation level of membrane proteins, such as cytoskeletal spectrin beta-subunit (band 2) and transmembrane band 3 protein. By contrast, the Tyr-phosphorylation state of band 3 protein is practically unaffected by okadaic acid, being unaffected both Tyr-protein kinase and P-Tyr-protein phosphatase activities.
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PMID:Effect of okadaic acid on membrane protein phosphorylation in human erythrocytes. 839 24

We report the molecular cloning and characterization of a 49-kDa form of casein kinase I from rat testis. A cDNA clone encoding the enzyme, designated casein kinase I delta, contained an open reading frame of 1284 nucleotides that predicts a polypeptide of 428 amino acids with a M(r) of 49,121. The predicted amino acid sequence shares 76% identity with casein kinase I alpha, a 37-kDa form recently cloned from bovine brain (Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9548-9552), and 65% identity with HRR25, a 57-kDa form of casein kinase I from yeast shown to be involved in DNA repair (Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. G., and Heffron, F. (1991) Science 253, 1031-1034). Northern analysis of rat or rabbit RNA revealed three hybridizing species of 3.5-4.1, 2.2, and 1.9 kilobase pairs (kb). The largest message was detected in all tissues examined, whereas the 1.9- and 2.2-kb species were found predominantly in testis. A probe corresponding to the 3'-untranslated region of the casein kinase I delta cDNA hybridized only to the 1.9-kb transcript. Expression of the casein kinase I delta cDNA in Escherichia coli resulted in active enzyme that phosphorylated casein, phosvitin, and the peptide substrate DDDDVASLPGLRRR. Enzyme activity was associated with a predominant polypeptide of 55-kDa, although COOH-terminal degradation products of 50 and 42 kDa were also present in partially purified enzyme. Recombinant casein kinase I delta was inhibited by the specific casein kinase I inhibitor, CKI-7, half-maximally at 12 microM. Heparin inhibited recombinant casein kinase I delta when phosvitin was the substrate, with half-maximal inhibition at 11.5 micrograms/ml. However, if the peptide substrate was used, heparin activated recombinant casein kinase I delta 4-5-fold, with half-maximal activation at 9.5 micrograms/ml. A truncated form of casein kinase I delta, lacking the COOH-terminal 111 amino acids, was no longer activated by heparin. Casein kinase I delta therefore represents a separate member of the casein kinase I family distinguished by its larger size and unique kinetic behavior with respect to heparin.
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PMID:Molecular cloning, expression, and characterization of a 49-kilodalton casein kinase I isoform from rat testis. 845 11

The three-dimensional structure for the catalytic region of the mammalian protein kinase, casein kinase I delta (CKI delta), has been solved by X-ray crystallography to a resolution of 2.3 A. A truncation mutant of CKI delta lacking the C-terminal autoinhibitory region was expressed in Escherichia coli, purified, and crystallized. The structure was solved by molecular replacement using the crystal structure of the catalytic domain of a CKI homolog from Schizosaccharomyces pombe, Cki1. A tungstate derivative confirmed the initial molecular replacement solution and identified an anion binding site which may contribute to the unique substrate specificity of CKI. Like other protein kinases, the catalytic domain of CKI is composed of two lobes with a cleft between them for binding ATP. Comparison of the mammalian and yeast CKI structures suggests that a rotation of the N-terminal domain occurs upon ATP binding. This domain motion is similar, but not identical, to that observed in cAMP-dependent protein kinase upon binding ATP. Although Cki1 has many similarities to CKI delta over the catalytic domain, these two forms of CKI likely perform different functions in vivo. Relating the primary sequences of other CKI enzymes to the three-dimensional architecture of CKI delta reveals a catalytic face that is especially conserved among the subset of CKI family members associated with the regulation of DNA repair.
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PMID:Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition. 864 28

Cyclin and cyclin-dependent kinase(cdk) complexes, and their inhibitors (CKIs) play important roles in growth regulation on the cells. p27/kip1 is a CKI associated with G1 arrest induced by cell to cell contact, transforming growth factor-beta and cyclic AMP. The abnormality of p27/Kip1 genes in human tumors usually appears as a steady level defect of expression, since mutations in them is rare. Thus it is important to estimate the expression level of this gene. To detect the change of p27/Kip1 mRNA level in blood cells, we developed the ribonuclease protection assay using nonradioactive riboprobe which was produced by reverse transcriptase-polymerase chain reaction (RT-PCR) with T7 promoter-added antisense primer and the in vitro transcription system. Our assay may be useful for clinical evaluation of the mRNA level.
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PMID:[Detection of p27/kip1 mRNA in blood cells by nonradioactive ribonuclease protection assay]. 867 70

Rabbit erythrocytes of progressively increasing age were isolated using an avidin-biotin affinity technique and the activity of protein kinases and other enzymes was analyzed in cytosols and membranes from the isolated cells. The activities of cytosolic protein kinase C (PKC), cAMP-dependent kinase (PKA), and casein kinase type I and II (CKI and II) were all found to undergo an age-dependent decrease of twofold to fourfold over the 8-week lifespan of the cells. Membrane-associated tyrosine kinase showed little or no decrease, but membrane-associated CKI showed a dramatic eightfold decrease over the 8-week period. By contrast, various cytosolic enzymes, including lactate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and acid phosphatase, showed no change in activity over the same time period. Density-separated human erythrocytes showed qualitatively similar decreases in cytosolic protein kinase activities in the densest fractions, which contain the oldest cells. Our results show that aging erythrocytes undergo progressive loss of protein kinases that may adversely affect various cellular processes. The age-dependent loss of kinase activity reported here is one of the most striking manifestations of erythrocyte senescence yet to be reported.
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PMID:Specific loss of protein kinase activities in senescent erythrocytes. 869 69


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