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

Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.
 ...
PMID:Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa. 216 Aug 33

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.
 ...
PMID:Specific loss of protein kinase activities in senescent erythrocytes. 869 69

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD+, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca2+/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.
 ...
PMID:The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide. 937 36

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of alpha-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes.
 ...
PMID:Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts. 1022 60

In the search for new markers of human endometrial hyperplasia and adenocarcinoma the method of quantitative two-dimensional gel electrophoresis was applied to study the protein expression profiles of metabolically [(35)S]-methionine-labelled proteins of endometrial explants. Approximately 1700 protein spots were resolved by the two-dimensional gel electrophoresis, and the expression pattern of each of these proteins was assessed for increased expression during hyperplasia or adenocarcinoma. In total, six protein spots showed increased expression in hyperplasia, 19 in carcinoma, and eight in both hyperplasia and carcinoma. Twelve of these 33 differentially expressed proteins were identified by peptide mass mapping combined with sequence database searching. Among the identified proteins were proteins involved in cellular transport and chaperoning, i.e. heat shock protein 27 kDa protein, heat shock 70 kDa protein, heat shock cognate 71 kDa protein, and serotransferrin. Other identified proteins were: regulatory chain protein of cAMP-dependent protein kinase, prohibitin, and heterogeneous nuclear ribonucleoprotein A2/B1. Finally we identified proteins associated with the cytoskeleton, vimentin and tropomyosin isoform 3, and the glycolytic pathway, alpha enolase, and phosphoglycerate kinase. The remaining unidentified proteins were either not contained in the database and must be assumed to be novel proteins, or were present in too low amounts to allow characterization.
 ...
PMID:Two-dimensional gel analysis of human endometrial proteins: characterization of proteins with increased expression in hyperplasia and adenocarcinoma. 1042 3

Primary human T lymphocytes were transduced at high efficiency with the Moloney murine leukemia virus (Mo-MuLV) vector, LNC-mB7-1, in which an internal cytomegalovirus (CMV) promoter drives expression of the murine B7-1 cDNA. Compared with transduced T cells expanded in IL-2 or reactivated with soluble antibodies to CD3 or CD28, transgene expression was significantly increased after activation on immobilized anti-CD3 antibodies (CD3i) or by simultaneous activation on immobilized anti-CD3 and anti-CD28 antibodies (CD3i/CD28i). A similar pattern of transgene expression was observed in T cells transduced with Mo-MuLV LNC-EGFP. Proviral copy number was maintained in LNC-mB7-1-transduced T cells expanded in IL-2 or reactivated on CD3i/CD28i. Substantial increases in LNC-mB7-1 steady state mRNA in reactivated T lymphocytes, compared with those maintained in IL-2, correlated with increased transcription of the LNC-mB7-1 proviral DNA. Furthermore, T cells transduced with the Mo-MuLV ZIPPGK-mADA, in which the mADA cDNA is driven by an internal human phosphoglycerate kinase (PGK) promoter, showed increases in steady state ZIPPGK-mADA RNA on reactivation. High levels of transgene expression were evident irrespective of cell cycle position in both CD4+ and CD8+ lymphocytes. After reactivation, increases in LNC-mB7-1 mRNA were observed in the presence of the protein synthesis inhibitor cycloheximide, indicating that proteins involved in upregulating transgene expression preexisted in transduced lymphocytes. Induction of transgene expression on CD3i/CD28i showed a dose-dependent decrease in transgene expression when incubated with selective protein kinase inhibitors. These data provide new insights into the mechanisms governing transgene expression driven by Mo-MuLV constructs containing internal promoters in transduced primary T lymphocytes.
 ...
PMID:Costimulation of transduced T lymphocytes via T cell receptor-CD3 complex and CD28 leads to increased transcription of integrated retrovirus. 1049 53

Drosophila larvae and adults exhibit a naturally occurring genetically based behavioural polymorphism in locomotor activity while foraging. Larvae of the rover morph exhibit longer foraging trails than sitters and forage between food patches, while sitters have shorter foraging trails and forage within patches. This behaviour is influenced by levels of cGMP-dependent protein kinase (PGK) encoded by the foraging (for) gene. Rover larvae have higher expression levels and higher PGK activities than do sitters. Here we discuss the importance of the for gene for studies of the mechanistic and evolutionary significance of individual differences in behaviour. We also show how structure-function analysis can be used to investigate a role for mushroom bodies in larval behaviour both in the presence and in the absence of food. Hydroxyurea fed to newly hatched larvae prevents the development of all post-embryonically derived mushroom body (MB) neuropil. This method was used to ablate MBs in rover and sitter genetic variants of foraging to test whether these structures mediate expression of the foraging behavioural polymorphism. We found that locomotor activity levels during foraging of both the rover and sitter larval morphs were not significantly influenced by MB ablation. Alternative hypotheses that may explain how variation in foraging behaviour is generated are discussed.
 ...
PMID:Foraging behaviour in Drosophila larvae: mushroom body ablation. 1123 55

Successful embryonic development is dependent on time and location-specific expression of appropriate genes. Unfortunately, information on stage-specific gene expression during early embryonic development in the bovine is lacking. In the present study, we compared gene expression between in vitro-produced Day 7-8 intact blastocysts (driver) and Day 9-10 hatched blastocysts (tester) using suppression-subtractive hybridization. Pools of 30 embryos for both driver and tester were used in the RNA extraction process. From limited amounts of starting material ( approximately 400 ng of total RNA), a reverse transcription-polymerase chain reaction (PCR) procedure was used to amplify the mRNA and generate sufficient cDNA to conduct suppression-subtractive hybridization. The subtracted cDNA products were cloned, and 126 cDNAs representing expressed mRNAs were isolated, sized, single-pass sequenced, and compared to known sequences in GenBank. Ninety-two clones provided sequence information for further analysis. Among these, 31 exhibited high homology to known genes. Three, 26S proteasomal ATPase (PSMC3), casein kinase 2 alpha subunit (CK2), and phosphoglycerate kinase (PGK) were selected and further characterized using real-time quantitative PCR to assess their differential expression in hatched blastocysts. Overall, a 1.3-, 1.6-, and 1.5-fold increase in expression level was observed in hatched blastocysts compared with intact blastocyst for PSMC3, CK2, and PGK, respectively. These results show that construction of subtracted cDNA libraries from small numbers of embryos is feasible and can provide information on gene expression patterns during preattachment embryogenesis.
 ...
PMID:Analysis of gene expression in the bovine blastocyst produced in vitro using suppression-subtractive hybridization. 1213 80

We have isolated and characterized the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene, and 1665 bp of full-length PGK2 cDNA were also compiled using modified rapid amplification 5'-RACE and 3'-RACE information. The results of genomic and cDNA sequences of the porcine PGK2 gene demonstrated that it is a single-exon intronless gene with a complete open reading frame of 1251 bp encoding a PGK protein of 417 amino acids. Real-time quantitative PCR results showed that PGK2 mRNA was solely expressed in the testis. There was a lower amount of PGK2 expression in the testis of a 10-month-old herniated boar and a very small amount of PGK2 expression in the testis of an 8-week-old cryptorchid piglet compared to an adult boar. Two SNPs in the PGK2 gene (SNP-A: T427C; SNP-B: C914A) resulting in amino acid substitutions (SNP-A: Ser102-Pro102; SNP-B: Thr264-Lys264) were detected and genotyped among six pig breeds. The nucleotide C at SNP-A responsible for the amino acid exchange to proline could lead to the loss of a casein kinase II (CK2) phosphorylation site in the PGK2 peptide. Association analyses between PGK2 genotypes and several traits of sperm quantity and quality were performed. The results showed that SNP-B has a positive significant effect on semen volume in the breed Pietrain (p = 0.08), i.e., boars carrying genotype CC revealed an increased volume of 49 ml compared with boars having the genotype AA.
 ...
PMID:Molecular characterization of the porcine testis-specific phosphoglycerate kinase 2 (PGK2) gene and its association with male fertility. 1559 58

The structural elements of the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling pathway have been described in the vestibular peripheral system. However, the functions of NO in the vestibular endorgans are still not clear. We evaluated the action of NO on the Ca(2+) currents in hair cells isolated from the semicircular canal crista ampullaris of the rat (P14-P18) by using the whole cell and perforated-cell patch-clamp technique. The NO donors 3-morpholinosydnonimine (SIN-1), sodium nitroprusside (SNP), and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide (NOR-4) inhibited the Ca(2+) current in hair cells in a voltage-independent manner. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO) prevented the inhibitory effect of SNP on the Ca(2+) current. The selective inhibitor of the soluble form of the enzyme guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), also decreased the SNP-induced inhibition of the Ca(2+) current. The membrane-permeant cGMP analogue 8-Br-cGMP mimicked the SNP effect. KT-5823, a specific inhibitor of cGMP-dependent protein kinase (PGK), prevented the inhibition of the Ca(2+) current by SNP and 8-Br-cGMP. In the presence of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent that prevents the S-nitrosylation reaction, the SNP effect on the Ca(2+) current was significantly diminished. These results demonstrated that NO inhibits in a voltage-independent manner the voltage-activated Ca(2+) current in rat vestibular hair cells by the activation of a cGMP-signaling pathway and through a direct action on the channel protein by a S-nitrosylation reaction. The inhibition of the Ca(2+) current by NO may contribute to the regulation of the intracellular Ca(2+) concentration and hair-cell synaptic transmission.
 ...
PMID:Modulation of voltage-gated Ca2+ current in vestibular hair cells by nitric oxide. 1718 10


1 2 Next >>