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)

Directional tag PCR subtractive hybridization was applied to construct a cDNA library generated from three different human osteosarcoma (OS) target cell lines (OHS, SaOS-2 and KPDXM) from which normal osteoblast (NO) sequences were subtracted. After two consecutive subtractive steps more than 98% of the common mRNAs species were depleted, leading to effective enrichment of the remaining target sequences. After differential screening of 960 clones, 81 candidates were further studied by Northern blot analysis and 73 represented separate mRNA species. Fifty-three of these showed enriched mRNA levels, of which 36 represented known and 17 not previously published cDNAs or EST sequences. The mRNAs showed a 1.4- to 504-fold enrichment compared to the mRNA levels in NO cells. The known mRNAs are: Ribosomal protein S11, KSP-37, Tethering factor SEC34, FXYD6, Alpha enolase, G-s-alpha, GPR85, DAF, RPL35A, GIF, TAPA-1, ANAPC11, DCI, hsp27, MRPS7 homolog, eIF p110 subunit, DPH2L, HMG-14, FB1 protein, chondroitin-6-sulphonase, calgizzarin, RNA polymerase II subunit, RPL13A, DHS, gp96, HHP2, acidic ribosomal phosphoprotein P2, ANT-2, ARF1, AFG3L2, SKD3, phosphoglucoisomerase, GST pi, CKI gamma 2, DNA polymerase delta small subunit and TRAP delta. Sections of human osteosarcoma biopsies and a xenograft were studied by in situ analysis. Seven cDNAs highly expressed in Northern blot analysis were tested. Their in situ expression differed between the xenograft and human sections as did that of collagen I. In the xenograft made from one of the target cell lines (OHS), a fair to strong representation of 3 cloned mRNAs was observed while collagen I mRNA was not detectable. We conclude that the molecular heterogeneity of these tumors is considerable. These results ought to have implications for future work to describe phenotypic subtypes with the aim of improving the diagnosis of human osteosarcomas.
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PMID:Molecular heterogeneity in human osteosarcoma demonstrated by enriched mRNAs isolated by directional tag PCR subtraction cloning. 1289 94

The authors have engineered plasmid constructs for developmental and constitutive expression of yeast-enhanced green fluorescent protein (yEGFP3) in Candida albicans. The promoter for the hyphae-specific gene Hyphal Wall Protein 1 (HWP1) conferred developmental expression of yEGFP3 in germ tubes and hyphae but not in yeasts or pseudohyphae when targeted to the ENO1 (enolase) locus in single copy. The pHWP1GFP3 construct allows for the easy visualization of HWP1 promoter activity in individual cells expressing true hyphae without having to prepare RNA for analysis. Constitutive expression of yEGFP was seen in all cell morphologies when the HWP1 promoter was replaced with the ENO1 promoter region. The use of the plasmids for expression of genes other than yEGFP3 was examined by substituting the putative C. albicans BCY1 (SRA1) gene, a component of the cAMP signalling pathway involved in yeast to hyphae transitions, for yEGFP3. Strains overexpressing BCY1 from the ENO1 promoter were inhibited in germ tube formation and filamentation in both liquid and solid media, a phenotype consistent with keeping protein kinase A in its inactive form by association with Bcy1p. The plasmids are suitable for studies of germ tube induction or assessing germ tube formation by measuring yEGFP3 expression, for inducible expression of genes concomitant with germ tube formation by the HWP1 promoter, for constitutive expression of genes by the ENO1 promoter, and for expressing yEGFP3 using a promoter of choice.
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PMID:Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans. 1452 29

The skeletal muscle specific Ca(2)+/calmodulin-dependent protein kinase (CaMKIIbeta(M)) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, alphaKAP, but its function remains to be defined. Protein interactions of CaMKIIbeta(M) indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKIIbeta(M) was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKIIbeta(M), and their phosphorylation was enhanced in a Ca(2+)- and calmodulin (CaM)-dependent manner. The CaMKIIbeta(M) could directly phosphorylate GAPDH and markedly increase ( approximately 3.4-fold) its activity in a Ca(2+)/CaM-dependent manner. These data suggest that the muscle CaMKIIbeta(M) isoform may serve to assemble the glycogen-mobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKIIbeta(M) in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.
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PMID:The muscle-specific calmodulin-dependent protein kinase assembles with the glycolytic enzyme complex at the sarcoplasmic reticulum and modulates the activity of glyceraldehyde-3-phosphate dehydrogenase in a Ca2+/calmodulin-dependent manner. 1519 64

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.
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PMID:Characterization of dual specificity protein kinase from maize seedlings. 1544 26

Thiol proteins are important in cellular antioxidant defenses and redox signalling. It is postulated that reactive oxidants cause selective thiol oxidation, but relative sensitivities of different cell proteins and critical targets are not well characterized. We exposed Jurkat cells to H2O2 for 10 min and measured changes in reversibly oxidized proteins by labelling with iodoacetamidofluorescein and two-dimensional electrophoresis. At 200 microM H2O2, which caused activation of the MAP (mitogen-activated protein) kinase ERK (extracellular-signal-regulated kinase), growth arrest and apoptosis, relatively few changes were seen. A total of 28 spots were reversibly oxidized (increased labelling intensity) and 24 decreased. The latter included isoforms of peroxiredoxins 1 and 2, which were irreversibly oxidized. Oxidation of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was striking, and other affected proteins included glutathione S-transferase P1-1, enolase, a regulatory subunit of protein kinase A, annexin VI, the mitotic checkpoint serine/threonine-protein kinase BUB1beta, HSP90beta (heat-shock protein 90beta) and proteosome components. At 20 microM H2O2, changes were fewer, but GAPDH and peroxiredoxin 2 were still modified. Dinitrochlorobenzene treatment, which inhibited cellular thioredoxin reductase and partially depleted GSH, caused reversible oxidation of several proteins, including thioredoxin 1 and peroxiredoxins 1 and 2. Most changes were distinct from those with H2O2, and changes with H2O2 were scarcely enhanced by dinitrochlorobenzene. Relatively few proteins, including deoxycytidine kinase, nucleoside diphosphate kinase and a proteosome activator subunit, responded only to the combined treatment. Thus most of the effects of H2O2 were not linked to thioredoxin oxidation. Our study has identified peroxiredoxin 2 and GAPDH as two of the most oxidant-sensitive cell proteins and has highlighted how readily peroxiredoxins undergo irreversible oxidation.
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PMID:Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells. 1580 6

One approach to the treatment of retinal diseases, such as retinitis pigmentosa, is to replace diseased or degenerating cells with healthy cells. Even if all of the problems associated with tissue transplant were to be resolved, the availability of tissue would remain an ongoing problem. We have previously shown that transformed human retinal cells can be grown in a NASA-developed horizontally rotating culture vessel (bioreactor) to form three-dimensional-like structures with the expression of several retinal specific proteins. In this study, we have investigated growth of non-transformed human retinal progenitors (retinal stem cells) in a rotating bioreactor. This rotating culture vessel promotes cell-cell interaction between similar and dissimilar cells. We cultured retinal progenitors (Ret 1-4) alone or as a co-culture with human retinal pigment epithelial cells (RPE, D407) in this system to determine if 3D structures can be generated from non-transformed progenitors. Our second goal was to determine if the formation of 3D structures correlates with the upregulation of neurotrophins, basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFalpha), ciliary neurotrophic factor (CNTF), and brain-delivered neurotrophic factor (BDNF). These factors have been implicated in progenitor cell proliferation, commitment, differentiation, and survival. We also investigated the expression of the following retinal specific proteins in this system: neuron specific enolase (NSE); tyrosine hydroxylase (TH); D(2)D(3), D(4) receptors; protein kinase-C alpha (PKCalpha), and calbindin. The 3D structures generated were characterized by phase and scanning transmission electron microscopy. Retinal progenitors, cultured alone or as a co-culture in the rotating bioreactor, formed 3D structures with some degree of differentiation, accompanied by the upregulation of bFGF, CNTF, and TGFalpha. Brain-derived neurotrophic factor, which is expressed in vivo in RPE (D407), was not expressed in monolayer cultures of RPE but expressed in the rotating bioreactor-cultured RPE and retinal progenitors (Ret 1-4). Upregulation of neurotrophins was noted in all rotating bioreactor-cultured cells. Also, upregulation of D(4) receptor, calbindin, and PKCalpha was noted in the rotating bioreactor-cultured cells. We conclude that non-transformed retinal progenitors can be grown in the rotating bioreactor to form 3D structures with some degree of differentiation. We relied on molecular and biochemical analysis to characterize differentiation in cells grown in the rotating bioreactor.
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PMID:Enhanced neurotrophin synthesis and molecular differentiation in non-transformed human retinal progenitor cells cultured in a rotating bioreactor. 1649 51

Several recent studies have proposed that astrocytes may contribute to neurogenesis, not only as a source of trophic substances regulating it, but also as stem cells themselves. In order to better understand these mechanisms, primary astrocyte cultures were established from human fetal brain. After 3-4 weeks in culture, astrocytes (about 95% GFAP+; neurofilament, NF-; neuro-specific enolase, NSE-) were treated with a cocktail of protein kinase activators and FGF-1. After 5 h of treatment, most cells showed morphological changes that increased progressively up to 24-48 h, exhibiting a round cell body with long processes. Immunocytochemistry showed that treatment-induced NF and NSE expression in about 40% of cells. Nestin expression increased after treatment, whereas GFAP immunostaining was not significantly modified. Western blot and RT-PCR confirmed the results. No neuronal electrophysiological properties were observed after treatment, suggesting an incomplete maturation under these experimental conditions. Understanding the regenerative capability and neurogenic potential of astrocytes might be useful in devising therapeutic approaches for a variety of neurological disorders.
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PMID:Human astrocytes can be induced to differentiate into cells with neuronal phenotype. 1671 98

Fertilization of the mammalian oocyte depends on the ability of spermatozoa to undergo a process known as capacitation as they ascend the female reproductive tract. A fundamental feature of this process is a marked increase in tyrosine phosphorylation by an unusual protein kinase A (PKA)-mediated pathway. To date, the identity of the intermediate PKA-activated tyrosine kinase driving capacitation is still unresolved. In this study, we have identified SRC as a candidate intermediate kinase centrally involved in the control of sperm capacitation. Consistent with this conclusion, the SRC kinase inhibitor SU6656 was shown to suppress both tyrosine phosphorylation and hyperactivation in murine spermatozoa. Moreover, SRC co-immunoprecipitated with PKA and this interaction was found to lead to an activating phosphorylation of SRC at position Y416. We have also used difference-in-2D-gel-electrophoresis (DIGE) in combination with mass spectrometry to identify a number of SRC substrates that become phosphorylated during capacitation including enolase, HSP90 and tubulin. Our data further suggest that the activation of SRC during capacitation is negatively controlled by C-terminal SRC kinase. The latter was localized to the acrosome and flagellum of murine spermatozoa by immunocytochemistry, whereas capacitation was associated with an inactivating serine phosphosphorylation of this inhibitory kinase.
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PMID:Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa. 1683 69

The transcription factor CREB (cyclic AMP response element binding protein) is implicated in diverse brain functions and represents a prospective target in gene therapy for human disorders. However, the transgenic expression and stability of exogenously expressed CREB within the cell remains poorly characterized. Here we found that transient expression of a CREB dominant interfering mutant A-CREB or the inducible cAMP early repressor, ICER, led to the dramatic decrease of exogenously co-expressed CREB in 293 human embryonic kidney cells. Elevation of protein kinase A activity within the cells restored CREB protein levels. A-CREB did not effect the transient expression of a truncated CREB lacking the leucine zipper domain demonstrating a specific effect of heterodimerization on CREB protein stability. Somatic gene transfer into the rat brain using a recombinant adeno-associated virus vector provided robust expression of both transgenic CREB and ICER mRNAs under the control of a constitutive neuron specific enolase (NSE) promoter. In contrast to ICER, the expression of the transgenic CREB mRNA did not result in elevation of CREB protein levels within dentate granule cells of the hippocampus, suggesting its prompt degradation under basal conditions. However, following tetanization of the perforant pathway, which is known to induce CREB phosphorylation, there was a significant increase in the amount of transgenic CREB protein within dentate granule cells. Hence, heterodimerization of unphosphorylated CREB with either A-CREB or ICER triggers CREB protein degradation, whereas phosphorylation prevents CREB from such degradation both in vitro and in vivo.
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PMID:Phosphorylation-dependent degradation of transgenic CREB protein initiated by heterodimerization. 1716 45

SRC-related tyrosine kinases are suggested to play a role in the increase of sperm protein phosphotyrosine content that occurs during capacitation. In our laboratory, we previously demonstrated that the SRC-related tyrosine kinase YES1 (also known as c-YES) is present in human spermatozoa. However, since it is negatively regulated by Ca(2+), whose intracellular concentration increases during capacitation, another kinase would most likely be involved in the capacitation-related increase in sperm protein tyrosine phosphorylation. The present study represents the first direct assessment of SRC tyrosine kinase activity in ejaculated mammalian sperm. By immunohistochemistry on human testis sections, it is clearly shown that SRC is expressed during spermatogenesis, mainly in round and elongating spermatids. Using an indirect immunofluorescence approach, SRC is detected in the acrosomal region of the head and in the sperm flagellum of ejaculated sperm. This tyrosine kinase is associated with the plasma membrane and with cytoskeletal elements, as suggested by its partial solubility in nonionic detergents. Despite its partial solubility, SRC kinase activity was assayed after immunoprecipitation using acid-denatured enolase as a substrate. It is clearly demonstrated that SRC activity is inhibited by SU6656 and PP1, selective SRC family tyrosine kinase inhibitors, and activated in a Ca(2+)-dependent manner. Furthermore, it is shown that SRC is activated in a cAMP/PRKA-dependent manner; SRC coimmunoprecipitates with the catalytic subunit of the cAMP-dependent protein kinase (PRKAC) and is phosphorylated by this latter kinase, resulting in an increase in enolase phosphorylation. All these results support the involvement of the tyrosine kinase SRC in the increase in sperm protein phosphotyrosine content observed during capacitation.
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PMID:Increased activity of the human sperm tyrosine kinase SRC by the cAMP-dependent pathway in the presence of calcium. 1856 2


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