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 presence of ATP in the genital tract fluid of mammals provokes questions regarding its function in the fertilization process. We investigated the effect of extracellular ATP (ATPe) on the activation of bovine spermatozoa. A signal transduction mechanism for ATP involving the receptor-mediated release of second messengers is described. Treatment of spermatozoa with ATP, uridine triphosphate (UTP), or 2-methylthio-ATP resulted in a concentration-dependent increase of acrosomal exocytosis, whereas treatment with either AMP or adenosine induced little exocytosis. This suggested that the receptor involved is of the P2 and not the P1 type. Several lines of evidence also suggest that the ATP purinoceptor is of the P2y and not the P2x type. First, the acrosome reaction was induced by the P2y-agonists ATP, UTP, or 2-methylthio-ATP, but no effects were shown by the P2x-agonists alpha,beta-methylene-ATP or beta,gamma-methylene-ATP. Second, ATP-induced acrosomal exocytosis was inhibited by the P2y antagonists, but not by the P2x antagonists. Third, enhanced Ca2+ uptake into the cells was observed with ATP and 2-methylthio-ATP, but not with beta,gamma-methylene-ATP. Additionally, ATP induced elevation of intracellular Ca2+ and cAMP, and the effect on cAMP was predominantly enhanced by including Ca2+ and the Ca2+-ionophore A23187 in the incubation medium. Extracellular ATP also activates protein kinase Calpha (PKCalpha), and the acrosome reaction, stimulated by ATPe, is inhibited by a PKC-specific inhibitor. In summary, we suggest that ATPe activates the P2 purinoceptor that elevates [Ca2+]i, which leads to PKCalpha activation and culminates in acrosomal exocytosis.
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PMID:Extracellular adenosine triphosphate stimulates acrosomal exocytosis in bovine spermatozoa via P2 purinoceptor. 1180 59

1. The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastomaxrat glioma hybrid cells. 2. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5'-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A(2) adenosine receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA). 3. In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of G(s)-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. 4. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. 5. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells.
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PMID:Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness. 1195 6

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl(-) secretion and inhibit amiloride-sensitive Na(+) transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na(+) channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl(-) transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N(2),2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl(-).
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PMID:No evidence for inhibition of ENaC through CFTR-mediated release of ATP. 1222 48

The reaction mechanism of the phosphoryl transfer catalyzed by dinucleoside diphosphate kinase from Dictyostelium discoideum is investigated by semiempirical AM1 molecular orbital computation of an active site model system on the basis of various X-ray crystallographic structures. The computational results suggest that the phosphoryl transfer from adenosine triphosphate to the His122 residue is accompanied by the simultaneous shift of a proton from the histidine residue to one of the oxygen atoms of the gamma phosphate group. This involves a doubly protonated His122 residue whilst this residue is neutral in its ternary complex with ADP and the transition state analogue AlF(3). The proposed mechanism is thus analogous to that of phosphoryl transfer by cyclic adenosine monophosphate dependent protein kinase and uridine/cytidine monophosphate kinase as found in our earlier work and clarifies the role of the ribose 3'-OH group. Furthermore, the energetics of phosphoryl transfer onto other nucleoside analogues such as 3'-azido-3'-deoxythymidine-diphosphate and 2',3'-dideoxy-2',3'-didehydro-thymidine-diphosphate are investigated. The calculated reaction barriers for the phosphorylation of the diphosphates by the enzyme are all within a range of 13.1 kJ mol(-1), which suggests that variations in the activation energies alone cannot account for the experimentally observed differences in enzymatic activity. Consequences for the design of new anti-HIV nucleoside analogues are discussed. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2002/f360_s.pdf or from the author.
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PMID:The mechanism of phosphorylation of natural nucleosides and anti-HIV analogues by nucleoside diphosphate kinase is independent of their sugar substituents. 1232 98

Recently we reported that the pyridinylimidazole class of p38 mitogen-activated protein (MAP) kinase inhibitors potently inhibited the facilitated transport of nucleosides and nucleoside analogs in K562 cells. These compounds competed with the binding of nitrobenzylthioinosine (NBMPR) to K562 cells, consistent with inhibition of the NBMPR-sensitive equilibrative transporter (ENT1). In this study we examined a large number of additional protein kinase inhibitors for their effects on nucleoside transport. We find that incubation of K562 cells with tyrosine kinase inhibitors (AG825, AG1517, AG1478, STI-571), protein kinase C (PKC) inhibitors (staurosporine, GF 109203X, R0 31-8220, arcyriarubin A), cyclin-dependent kinase inhibitors (roscovitine, olomoucine, indirubin-3'-monoxime), or rapamycin resulted in a dose-dependent reduction of intracellular uptake of [3H]uridine. In contrast, neither the MAP kinase kinase inhibitors (U0126, PD 98059) nor the phosphatidyl inositol-3 kinase inhibitors (wortmannin, LY 294002) affected this process. Furthermore, both transient uptake and prolonged [3H]thymidine incorporation in K562 cells were inhibited by protein kinase inhibitors, inactive analogs of kinase inhibitors (R0 31-6045, SB202474), and NBMPR, independently of effects on cell proliferation as determined by MTT assay. These studies demonstrate that a wide variety of protein kinase inhibitors affect nucleoside uptake through selective inhibition of nucleoside transporters, independently of kinase inhibition.
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PMID:Inhibition of nucleoside transport by protein kinase inhibitors. 1253 31

Glycogen synthase (GS) catalyses the incorporation of uridine diphosphate-glucose into glycogen in skeletal muscle. In concert with the glucose transport step, GS activity is thought to be rate-limiting in the disposal of glucose as muscle glycogen. Glycogen synthase is regulated by both allosteric factors (primarily glucose 6-phosphate) and covalent modification by reversible phosphorylation and dephosphorylation leading to inactivation and activation of GS, respectively. Exercise activates both stimulatory and inhibitory regulators of GS and it is thought that the resultant activity of GS during exercise depends on the relative strength of opposing signals. However, the mechanisms by which exercise regulates GS activity are not fully understood. Glycogen breakdown, the GM-protein phosphatase 1 complex and possibly cellular relocalization of GS may be considered important factors involved in the stimulation of GS activity during exercise, while adenosine monophosphate-activated protein kinase and plasma adrenaline (via protein kinase A) can be considered as essential for the exercise-induced inhibitory signals to GS.
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PMID:Regulation of glycogen synthase in skeletal muscle during exercise. 1286 35

For many years, a large body of circumstantial evidence supported the notion that the synovial membrane produced the hyaluronan-rich synovial fluid. A quantitative cytochemical technique for uridine-diphospho glucose dehydrogenase (UDPGD) activity established that fibroblast-like cells on the intimal surface of the synovial lining made a specific contribution to maintaining these glycosaminoglycan levels. Our studies have aimed to determine the mechanisms that control the attainment and persistence of this differentiated phenotype, and have recently focused on their appearance during joint cavity development in the embryonic limb; a process that is dependent upon skeletal movement. These in situ micro-biochemical studies have shown that cells bordering the presumptive joint cavity exhibit raised UDPGD activity, are associated with a matrix rich in hyaluronan and show immobilization-induced loss in such characteristics. Together with complimentary studies in adult joints, this suggests that mechanical stimuli promote the acquisition of this joint line-forming phenotype. For this reason our studies have attempted to identify the 'up-stream' mechano-dependent factors that control these events. Endothelial cells respond to mechanical stimuli by activating, via phosphorylation, mitogen activated protein kinase/extracellular signal-regulated kinase (MAPkinase/ERK). Using phospho-specific anti-ERK-1/2 antibodies we have shown that immunolabelling of developing limbs shows a clear joint line-selective activation during cavitation, with little if any labelling within neighbouring elements, and that this is abolished in immobilized limbs. In an attempt to facilitate the final mechanistic deciphering of these responses we have used an in vitro-based approach and found by Western blotting that active ERK-1/2 expression was increased in cultured articular surface cells following application of dynamic mechanical strain. Intriguingly, the use of a selective inhibitor (PD98059) of ERK activation by its classical activating kinase, Mek, to restrict such strain-induced increases, produced an enhanced strain-related increase in UDPGD mRNA expression. This suggests that mechano-dependent ERK activation serves a feedback regulatory role during differentiation of these cells. Whilst it is clear that these in vitro experiments serve a useful function, it is clear that they generally take little regard of the influence that might be provided by cell-cell and cell-matrix interactions within the developing limb's complex and dynamic environment and architecture. It is therefore imperative that we attempt to bridge the gap between the cell biology of such phenomena on the one hand, and the morphological approach to this same problem on the other.
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PMID:Identifying and characterizing the joint cavity-forming cell. 1291 Apr 76

The uptake of nucleosides and nucleoside analogs into human leukemia K562 cells is facilitated by the equilibrative transporters ENT1 and ENT2. Incubation of K562 cells with a variety of protein kinase inhibitors inhibited the transport of both uridine (ARA-C) and cytidine (CPEC) analogs. These inhibitory effects were observed for a large number of kinase inhibitors including those against p38 MAPK, the EGF receptor kinase, protein kinase C, TOR and others. Thus these results suggest that the nucleoside transporters are unexpected targets for kinase inhibitors and may influence the design and application of combinatorial approaches of nucleoside analogs and kinase inhibitors in clinical applications.
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PMID:Regulation of equilibrative nucleoside uptake by protein kinase inhibitors. 1557 Dec 74

The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.
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PMID:Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells. 1611 17

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral interferon-alpha response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the 3'-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the 3'-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nM range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.
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PMID:Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein. 1612 20


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