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)

Extracellular ATP suppressed the growth of HL-60 leukemia cells and induced their differentiation as revealed by N-formyl-methionyl-leucyl-phenylalanine-induced beta-glucuronidase release. ATP degraded to ADP, AMP, and adenosine, and the effect of ATP on cell growth was mimicked by these metabolites added to the cultures. The stable analog alpha,beta-methylene ATP, however, had only a weak inhibitory effect on cell growth. Adenine nucleotide-induced growth suppression was reversed by uridine, suggesting the involvement of intracellular pyrimidine starvation secondary to adenosine accumulation. Consistent with this, ATP induced intracellular starvation of pyrimidine nucleotides, and this effect was also prevented by pretreatment of cells with uridine. The order of effectiveness of ATP-induced differentiation of HL-60 cells, unlike that for growth suppression, was ATP > ADP > AMP, and adenosine had no effect. Furthermore, uridine had no effect and the stable analog, alpha,beta-methylene ATP also induced HL-60 cell differentiation, suggesting that differentiation was due to ATP per se. We tested the hypothesis that ATP-induced differentiation arises from activation of adenylyl cyclase by the novel P2Y(11) receptor using the cell-permeable inhibitor of protein kinase A, Rp-CPT-cAMPS (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp isomer). Rp-CPT-cAMPS (1-100 microM) prevented ATP-induced differentiation of HL-60 cells as assessed by fMLP-induced beta-glucuronidase release. However, Rp-CPT-cAMPS did not prevent ATP-induced growth suppression. Taken together, the data indicate that extracellular ATP suppresses HL-60 growth and induces their differentiation by distinct mechanisms. Growth suppression arises from adenosine generation and consequent pyrimidine starvation. Differentiation arises, at least in part, from a distinct mechanism involving the activation of cell surface P2 receptors coupled to cAMP generation and activation of protein kinase A.
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PMID:Extracellular ATP-dependent suppression of proliferation and induction of differentiation of human HL-60 leukemia cells by distinct mechanisms. 1107 40

Mammalian cells express at least two subtypes of equilibrative nucleoside transporters, i.e. ENT1 and ENT2, which can be distinguished functionally by their sensitivity and resistance respectively to inhibition by nitrobenzylthioinosine. The ENT1 transporters exhibit distinctive species differences in their sensitivities to inhibition by dipyridamole, dilazep and draflazine (human>mouse>rat). A comparison of the ENT1 structures in the three species would facilitate the identification of the regions involved in the actions of these cardioprotective agents. We now report the molecular cloning and functional expression of the murine (m)ENT1 and mENT2 transporters. mENT1 and mENT2 encode proteins containing 458 and 456 residues respectively, with a predicted 11-transmembrane-domain topology. mENT1 has 88% and 78% amino acid identity with rat ENT1 and human ENT1 respectively; mENT2 is more highly conserved, with 94% and 88% identity with rat ENT2 and human ENT2 respectively. We have also isolated two additional distinct cDNAs that encode proteins similar to mENT1; these probably represent distinct mENT1 isoforms or alternative splicing products. One cDNA encodes a protein with two additional amino acids (designated mENT1b) that adds a potential protein kinase CK2 phosphorylation site in the central intracellular loop of the transporter, and is similar, in this regard, to the human and rat ENT1 orthologues. The other cDNA has a 5'-untranslated region sequence that is distinct from that of full-length mENT1. Microinjection of mENT1, mENT1b or mENT2 cRNA into Xenopus oocytes resulted in enhanced uptake of [(3)H]uridine by the oocytes relative to that seen in water-injected controls. mENT1-mediated, but not mENT2-mediated, [(3)H]uridine uptake was inhibited by nitrobenzylthioinosine and dilazep. Dipyridamole inhibited both mENT1 and mENT2, but was significantly more effective against mENT1. Adenosine inhibited both systems with a similar potency, as did a range of other purine and pyrimidine nucleosides. These results are compatible with the known characteristics of the native mENT1 and mENT2 transporters.
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PMID:Molecular cloning and functional characterization of inhibitor-sensitive (mENT1) and inhibitor-resistant (mENT2) equilibrative nucleoside transporters from mouse brain. 1108 29

We have isolated a mouse cDNA clone corresponding to a novel isoform of the NBMPR-sensitive equilibrative nucleoside transporter (ENT1). The cDNA contains a 6 bp deletion in the open reading frame that changes the amino acid composition in a consensus casein kinase II (CKII) phosphorylation site at Ser-254. The clone containing Ser-254 is termed mENT1.1 and the clone lacking the serine termed mENT1.2. The deduced amino acid sequence of mENT1.1 corresponds to the previously cloned human and rat ENT1 proteins at Ser-254. Tissue distribution studies show that mRNA for both ENT1 isoforms are ubiquitously co-expressed in mouse. Analysis of genomic DNA corresponding to mouse ENT1 indicates the isoforms can be produced by alternative splicing at the end of exon 7. CEM/C19 cells stably expressing mENT1.1 and mENT1.2 show similar dose response curves for NBMPR and dipyridamole inhibition of [(3)H]adenosine uptake as well as exhibiting comparable selectivity for both purine and pyrimidine nucleosides but not the corresponding nucleobases.
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PMID:Cloning of a novel isoform of the mouse NBMPR-sensitive equilibrative nucleoside transporter (ENT1) lacking a putative phosphorylation site. 1117 96

The effects of extracellular nucleotide triphosphates on the stimulation of mucin production by airway epithelial cells were examined. The order of potency in stimulating mucin secretion in primary cultures of human tracheobronchial epithelial cells is: uridine 5'-triphosphate (UTP) approximately equal to adenosine 5'-triphosphate (ATP) approximately equal to ATP-gamma-S > uridine 5'-diphosphate approximately equal to adenosine 5'-diphosphate > alpha,beta-methylene ATP >> adenosine. However, only UTP can increase mucin gene (MUC5AC, MUC5B) expression; ATP and other analogues have no stimulatory effect. The stimulation of MUC5AC and MUC5B expression by UTP is time- and dose-dependent. A similar effect on the elevation of mucous cell population in mouse airway epithelium can be demonstrated in vivo by an intratracheal instillation of UTP-saline solution. The stimulatory effect of UTP or ATP on mucin secretion was inhibited by pertussis toxin, U73122, and Calphostin C, but not by PD98059, suggesting a G-protein/ phospholipase (PL) C/protein kinase (PK) C-dependent and mitogen-activated protein kinase (MAPK)-independent signaling pathway. However, the stimulatory effect of UTP on mucin gene expression was sensitive to pertussis toxin and PD98059, but not to Calphostin C and U73122, suggesting a G-protein/MAPK-dependent and PLC/PKC-independent signaling pathway. These findings are the first demonstration that UTP, a pyrimidine nucleotide triphosphate, can enhance both mucin secretion and mucin gene expression through different signaling pathways.
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PMID:Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates. 1169 42

Binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase (PKA) is an essential step in cyclic AMP-mediated intracellular signal transduction. This binding is, however, rapidly inhibited in the acute phase of cerebral ischemia, indicating that the signal transduction via PKA is very vulnerable to ischemia, although this signal pathway is very important for neuronal survival in the brain. Several lines of evidence suggest that the activation of voltage-sensitive Na+ and Ca(2+) channels is an important mediator of acute ischemic brain damage. In the present study, therefore, we examined the effect of a novel Na+ and Ca(2+) channel blocker, NS-7 (4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride), on changes in the binding activity of PKA to cyclic AMP in permanent focal cerebral ischemia, which was induced by occlusion of the middle cerebral artery by the intraluminal suture method for 5 h in the rat. NS-7 (1 mg/kg) or saline was intravenously infused 5 min after occlusion. The binding activity of PKA to cyclic AMP and local cerebral blood flow were assessed by the in vitro [(3)H]cyclic AMP binding and the [(14)C]iodoantipyrine methods, respectively. NS-7 significantly suppressed inhibition of the binding activity of PKA to cyclic AMP in the ischemic regions such as the frontal and parietal cortices and the medial region of the caudate-putamen without affecting cerebral blood flow or arterial blood pressure. Infarct area measured in the brain slices stained with cresyl violet was significantly smaller in animals treated with NS-7 than in those treated with saline. Blockade of voltage-sensitive Na+ and Ca(2+) channels by NS-7 was expected to reduce ischemia-induced depolarization and thus prevent a massive formation of free radicals, which is known to inhibit the binding activity of PKA to cyclic AMP. These data clearly indicate that NS-7 provides very efficient neuroprotection in the acute phase of cerebral ischemia, and sustains the normal function of PKA.
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PMID:A novel voltage-sensitive Na(+) and Ca(2+) channel blocker, NS-7, prevents suppression of cyclic AMP-dependent protein kinase and reduces infarct area in the acute phase of cerebral ischemia in rat. 1174

Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y(1), P2Y(2), and P2Y(4) receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y(6) receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells (vascular smooth muscle cells; VSMC), UDP and UTP stimulated (3)H-labeled thymidine incorporation with similar pEC(50) values (5.96 and 5.69). Addition of hexokinase did not reduce the mitogenic effect of UDP. In cells transfected with P2Y receptors the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDPbetaS) was specific for P2Y(6) with no effect on P2Y(1), P2Y(2), or P2Y(4) receptors. UDPbetaS stimulated [(3)H]thymidine and [(3)H]leucine incorporation and increased cell number in VSMC. Flow cytometry demonstrated that UDP stimulated cell cycle progression to both the S and G(2) phases. The intracellular signal pathways were dependent on phospholipase C, possibly protein kinase C-delta, and a tyrosine kinase pathway but independent of G(i) proteins, eicosanoids, and protein kinase A. The half-life of P2Y(6) receptor mRNA was <1 h by competitive RT-PCR. The mitogen-activated protein kinase kinase inhibitor PD-098059 significantly suppressed, whereas ATP and interleukin-1beta upregulated, expression of P2Y(6) receptor mRNA. The results demonstrate that UDP stimulates mitogenesis through activation of P2Y(6) receptors and that the receptor is regulated by factors important in the development of vascular disease.
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PMID:UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y(6) receptors. 1178 30

The carbamoyl phosphate synthetase domain of the multifunctional protein CAD catalyzes the initial, rate-limiting step in mammalian de novo pyrimidine biosynthesis. In addition to allosteric regulation by the inhibitor UTP and the activator PRPP, the carbamoyl phosphate synthetase activity is controlled by mitogen-activated protein kinase (MAPK)- and protein kinase A (PKA)-mediated phosphorylation. MAPK phosphorylation, both in vivo and in vitro, increases sensitivity to PRPP and decreases sensitivity to the inhibitor UTP, whereas PKA phosphorylation reduces the response to both allosteric effectors. To elucidate the factors responsible for growth state-dependent regulation of pyrimidine biosynthesis, the activity of the de novo pyrimidine pathway, the MAPK and PKA activities, the phosphorylation state, and the allosteric regulation of CAD were measured as a function of growth state. As cells entered the exponential growth phase, there was an 8-fold increase in pyrimidine biosynthesis that was accompanied by a 40-fold increase in MAPK activity and a 4-fold increase in CAD threonine phosphorylation. PRPP activation increased to 21-fold, and UTP became a modest activator. These changes were reversed when the cultures approach confluence and growth ceases. Moreover, CAD phosphoserine, a measure of PKA phosphorylation, increased 2-fold in confluent cells. These results are consistent with the activation of CAD by MAPK during periods of rapid growth and its down-regulation in confluent cells associated with decreased MAPK phosphorylation and a concomitant increase in PKA phosphorylation. A scheme is proposed that could account for growth-dependent regulation of pyrimidine biosynthesis based on the sequential action of MAPK and PKA on the carbamoyl phosphate synthetase activity of CAD.
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PMID:Growth-dependent regulation of mammalian pyrimidine biosynthesis by the protein kinase A and MAPK signaling cascades. 1187 54

CAD, a large multifunctional protein that carries carbamoyl phosphate synthetase (CPSase), aspartate transcarbamoylase, and dihydroorotase activities, catalyzes the first three steps of de novo pyrimidine biosynthesis in mammalian cells. The CPSase component, which catalyzes the initial, rate-limiting step, exhibits complex regulatory mechanisms involving allosteric effectors and phosphorylation that control the flux of metabolites through the pathway. Incubation of CAD with ATP in the absence of exogenous kinases resulted in the incorporation of 1 mol of P(i)/mol of CAD monomer. Mass spectrometry analysis of tryptic digests showed that Thr(1037) located within the CAD CPS.B subdomain was specifically modified. The reaction is specific for MgATP, ADP was a competitive inhibitor, and the native tertiary structure of the protein was required. Phosphorylation occurred after denaturation, further purification of CAD by SDS gel electrophoresis, and renaturation on a nitrocellulose membrane, strongly suggesting that phosphate incorporation resulted from an intrinsic kinase activity and was not the result of contaminating kinases. Chemical modification with the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine, showed that one or both of the active sites that catalyze the ATP-dependent partial reactions are also involved in autophosphorylation. The rate of phosphorylation was dependent on the concentration of CAD, indicating that the reaction was, at least in part, intermolecular. Autophosphorylation resulted in a 2-fold increase in CPSase activity, an increased sensitivity to the feedback inhibitor UTP, and decreased allosteric activation by 5-phosphoribosyl-1-pyrophosphate, functional changes that were distinctly different from those resulting from phosphorylation by either the protein kinase A or mitogen-activated protein kinase cascades.
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PMID:Autophosphorylation of the mammalian multifunctional protein that initiates de novo pyrimidine biosynthesis. 1198 31

Analysis of the role of specific protein kinases in signal transduction networks has relied heavily on ATP analog inhibitors. Currently used agents, however, often do not distinguish between kinase family members. Genetic approaches can also be used to inactivate a specific kinase, but these techniques do not afford the rapid kinetics possible with pharmacological inhibitors. To circumvent this problem, modification of the structure of a particular protein kinase can be performed to engineer a drug-target interaction of choice. We have used this method to create protein kinase A (PKA) catalytic subunits with modifications that confer sensitivity to novel ATP analog inhibitors. Mutation of methionine 120 to alanine or glycine in either the Calpha or Cbeta subunits of PKA induces sensitivity to a series of C-3 derivatized pyrazolo[3,4-d]pyrimidine-based inhibitors. Modification of threonine 183 enhances this inhibitor sensitivity. The IC(50) values in cell culture of the most broadly effective agent, 1-NM, ranged from 25 to 200 nm depending upon the combination of modified amino acids and were significantly higher than the potencies observed with H-89. Despite their high sequence conservation, Cbeta enzymes with inhibitor-sensitive amino acids at position 120 showed a substantial loss of overall catalytic activity when used to induce reporter gene transcription in transfected cells. Conversion of position 46 (lysine to isoleucine) rescued the ability of position 120 mutated Cbeta enzymes to induce gene transcription. Application of this combined genetic and pharmacological approach should allow analysis of the specific roles of PKA isoforms in cell culture and in vivo.
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PMID:Protein engineering of protein kinase A catalytic subunits results in the acquisition of novel inhibitor sensitivity. 1203 35

In the present study, we show that adenosine (Ado) affects vaccinia virus (VV) replication in BSC-40 cells. In order to identify its effects on VV replicative cycle we analyzed the synthesis of virus macromolecules in cells incubated with 0.5 mM Ado. A 50% reduction in the steady-state level of virus DNA synthesis was observed. Consequently, virus post-replicative gene expression was also affected. A prolonged synthesis of the F11L early virus protein was also observed and it is likely related to a slow decline in the steady-state level of early mRNAs, as suggested by northern blot analysis of the VGF early transcript. The involvement of cAMP-signaling pathway as mediator of Ado response was also evaluated. Ado stimulated a three-fold increase in cAMP levels in BSC-40 cells and cAMP-mimetic agents reduced virus yield in a dose-dependent manner. Co-incubation of infected cells with H-89 reduced the inhibitory effects of 8-Br-cAMP and Ado on VV yields suggesting PKA involvement. A(2) receptor-mediated activation of PKA was indicated by antagonism of Ado response by theophylline and DMPX. Together, these results indicate that virus DNA replication is the main target of Ado. The mechanism involved is not related to reduction of the pyrimidine nucleotide synthesis. Furthermore, Ado-induced PKA activation modulates negatively an unidentified step of the virus replicative cycle.
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PMID:Inhibition of vaccinia virus replication by adenosine in BSC-40 cells: involvement of A(2) receptor-mediated PKA activation. 1211 15


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