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)

In response to formyl-Met-Leu-Phe (fMLP), human neutrophils (PMN) generate superoxide anion (O2-) by the enzyme complex NADPH oxidase. The modulation of phosphoinositide (PPI) turnover and the activation of phospholipases C (PLC) and D (PLD) have been shown to be early steps in the oxidative response of fMLP-stimulated PMN. Although the physiological nonapeptide bradykinin (BK) is involved in inflammation, its participation in PMN activation has not been properly studied. In this work, activation of signal transduction pathways that mediate the oxidative response, and the modulation of the NADPH oxidase activity by BK, are analyzed. A direct comparison between the signal transduction pathway induced by BK and fMLP is also made. BK was not able to elicit O2- production by PMN. Nevertheless, several signal transduction pathways associated with PMN activation were triggered by BK. The nonapeptide induced the phosphorylation of prelabeled membrane PPI. This phenomenon was imitated by PMA and inhibited by H7 and staurosporine, thus suggesting the participation of protein kinase c (PKC). A loss of labeled [32P]PPI was triggered by fMLP. The fact that both PMA and fMLP stimulated O2- production but modulated PPI turnover in different ways, indicates that PPI labeling does not correlate with the oxidative response. Because PKC activation seemed to be a prerequisite for BK-induced modulation of PPI turnover, PLC activation could act as an intermediate step in this mechanism. Our results show that BK activated a PIP2-PLC measured as the release of [3H]IP3. On the contrary, a PC-PLD was highly stimulated by fMLP but not by BK. The fact that BK induced PLC activity but neither that of PLD nor NADPH oxidase, whereas fMLP triggered the activation of both phospholipases and evoked the PMN respiratory burst, suggests that diacylglycerol (DAG) from PIP2 as well as PA or PA-derived DAG, synergize to trigger the PMN oxidative response. Finally, BK inhibited O2- production by fMLP-activated PMN in a time-dependent manner. Since BK did not induce NO production by PMN, the inhibitory effect on the oxidative function was not due to ONOO- formation. These data show that BK plays an important role in inflammation by modulating the PMN function.
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PMID:Bradykinin stimulates phosphoinositide turnover and phospholipase C but not phospholipase D and NADPH oxidase in human neutrophils. 861 9

In the preceding report (Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J. (1996) J. Biol. Chem. 271, 7745-7751), we identified two distinct isoforms of dUTPase in human cells. These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S. (1990) J. Cell. Biochem. 43, 339-353). To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken. [32P]Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated. Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo. Analysis of the wild type and Ser-11 --> Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 --> Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase. The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34(cdc2). Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34(cdc2). Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo.
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PMID:Identification of a consensus cyclin-dependent kinase phosphorylation site unique to the nuclear form of human deoxyuridine triphosphate nucleotidohydrolase. 863 17

In eukaryotes, the guanine nucleotide exchange factor (eIF-2B) is a key protein in the control of polypeptide chain initiation. It catalyzes the exchange of chain initiation factor (eIF)-2-bound GDP for GTP and facilitates the formation of a ternary complex (eIF-2.GTP.Met-tRNAf). The activity of eIF-2B is inhibited indirectly by phosphorylation of the smallest subunit of eIF-2 which sequesters eIF-2B into an inactive eIF-2(alpha P).eIF-2B complex. On the other hand, eIF-2B activity may be regulated directly by covalent modification of its largest subunit with different kinases, such as casein kinase (CK)-I, CK-II and glycogen synthase kinase (GSK)-3. After stimulation of mammalian cells by insulin or growth factors, the allosteric activation of eIF-2B activity by sugar phosphates and inositol phosphates may also provide an important parameter in the regulation of protein synthesis.
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PMID:Regulation of protein synthesis in eukaryotic cells by the guanine nucleotide exchange factor and chain initiation factor 2. 865 27

The activation of the serine/threonine kinase, Raf-1, serves to connect upstream protein tyrosine kinases to downstream signaling events. We previously reported that FcgammaRI stimulation of interferon gamma-differentiated U937 cells (termed U937IF cells) induces a mobility shift in Erk2. Herein, we report that cross-linking of FcgammaRI receptor in U937IF cells induces a marked tyrosine phosphorylation of Raf-1 (10-fold increase). Tyrosine phosphorylation of Raf-1 is induced by FcgammaRI activation and not by PMA (1 microg/ml), N-formyl-Met-Leu-Phe (1 microM), calcium ionophore (1 microM), thrombin (0.05 unit/ml), FcgammaRII, or FcgammaRIII stimulation. The kinetics of Raf-1 tyrosine phosphorylation is rapid, reaching peak levels 1-2 min after FcgammaRI activation, and the tyrosine phosphorylation of Raf-1 precedes the activation of the respiratory burst. FcgammaRI cross-linking induces the tyrosine phosphorylation of Shc; tyrosine-phosphorylated Shc binds to Grb2 forming a Shc-Grb2 complex. The data provide evidence that the FcgammaRI receptor signals via the upstream activation of nonreceptor protein tyrosine kinases, which leads to the subsequent activation of Ras family GTPases and serine/threonine kinases, Raf-1 and mitogen-activated protein kinase.
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PMID:A role for Shc, Grb2, and Raf-1 in FcgammaRI signal relay. 866 46

All known 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes contain a sequence (GX4GK(S/T)) in the 6-phosphofructo-2-kinase domain corresponding to the so-called nucleotide binding fold signature or Walker A motif. Mutagenesis and crystal structure data from several nucleotide binding proteins, which also contain this sequence, showed the importance of the lysine and serine/threonine residues in nucleotide binding. We have studied the role of Lys-54 and Thr-55 in MgATP binding in the 6-phosphofructo-2-kinase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis. Lys-54 was mutated to methionine, whereas Thr-55 was mutated to valine, serine, and cysteine. Three mutants, Lys-54 to Met and Thr-55 to Cys or Val, displayed more than a 5000-fold decrease in 6-phosphofructo-2-kinase activity compared with the wild type. The mutations had no effect on fructose-2, 6-bisphosphatase activity and did not affect the activation of fructose-2,6-bisphosphatase after phosphorylation by cyclic 3', 5'-AMP-dependent protein kinase. Binding experiments with ATP, ADP, and their analogs (3'-N-methylanthraniloyl derivatives) showed that these two residues do not play the same role. Lys-54 is involved in ATP binding, whereas Thr-55 is important for catalysis.
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PMID:The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis. 866 45

Achatin-I (Gly-D-Phe-Ala-Asp), a tetrapeptide having a D-phenylalanine residue and isolated from Achatina ganglia, has been proposed as an excitatory neurotransmitter of Achatina neurones. In the present study, it was demonstrated using Achatina giant neurones that achetin-I, perfused at alow concentration, enhanced an inward current (Iin) caused by 5-hydroxytryptamine (fast component) and an outward current (Iout) caused by FMRFamide (Phe-Met-Arg-Phe-NH2), and that this peptide suppressed an Iin caused by oxytocin, and Iout caused by acetylcholine and APGW-amide (Ala-Pro-Gly-Trp-NH2). These findings indicate that achatin-I acts not only as a neurotransmitter but also as a neuromodulator for these neurones. In the preliminary experiments, it was shown that an Iin caused by achatin-I on an Achatina giant neurone type, PON (periodically oscillating neurone), was suppressed by H-89 (a PKA inhibitor) and W-7 (calmodulin inhibitor), and that an Iin caused by achatin-I on v-RCON (ventral-right cerebral distinct neurone) was suppressed by KT5823 (PKG inhibitor), suggesting that achatin-I acts on PON via the cyclic AMP-PKA system and on v-RCON via the cyclic GMP-PKG system. Moreover, calmodulin would play a role to produce the Iin for achatin-I on PON via the system mentioned.
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PMID:Further study on the effects of achatin-I, an Achatina endogenous neuroexcitatory tetrapeptide having a D-phenylalanine residue, on Achatina neurones. 885 10

Using the reverse transcription polymerase chain reaction (RT-PCR) we found that human neutrophils express mRNA for both A2A and A2B adenosine receptors, and using selective adenosine receptor agonists and antagonists we have characterized the type of adenosine receptor mediating inhibition of formyl-Met-Leu-Phe (fMLP)-induced oxidative burst. The order of potency of agonists was 5'-N-ethyl-carboxamidoadenosine (NECA) > 2-phenylaminoadenosine > 2-[p-(2-carbonyl-ethyl)-phenyl-ethylamino]-5'-N-ethyl-carboxamido adenosine (CGS 21680) > adenosine > N6-cyclopentyl-adenosine. This agrees with the agonist potency at human A2A receptors. The effect of adenosine was antagonized by 30 microM theophylline > caffeine = paraxanthine, i.e. concentrations close to those occurring in plasma after consumption of caffeine-containing beverages. The effect of NECA was unaltered by the A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine, but inhibited by the A2A receptor selective antagonists 4-amino-8-chloro-1-phenyl-[1,2,4]-triazolo[4,3-a]quinoxaline (CP 66,713), 1,3-dipropyl-8-(3,4-dimethoxystyryl) -7-methylxanthine (KF 17387) and 8-(3-chlorostyryl)caffeine as well as by the non-selective, non-xanthine antagonist 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo-[1,5-c]quinazoline methane sulphonate (CGS 15943). The adenosine receptor mediated responses were antagonized by the protein kinase A blocker Rp-cyclic adenosine 3',5'-phosphorothioate (Rp-cAMP). In conclusion, the adenosine-induced inhibition of neutrophil activation is mediated by adenosine A2A receptors.
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PMID:Adenosine A2A receptors mediate the inhibitory effect of adenosine on formyl-Met-Leu-Phe-stimulated respiratory burst in neutrophil leucocytes. 887 55

The 5'-AMP-activated protein kinase (AMPK) mediates several cellular responses to metabolic stress. Rat liver contains at least two isoforms of this enzyme, either alpha1 or alpha2 catalytic subunits together with beta and gamma noncatalytic subunits in a trimeric complex. The alpha1 isoform is purified using a peptide substrate affinity chromatography column with ADR1 (222-234)P229 (LKKLTRRPSFSAQ), corresponding to the cAMP-dependent protein kinase phosphorylation site in the yeast transcriptional activator of the ADH2 gene, ADR1. This peptide is phosphorylated at Ser230 by AMPK alpha1 with a Km of 3.8 microM and a Vmax of 4.8 micromol/min/mg compared to the commonly used rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide substrate, HMRSAMSGLHLVKRR, with a Km of 33.3 microM and a Vmax of 8.1 micromol/min/mg. Thus, the AMPK exhibits some overlapping specificity with the cAMP-dependent protein kinase. The rat liver AMPK alpha1 isoform has a Kcat approximately 250-fold higher than the AMPK alpha2 isoform isolated from rat liver. The AMPK alpha1 isoform readily phosphorylates peptides corresponding to the reported AMPK phosphorylation sites in rat, chicken, and yeast acetyl-CoA carboxylase and rat hydroxymethylglutaryl-CoA reductase but not phosphorylase kinase. Based on previous peptide substrate specificity studies (Dale, S., Wilson, W. A., Edelman, A. M., and Hardie, G. (1995) FEBS Lett. 361, 191-195) using partially purified enzyme and variants of the peptide AMARAASAAALARRR, it was proposed that the AMPK preferred the phosphorylation site motif Phi(X, beta)XXS/TXXXPhi (Phi, hydrophobic; beta, basic). In good AMPK alpha1 peptide substrates, a hydrophobic residue at the P-5 position is conserved but not at the P+4 position. Oxidation of the Met residues in the rat acetyl-CoA carboxylase (73-87)A77R86-87 peptide increased the Km 6-fold and reduced the Vmax to 4% of the reduced peptide.
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PMID:Isoform-specific purification and substrate specificity of the 5'-AMP-activated protein kinase. 891 Apr 70

We studied the effect of the adenylate cyclase activator forskolin, of protein kinase C-activating phorbol esters and of prolonged preganglionic input activation on the inhibitory response of the perfused superior cervical ganglion of the cat to exogenous met-enkephalin (Met-ENK). Met-ENK inhibited, in a concentration-dependent manner, the postganglionic compound action potential evoked by cervical sympathetic trunk stimulation. The inhibition was reversible, was blocked by naloxone as well as by pertussis toxin and showed no homologous desensitization in the concentration range 0.01-10 microM. Pretreatment of the ganglion with 4 beta-phorbol 12,13-dibutyrate or 4 beta-phorbol 12,13-diacetate depressed the Met-ENK response for several hours, while pretreatment with forskolin had no effect. This action of phorbol esters was prevented by the protein kinase inhibitor H-7 but not by the calmodulin antagonist W-7 or the protein kinase A inhibitor HA 1004 and was calcium-dependent. Recovery of the response from the depression produced by phorbol esters was not affected by a protein synthesis inhibitor. A 40 Hz 20 min stimulus train to the cervical sympathetic trunk mimicked the effect of phorbol esters, depressing for several hours the inhibition produced by Met-ENK. Stimulus trains of duration shorter than 5 min or frequency lower than 5 Hz were ineffective. This effect of prolonged preganglionic stimulation occurred even when the stimulus train was delivered during complete block of nicotinic and muscarinic ganglionic transmission but was lost when the stimulus train was delivered during perfusion with calcium-free Krebs. The protein kinase inhibitor H-7 prevented the depression of the Met-ENK response by the train, while W-7 and HA 1004 had no effect. These findings suggest that, in the superior cervical ganglion of the cat, a kinase, activated by phorbol esters and inhibited by H-7, exerts a long-term control of the ganglion cell responsiveness to opiate receptor activation. A similar mechanism can be synaptically activated by a non-cholinergic transmitter, released by the preganglionic axons during prolonged, high frequency, activity.
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PMID:Long-term depression of a sympathetic ganglionic response to opioids by prolonged synaptic activity and by phorbol esters. 896 46

1. The possible mechanisms of the inhibitory effect of ethyl 2-(3-hydroxyanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate (HAJ11) on the respiratory burst of rat neutrophils in vitro was investigated. 2. HAJ11 caused a reversible and a concentration-dependent inhibition of formyl-Met-Leu-Phe (fMLP)-induced superoxide anion (O2.-) generation (IC50 4.9 +/- 0.7 microM) and O2 consumption (IC50 4.9 +/- 1.5 microM). Concanavalin A (Con A)- and NaF-induced O2.- generation were also suppressed by HAJ11. However, HAL11 was a weak inhibitor of the phorbol 12-myristate 13-acetate (PMA)-induced responses. 3. HAJ11 did not scavenge the /2.- generation in the xanthine-xanthine oxidase system and dihydroxyfumaric acid (DHF) autoxidation. 4. HAJ11 showed no activity on fMLP-induced inositol phosphates formation and [Ca2+]i elevation in intact neutrophils. In addition, HAJ11 had no effect on neutrophil cytosolic phospholipase C (PLC) activity. 5. HAJ11 reduced fMLP-induced phosphatidic acid (PA) (IC50 29.1 +/- 6.5 microM) and phosphatidylethanol (PE+) (IC50 22.6 +/- 1.9 microM) formation in a concentration-dependent manner. HAJ11 also reduced protein tyrosine phosphorylation in neutrophils stimulated by fMLP. 6. HAJ11 was a weak inhibitor of neutrophil cytosolic protein kinase C (PKC) activity, and had a negligible effect on brain PKC. Cellular cyclic nucleotides levels were not altered by HAJ11. In addition, HAJ11 did not affect protein kinase A (PKA) activity. 7. HAJ11 had not effect on the O2.- generation of PMA-activated and arachidonic acid (AA)-activated NADPH oxidase preparations. 8. Taken together these results indicate that the inhibition of respiratory burst by HAJ11 probably mainly occurs through inhibition of protein tyrosine phosphorylation and phospholipase D (PLD) activity.
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PMID:Inhibition by HAJ11 of respiratory burst in neutrophils and the involvement of protein tyrosine phosphorylation and phospholipase D activation. 911 3


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