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)

The intracellular signaling pathways involved in human monocyte chemotaxis toward a variety of chemoattractant molecules were evaluated using selected pharmacological agents. Neither phosphatidylinositol-3-kinase (P13K) or extracellular signal-regulated kinase (ERK) activity were required for monocyte migration toward monocyte chemoattractant protein-1 (MCP-1), RANTES (Regulated on Activation, Normal T cell Expressed and Secreted), macrophage inflammatory protein-1alpha (MIP-1alpha) or formyl-Met-Leu-Phe (fMLP), since pretreatment with wortmannin or LY294002, or with PD098059, had no effect on the chemotactic response. Addition of forskolin and IBMX significantly attenuated chemotaxis to each of these chemoattractants and was reversed by co-treatment with Rp-cAMP, a competitive inhibitor of cAMP-dependent protein kinase A. Incubation with the protein kinase C (PKC) inhibitor GF109203X-HCl (GF109) did not affect monocyte migration, but pretreatment of monocytes with PMA significantly impaired the response to each of these chemotactic agents. Inhibition by PMA was reversed by co-treatment with GF109, implying that heterologous PKC activation is capable of desensitizing chemokine and fMLP-induced monocyte chemotaxis. These results help to define the signalling pathways involved in human monocyte chemotaxis and suggest pharmacological approaches to evaluating the cross-desensitization of chemoattractant-induced leukocyte migration.
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PMID:Evaluation of signal transduction pathways in chemoattractant-induced human monocyte chemotaxis. 1132 60

A novel class of chemical microchips consisting of glass microscope slides was prepared for the covalent attachment of small molecule ligands and peptides through site-specific oxime bond or thiazolidine ring ligation reaction. Commercially available microscope slides were thoroughly cleaned and derivatized with (3-aminopropyl)triethoxysilane (APTES). The amino slides were then converted to glyoxylyl derivatives via two different routes: (1) coupling of Fmoc-Ser followed by deprotection and oxidation, or (2) coupling with protected glyoxylic acid and final deprotection with HCl. Biotin or peptide ligands derivatized at the carboxyl terminus with a 4,7,10-trioxa-1,13-tridecanediamine succinimic acid linker and an amino-oxy group or a 1,2-amino-thiol group (e.g., cysteine with a free N(alpha)-amino group) were printed onto these slides using a DNA microarray spotter. After chemical ligation, the microarray of immobilized ligands was analyzed with three different biological assays: (1) protein-binding assay with fluorescence detection, (2) functional phosphorylation assay using [gamma(33)P]-ATP and specific protein kinase to label peptide substrate spots, and (3) adhesion assay with intact cells. In the cell adhesion assay, not only can we determine the binding specificity of the peptide against different cell lines, we can also determine functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip. This chemical microchip system enables us to rapidly analyze the functional properties of numerous ligands that we have identified from the "one-bead one-compound" combinatorial library method.
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PMID:Peptide and small molecule microarray for high throughput cell adhesion and functional assays. 1135 31

Gastric gland stimulation triggers H(+),K(+)-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLO-permeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H(+),K(+)-ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H(+),K(+)-ATPase-containing tubulovesicles to apical plasma membrane.
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PMID:Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model. 1175 Nov 54

Lasp-1 has been identified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in pancreas, intestine and gastric mucosa and is selectively expressed in cells within epithelial tissues. In the gastric parietal cell, cAMP-dependent phosphorylation induces the partial translocation of lasp-1 to the apically directed F-actin-rich canalicular membrane, which is the site of active HCl secretion. Lasp-1 is an unusual modular protein that contains an N-terminal LIM domain, a C-terminal SH3 domain and two internal nebulin repeats. Domain-based analyses have recently categorized this protein as an epithelial representative of the nebulin family, which also includes the actin binding, muscle-specific proteins, nebulin, nebulette and N-RAP. In this study, we show that lasp-1 binds to non-muscle filamentous (F) actin in vitro in a phosphorylation-dependent manner. In addition, we provide evidence that lasp-1 is concentrated within focal complexes as well as in the leading edges of lamellipodia and the tips of filopodia in non-transformed gastric fibroblasts. In actin pull-down assays, the apparent K(d) of bacterially expressed his-tagged lasp-1 binding to F-actin was 2 micro M with a saturation stoichiometry of approximately 1:7. Phosphorylation of recombinant lasp-1 with recombinant PKA increased the K(d) and decreased the B(max) for lasp-1 binding to F-actin. Microsequencing and site-directed mutagenesis localized the major in vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to S(99) and S(146). BLAST searches confirmed that both sites are conserved in human and chicken homologues. Transfection of lasp-1 cDNA encoding for alanine substitutions at S(99) and S(146), into parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1 to the intracellular canalicular region. In gastric fibroblasts, exposure to the protein kinase C activator, PMA, was correlated with the translocation of lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC, these data suggest that other mechanisms in addition to cAMP-dependent phosphorylation can mediate the translocation of lasp-1 to regions of dynamic actin turnover. The localization of lasp-1 to these subcellular regions under a range of experimental conditions and the phosphorylation-dependent regulation of this protein in F-actin rich epithelial cells suggests an integral and possibly cell-specific role in modulating cytoskeletal/membrane-based cellular activities.
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PMID:Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo. 1243 67

Protein phosphorylation is a major mechanism for regulation of N-methyl-D-aspartate (NMDA) receptor function. The NMDA receptor 1 subunit (NR1) is phosphorylated by protein kinase A (PKA) on serine 890 and 897. We have recently reported that there is enhanced phosphorylation of NR1 on serine 897 in dorsal horn and spinothalamic tract (STT) neurons after intradermal injection of capsaicin (CAP) in rats [Zou et al. (2000) J. Neurosci. 20, 6989-6997]. Whether or not this phosphorylation, which develops during central sensitization following CAP injection, is mediated by PKA remains to be determined. In this study, western blots and immunofluorescence staining were employed to observe if pretreatment with a PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H89), blocks the enhanced phosphorylation of NR1 on serine 897 following injection of CAP into the glabrous skin of one hind paw of anesthetized rats. Western blots showed that pretreatment with H89 caused a decrease in CAP-induced phosphorylation of NR1 protein in spinal cord segments L(4)-S(1). In experiments using immunofluorescence staining, the numbers of phospho-NR1-like immunoreactive (p-NR1-LI) neurons seen after CAP injection were significantly decreased in the dorsal horn of the L(4)-L(5) segments on the side ipsilateral to the injection after PKA was inhibited. When STT cells were labeled by microinjection of the retrograde tracer, fluorogold, we found that the proportion of p-NR1-LI STT cells on the side ipsilateral to the injection in the superficial laminae of spinal cord segments L(4)-L(5) was markedly reduced when H89 was administered intrathecally before CAP injection. However, the proportion of p-NR1-LI STT cells in deep laminae was unchanged unless the PKC inhibitor, chelerythrine chloride, was co-administered with H89. Combined with our previous findings, the present results indicate that NR1 in spinal dorsal horn neurons, including the superficial dorsal horn STT cells, is phosphorylated following CAP injection and that this phosphorylation is due to the action of PKA. However, the phosphorylation of deep STT cells involves both PKA and PKC.
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PMID:Role of protein kinase A in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats. 1243 16

We examined the effect of several protein kinase inhibitors, such as staurosporine for protein kinase C (PKC), H-89 for protein kinase A (PKA) and genistein for tyrosine kinase (TK) on acid-induced duodenal bicarbonate secretion (DBS) in rats. HCO(-)(3) secretion was measured using the pH-stat method. Mucosal acidification was performed by perfusing the duodenal loop for 10 min with pH 2.2 HCl. Indomethacin, staurosporine and genistein were added to acidified saline and then perfused, respectively. In some cases, genistein and phorbol 12-myristate 13-acetate (PMA) were added to the luminal solution to examine the effect on basal duodenal HCO(-)(3) secretion. PGE(2) (PKA pathway) and PMA (PKC pathway) stimulate basal DBS. Indomethacin, H-89, staurosporine and genistein inhibit acid-induced DBS, indicating involvement of the cyclooxygenase, PKA, PKC and TK pathways.
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PMID:Role of protein kinases on acid-induced duodenal bicarbonate secretion in rats. 1256 54

It has been suggested that the cannabinoid receptor type 1 (CB1), a G protein-coupled receptor, is internalized after agonist binding and activation of the second messenger pathways. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor, thus contributing to tolerance. Alterations in phosphorylation of proteins in the signal transduction cascade after CB1receptor activation could also alter tolerance to cannabinoids. We addressed our hypothesis by evaluating the role of several kinases in antinociceptive tolerance to Delta(9)-tetrahydrocannabinol (THC). We evaluated cAMP-dependent protein kinase (PKA) using KT5720, a PKA inhibitor; protein kinase C (PKC) using bisindolylmaleimide I, HCl (bis), a PKC inhibitor; cGMP-dependent protein kinase (PKG) using KT5823, a PKG inhibitor; beta-adrenergic receptor kinase (beta-ARK) using low molecular weight heparin (LMWH), a beta-ARK inhibitor; and phosphatidylinositol-3 kinase (PI3-K) using 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI3-K inhibitor and PP1, a Src family tyrosine kinase inhibitor. The cAMP analog used was dibutyryl-cAMP and the cGMP analog used was dibutyryl-cGMP. Our data indicate that selective kinases may be involved in cannabinoid tolerance. Mice and rats were rendered tolerant to Delta(9)-THC. The PKG inhibitor KT5823, the beta-ARK inhibitor LMWH, the PI3-K inhibitor LY294002, and inhibition of PKC by bis had no effect on tolerance. At a higher dose, bis attenuated the antinociceptive effect of delta(9)-THC in nontolerant mice. PP1, the Src family tyrosine kinase inhibitor, and KT5720, the PKA inhibitor, reversed THC-induced tolerance. In addition, inhibition of PKA reversed a decrease in dynorphin release shown to accompany THC tolerance in rats. These data support a role for PKA and Src tyrosine kinase in phosphorylation events in delta(9)-THC-tolerant mice.
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PMID:The role of several kinases in mice tolerant to delta 9-tetrahydrocannabinol. 1260 57

When capsaicin, the pungent compound in hot pepper, is applied to epithelia it produces pain, allodynia, and hyperalgesia. We investigated, using whole cell path clamp, whether some of these responses induced by capsaicin could be a consequence of capsaicin blocking I(A) currents, a reduction in which, such as occurs in injury, increases neuronal excitability. In capsaicin-sensitive (CS) rat trigeminal ganglion (TG) neurons, capsaicin inhibited I(A) currents in a dose-dependent manner. I(A) currents were reduced 49% by 1 microM capsaicin. In capsaicin-insensitive (CIS) rat TG neurons, or small-diameter mouse VR1-/- neurons, 1 microM capsaicin inhibited I(A) currents 9 and 3%, respectively. These data suggest that in CS neurons the vast majority of the capsaicin-induced inhibition of I(A) currents occurs as a consequence of the activation of vanilloid receptors. Capsaicin (1 microM) did not alter the I(A) conductance-voltage relationship but shifted the inactivation-voltage curve about 15 mV to hyperpolarizing voltages, thereby increasing the number of inactivated I(A) channels at the resting potential. I(A) currents were relatively unaffected by 1 mM CTP-cAMP or 500 nM phorbol-12, 13-dibuterate (a protein kinase C agonist) but were inhibited by 20-30% with either 1 mM CTP-cGMP or 25 microM N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide HCl (a calcium-calmodulin kinase inhibitor). In the presence of 0.5 microM KT5823, an inhibitor of protein kinase G (PKG) pathways, 1 microM capsaicin inhibited I(A) by only 26%. In summary, in CS neurons, capsaicin decreases I(A) currents through the activation of vanilloid receptors. That activation, partially through the activation of cGMP-PKG and calmodulin-dependent pathways should result in increased excitability of capsaicin-sensitive nociceptors.
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PMID:Modulation of IA currents by capsaicin in rat trigeminal ganglion neurons. 1262 18

Our previous in vitro microperfusion studies established that dopamine inhibits sodium chloride transport in the rat medullary thick ascending limb. The present study was designed to determine the intracellular signaling pathway mediating this response. The dopamine D1 receptor agonist fenoldopam (1 microM) inhibited sodium chloride transport in the thick ascending limb by 42+/-5%. The dopamine D1 receptor antagonist R-(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine-HCl (SCH-23390) completely blocked this effect of fenoldopam. Suppression of protein kinase A activity using either myristoylated protein kinase inhibitor (PKI) or N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89), as well as suppression of phospholipase C activity using 1-(6-((17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), had no effect on fenoldopam-dependent inhibition of transport. In contrast, inhibition of phospholipase A2 activity using E-6-(Bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) significantly attenuated the effect of fenoldopam by 74%. The cytochrome P-450 monooxygenase inhibitor 17-octadecynoic acid (17-ODYA) and the protein kinase C inhibitor staurosporine both significantly attenuated the effects of fenoldopam by 67%. Exposure to 20-Hydroxy-(5Z, 8Z, 11Z, 14Z)-eicosatetraenoic acid (20-HETE) inhibited transport by 31+/-5%, and this effect was significantly attenuated by 66% in the presence of staurosporine. We propose a signaling pathway in which dopamine activates a calcium-independent phospholipase A2 in the medullary thick ascending limb. Released arachidonic acid is then metabolized to 20-HETE which subsequently increases protein kinase C activity that acts as a final transport effector.
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PMID:Dopamine D1 receptor-dependent inhibition of NaCl transport in the rat thick ascending limb: mechanism of action. 1289 37

In humans, thromboxane A2 signals through two thromboxane A2 receptor (TP) isoforms termed TP alpha and TP beta. Signaling by TP alpha, but not TP beta, is subject to prostacyclin-induced desensitization mediated by direct protein kinase (PK) A phosphorylation where Ser329 represents the phosphotarget (Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412-20423). In the current study, the effect of the vasodilator nitric oxide (NO) on intracellular signaling by the TP isoforms was investigated. The NO donor 3-morpholinosydnonimine, HCl (SIN-1) and 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) functionally desensitized U46619-mediated calcium mobilization and inositol 1,4,5-trisphosphate generation by TP alpha whereas signaling by TP beta was unaffected by either agent. NO-mediated desensitization of TP alpha signaling occurred through a PKG-dependent, PKA- and PKC-independent mechanism. TP alpha, but not TP beta, was efficiently phosphorylated by PKG in vitro and underwent NO/PKG-mediated phosphorylation in whole cells. Deletion/site-directed mutagenesis and metabolic labeling studies identified Ser331 as the target residue of NO-induced PKG phosphorylation of TP alpha. Although TP alpha S331A was insensitive to NO/PKG-desensitization, similar to wild type TP alpha its signaling was fully desensitized by the prostacyclin receptor agonist cicaprost occurring through a PKA-dependent mechanism. Conversely, signaling by TP alpha S329A was insensitive to cicaprost stimulation whereas it was fully desensitized by NO/PKG signaling. In conclusion, TP alpha undergoes both NO- and prostacyclin-mediated desensitization that occur through entirely independent mechanisms involving direct PKG phosphorylation of Ser331, in response to NO, and PKA phosphorylation of Ser329, in response to prostacyclin, within the unique carboxyl-terminal tail domain of TP alpha. On the other hand, signaling by TP beta is unaffected by either NO or prostacyclin.
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PMID:The alpha, but not the beta, isoform of the human thromboxane A2 receptor is a target for nitric oxide-mediated desensitization. Independent modulation of Tp alpha signaling by nitric oxide and prostacyclin. 2761 56


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