Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.12 (PKG)
 2,515 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the dynamics of guanosine 3',5'-cyclic monophosphate (cGMP) in single living cells, we constructed genetically encoded, fluorescent cGMP indicators by bracketing cGMP-dependent protein kinase (cGPK), minus residues 1-77, between cyan and yellow mutants of green fluorescent protein. cGMP decreased fluorescence resonance energy transfer (FRET) and increased the ratio of cyan to yellow emissions by up to 1.5-fold with apparent dissociation constants of approximately 2 microM and >100:1 selectivity for cGMP over cAMP. To eliminate constitutive kinase activity, Thr(516) of cGPK was mutated to Ala. Emission ratio imaging of the indicators transfected into rat fetal lung fibroblast (RFL)-6 showed cGMP transients resulting from activation of soluble and particulate guanylyl cyclase, respectively, by nitric oxide (NO) and C-type natriuretic peptide (CNP). Whereas all naive cells tested responded to CNP, only 68% responded to NO. Both sets of signals showed large and variable (0.5-4 min) latencies. The phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not elevate cGMP on its own but consistently amplified responses to NO or CNP, suggesting that basal activity of guanylate cyclase is very low and emphasizing the importance of PDEs in cGMP recycling. A fraction of RFL cells showed slowly propagating tides of cGMP spreading across the cell in response to delocalized application of NO. Biolistically transfected Purkinje neurons showed cGMP responses to parallel fiber activity and NO donors, confirming that single-cell increases in cGMP occur under conditions appropriate to cause synaptic plasticity.
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PMID:Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. 1122 57

Imidazoline compounds have been considered for the treatment of type 2 diabetes. We have now investigated the effects of imidazolines on interleukin (IL)-1beta-induced beta-cell apoptosis and the signal transduction pathways involved. Inhibition of Ca2+ influx into beta-cells by D-600, a blocker of voltage-gated L-type Ca2+ channels, suppressed IL-1beta-induced apoptosis. Our data show that calcineurin, Ca2+/calmodulin-dependent serine/threonine protein phosphatase 2B, is responsible for the effect of Ca2+ on beta-cell apoptosis. We also demonstrate that IL-1beta-mediated apoptosis correlates with expression of inducible nitric oxide synthase (iNOS) and the increase in intracellular production of nitric oxide. An inhibitor of cGMP-dependent protein kinase (PKG), KT5823, suppressed IL-1beta-induced apoptosis, suggesting the involvement of a PKG-dependent pathway in the apoptotic process. One of the major findings in this study is that imidazoline compounds RX871024 and efaroxan, suggested as prototypes of a new generation of drugs against type 2 diabetes, can protect against IL-1beta-induced apoptosis in pancreatic beta-cells, possibly by their inhibition of the expression of iNOS, a key element in the IL-1beta-induced apoptotic pathway in pancreatic beta-cells. These data suggest that imidazoline compounds should be explored as a potential therapeutic agent for the treatment of both type 1 and type 2 diabetes.
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PMID:Imidazoline compounds protect against interleukin 1beta-induced beta-cell apoptosis. 1127 6

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.
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PMID:Heat shock protein 27 is a substrate of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin polymerization caused by HSP27 mutants. 1138 10

Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.
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PMID:Serine 331 is the major site of receptor phosphorylation induced by agents that activate protein kinase G in HEK 293 cells overexpressing thromboxane receptor alpha. 1151 66

The recently discovered 3'-phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase which phosphorylates several members of the conserved AGC kinase superfamily (comprising the prototypes protein kinases A (PKA), G (PKG) and C (PKC)). Phosphorylation of a threonine or serine residue in the activation loop (also known as the T-loop) of these kinases is a critical step in their activation, and is typically accompanied by additional phosphorylations elsewhere in the molecule. Phosphorylation of the activation loop is a common regulatory mechanism shared by most serine/threonine as well as tyrosine kinases as it facilitates alignment of amino acid residues in the active site which are involved in the phosphotransferase reaction. Therefore the discovery of PDK-1 as the enzyme which mediates this event in many protein kinases introduced a new and important step in signaling pathways which regulate numerous important cellular processes including cellular survival, glucose transport and metabolism, tumor progression as well as protein translation. Moreover, the finding that PDK-1 function is mediated in part by the phosphoinositide 3'-OH-kinase (PI 3-K) pathway also provided an explanation as to how the lipid products of PI 3-K, namely phosphatidylinositol-3,4-bisphosphate (PtdIns-3,4-P2) and phosphatidylinositol-3,4-5-trisphosphate (PtdIns-3,4,5-P3) stimulate the activation of protein kinase-dependent signaling pathways. These initial landmark observations were followed by many important studies which provided additional mechanistic insight into both PDK-1 regulation as well as the role of this kinase in cellular function. This review will focus on the regulation of PDK-1 and the various mechanisms which it uses to contribute to the activation of target kinases.
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PMID:3'-phosphoinositide-dependent kinase-1 (PDK-1) in PI 3-kinase signaling. 1189 68

We have evaluated the importance of the Ser/Thr protein phosphorylation and dephosphorylation for chondrogenesis in high-density chicken limb bud mesenchymal cell cultures (HDCs) by using H89, a cell-permeable protein kinase inhibitor, and okadaic acid (OA), a phosphoprotein phosphatase (PP)-specific inhibitor molecule. When 20 nM OA was applied to the HDCs on Days 2 and 3 of culturing, it significantly inhibited protein phosphatase 2A (PP2A), enhanced cartilage formation, and elevated the activity of cAMP-dependent protein kinase (PKA). Application of 20 microM H89 significantly decreased the activity of PKA and blocked the chondrogenesis in HDCs. Furthermore, OA enhanced cartilage formation and elevated the suppressed activity of PKA even in the H89-pretreated HDCs. cGMP-dependent protein kinase was not detected in HDCs, while protein kinase Cmu (PKCmu), which is also inhibited by nanomolar concentrations of H89, was present throughout the culturing period. Neither OA nor H89 influenced the expression of the catalytic subunit of PKA or the cAMP response element binding protein, CREB. However, a significantly elevated amount of Ser-133-phosphorylated-CREB (P-CREB) was detected following addition of OA, while H89 treatment resulted in a decrease of the amount of P-CREB. Our results demonstrate that PP2A plays a role in the regulation of the PKA signaling pathway and that the phosphorylation level of CREB is influenced by the activity of both enzymes during in vitro chondrogenesis.
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PMID:Protein phosphatase 2A is involved in the regulation of protein kinase A signaling pathway during in vitro chondrogenesis. 1192

The human protein kinase X gene (PRKX) is a member of an ancient family of cAMP-dependent serine/threonine kinases here shown to be phylogenetically distinct from the classical PKA, PKB/Akt, PKC, SGK, and PKG gene families. Renal expression of the PRKX gene is developmentally regulated and restricted to the ureteric bud epithelium of the fetal metanephric kidney. Aberrant adult kidney expression of PRKX was found in autosomal dominant polycystic kidney disease. PRKX kinase expression markedly activated migration of cultured renal epithelial cells in the presence of cAMP; this effect was blocked by cell treatment with the PKA inhibitor H89 and was not observed in PKA-transfected cells. In addition, expression of PRKX kinase activated branching morphogenesis of Madin-Darby canine kidney cells in collagen gels even in the absence of cAMP and/or hepatocyte growth factor, an effect not seen with either PKA expression or expression of a mutant, kinase-inactivated PRKX. These results suggest that the PRKX kinase may regulate epithelial morphogenesis during mammalian kidney development. Because another member of the PRKX gene family (the Dictyostelium discoideum gene KAPC-DICDI) also plays a role in cellular migration, these studies suggest that regulation of morphogenesis may be a distinctive property of these genes that has been conserved in evolution that is not shared with PKA family genes.
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PMID:PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis. 1208 74

The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.
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PMID:Activation of cGMP-dependent protein kinase by protein kinase C. 1260 95

The slow EPSP (sEPSP) or slow EPSC (sEPSC) at parallel fiber to Purkinje neuron synapses is attributable to a nonselective cation channel coupled to activation of metabotropic type 1 glutamate receptors (mGluR1s). Photorelease of L-glutamate in 1 msec from 4-methoxy-7-nitroindolinyl-or 7-nitroindolinyl-caged glutamate in cerebellar slices was used to isolate and study postsynaptic mechanisms coupling mGluR1 to the cation channel. L-Glutamate immediately activated a glutamate transporter current, followed by the slow mGluR1-activated conductance. Inhibitors of kinases, phosphatases, and G-proteins were tested on the peak glutamate-evoked currents. No effects of the inhibitors were seen on the initial glutamate transporter currents. In contrast, the later mGluR1 currents were either unaffected or enhanced by the protein tyrosine kinase (PTK) inhibitors PP1, K252a, and staurosporine were diminished or blocked by phosphatase inhibitors but were unaffected by inhibitors of serine-threonine kinases PKA, PKC, or PKG. The selective src-PTK inhibitor PP1 (10 microm intracellularly) potentiated submaximal mGluR1 currents evoked by low L-glutamate concentrations but had no effect on maximal responses (80 or 160 microm L-glutamate). L-Glutamate-evoked mGluR1 currents and parallel fiber sEPSCs were reversibly and completely inhibited by protein tyrosine phosphatase (PTP) inhibitor bpV(phen) (50-200 microm) and by nonselective phosphatase inhibitor orthovanadate (0.5 or 1 mm). mGluR1 currents were completely inhibited by GDPbetaS applied intracellularly (5 mm). The results confirm a role for a GTPase postsynaptically, show that tyrosine phosphorylation inhibits mGluR1 coupling to the channel, and show that PTPs increase activation by tyrosine dephosphorylation most likely upstream of the sEPSP cation channel.
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PMID:Evidence for protein tyrosine phosphatase, tyrosine kinase, and G-protein regulation of the parallel fiber metabotropic slow EPSC of rat cerebellar Purkinje neurons. 1276 93

Ena/VASP proteins are a conserved family of actin regulatory proteins made up of EVH1, EVH2 domains, and a proline-rich central region. They have been implicated in actin-based processes such as fibroblast migration, axon guidance, and T cell polarization and are important for the actin-based motility of the intracellular pathogen Listeria monocytogenes. Mechanistically, these proteins associate with barbed ends of actin filaments and antagonize filament capping by capping protein (CapZ). In addition, they reduce the density of Arp2/3-dependent actin filament branches and bind Profilin at sites of actin polymerization. Vertebrate Ena/VASP proteins are substrates for PKA/PKG serine/threonine kinases. Phosphorylation by these kinases appears to modulate Ena/VASP function within cells, although the mechanism underlying this regulation remains to be determined.
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PMID:Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. 1457 May 81


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