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

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

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca(2+)] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca(2+) by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca(2+). In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-beta2 and PLC-beta3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-beta3 by PKG (Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PLC-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca(2+)] by the NO-cGMP-dependent signaling pathway.
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PMID:Phosphorylation and regulation of G-protein-activated phospholipase C-beta 3 by cGMP-dependent protein kinases. 1127 98

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

Protein kinase G-I (PKG-I) activation is essential for vascular relaxation; however, its quantitative analysis in intact cells has been difficult. To overcome this difficulty, a monoclonal antibody, VASP-16C2, was recently developed that detects phosphorylated serine residue 239 of vasodilator-stimulated phosphoprotein (VASP), a substrate of PKG-I. In this study, we used this antibody to examine (i) possible functional differences between the alpha and beta isoforms of PKG-I, (ii) ability of cAMP to activate PKG-I, as compared to cGMP, the principal PKG-I-activating cyclic nucleotide, and (iii) time course and levels of PKG-I activation in vascular smooth muscle cells (VSMC) of young and old rats. We created COS-7 cell clones that overexpressed PKG-Ialpha or PKG-Ibeta, treated them with cAMP or cGMP, and analyzed their cell lysates for reactivity with VASP-16C2. The results showed that PKG-Ialpha phosphorylated VASP at a higher level than PKG-Ibeta, and cAMP was slightly weaker than cGMP in PKG-I activation. VSMC of young rats responded to cAMP or cGMP stimulation in a dose-dependent manner with increasing levels of PKG-I activation. The response was detected within 10 min and continued for at least 24 h. In contrast, VSMC of old rats showed no PKG-I activation during the first hour of cAMP or cGMP stimulation and, at 24 h these cells showed only low-level PKG-I activation. We propose that the reduced PKG-I activation may explain why vascular relaxation is decreased in older individuals.
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PMID:Age-related decrease of protein kinase G activation in vascular smooth muscle cells. 1154 81

Potassium channels activated by membrane stretch may contribute to maintenance of relaxation of smooth muscle cells in visceral hollow organs. Previous work has identified K(+) channels in murine colon that are activated by stretch and further regulated by NO-dependent mechanisms. We have screened murine gastrointestinal, vascular, bladder, and uterine smooth muscles for the expression of TREK and TRAAK mRNA. Although TREK-1 was expressed in many of these smooth muscles, TREK-2 was expressed only in murine antrum and pulmonary artery. TRAAK was not expressed in any smooth muscle cells tested. Whole cell currents from TREK-1 expressed in mammalian COS cells were activated by stretch, and single channel recordings showed that the stretch-dependent conductance was due to 90 pS channels. Sodium nitroprusside (10(-6) or 10(-5) m) and 8-Br-cGMP (10(-4) or 10(-3) m) increased TREK-1 currents in perforated whole cell and single channel recordings. Mutation of the PKG consensus sequence at serine 351 blocked the stimulatory effects of sodium nitroprusside and 8-Br-cGMP on open probability without affecting the inhibitory effects of 8-Br-cAMP. TREK-1 encodes a component of the stretch-activated K(+) conductance in smooth muscles and may contribute to nitrergic inhibition of gastrointestinal muscles.
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PMID:TREK-1 regulation by nitric oxide and cGMP-dependent protein kinase. An essential role in smooth muscle inhibitory neurotransmission. 1156 Sep 40

Cyclic AMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are autoinhibited through multiple interactions between their respective regulatory and catalytic domains. A large portion of this autoinhibition occurs through interactions between residues within the catalytic domain and those within either a substrate-like sequence (-RRXSX-) or pseudosubstrate sequence (-RRXAX-) in the regulatory domains. These contacts effectively inhibit catalysis by blocking substrate binding. Particularly important contacts involve the P-2, P-3, and P+1 residues where either serine, which is potentially autophosphorylated, or alanine occupies the P0 position. The primary sequence is apparently less important for autoinhibition in PKGs than in PKAs, and a conserved serine at P+2 in PKGs is important for autoinhibitory contacts. Elements outside the substrate-related sequences also contribute to autoinhibition in both PKA and PKG. For example, synthetic peptides with relatively short pseudosubstrate sequences are weak inhibitors; while heat-denatured RII subunit does not inhibit catalytic subunit, it is still rapidly autophosphorylated; and truncated PKGs lacking the substrate-like sequence are still partially autoinhibited. Thus, capacity for autoinhibition of PKA or PKG is provided by contacts involving direct interactions with the catalytic site and by contacts that stabilize an inactive conformation.
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PMID:Mechanisms of autoinhibition in cyclic nucleotide-dependent protein kinases. 1186 Dec 9

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

Presynaptic metabotropic glutamate receptors (mGluRs) often act as feedback inhibitors of synaptic transmission and serve important roles in defining the activity of glutamatergic synapses. Recent investigations have begun to identify novel interactions of presynaptic mGluRs, especially mGluR7, with multiple protein kinases and putative regulatory proteins that probably serve to further shape the overall activity of glutamatergic synapses. In the present study, we report that in addition to protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) can inhibit calmodulin (CaM) interactions with the carboxyl-terminal tail of mGluR7. These actions are mediated by PKC-, PKA-, or PKG-dependent phosphorylation of mGluR7 at a single serine residue, Ser(862), in the carboxyl terminus of the receptor. Mutation of this residue inhibits kinase-mediated phosphorylation of the mGluR7 carboxyl terminus and reverses kinase-mediated inhibition of CaM binding to mGluR7. However, PKC-mediated inhibition of the functional coupling of mGluR7 to G protein-coupled inward rectifier potassium (GIRK) currents in a heterologous expression system is not affected by mutating Ser(862). Furthermore, mutation of Ser(862) to glutamate to mimic receptor phosphorylation and inhibit CaM interactions with mGluR7 does not affect receptor function. These studies demonstrate that the ability of these second messenger-dependent kinases to inhibit mGluR7-mediated activation of GIRK current is not dependent on the phosphorylation of Ser(862) or the regulation of CaM binding to mGluR7. Furthermore, our studies suggest that CaM binding is not required for mGluR7-mediated activation of GIRK current.
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PMID:Dissociation of protein kinase-mediated regulation of metabotropic glutamate receptor 7 (mGluR7) interactions with calmodulin and regulation of mGluR7 function. 1202 91

Serines 64 and 79 are homologous residues that are juxtaposed to the autoinhibitory pseudosubstrate site in cGMP-dependent protein kinase type Ialpha and type Ibeta (PKG-Ialpha and PKG-Ibeta), respectively. Autophosphorylation of this residue is associated with activation of type I PKGs. To determine the role of this conserved serine, point mutations have been made in PKG-Ialpha (S64A, S64T, S64D, and S64N) and PKG-Ibeta (S79A). In wild-type PKG-Ialpha, basal kinase activity ratio (-cGMP/+cGMP) is 0.11, autophosphorylation increases this ratio 3-fold, and the K(a) and K(D) values for cGMP are 127 and 36 nm, respectively. S64A PKG-Ialpha basal kinase activity ratio increases 2-fold, cGMP binding affinity increases approximately 10-fold in both K(a) and K(D), and activation by autophosphorylation is slight. S64D and S64N mutants are nearly constitutively active in the absence of cGMP, cGMP binding affinity in each increases 18-fold, and autophosphorylation does not affect the kinase activity of these mutants. Mutation of the homologous site in PKG-Ibeta (S79A) increases the basal kinase activity ratio 2-fold and cGMP binding affinity 5-fold over that of wild-type PKG-Ibeta. The combined results demonstrate that a conserved serine juxtaposed to the pseudosubstrate site in type I PKGs contributes importantly to enzyme function by increasing autoinhibition and decreasing cGMP binding affinity.
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PMID:A conserved serine juxtaposed to the pseudosubstrate site of type I cGMP-dependent protein kinase contributes strongly to autoinhibition and lower cGMP affinity. 1208 49

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


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