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)

Splicing factor 1 (SF1) functions at early stages of pre-mRNA splicing and contributes to splice site recognition by interacting with the essential splicing factor U2AF65 and binding to the intron branch site. We have identified an 80 kDa substrate of cGMP-dependent protein kinase-I (PKG-I) isolated from rat brain, which is identical to SF1. PKG phosphorylates SF1 at Ser20, which inhibits the SF1-U2AF65 interaction leading to a block of pre-spliceosome assembly. Mutation of Ser20 to Ala or Thr also inhibits the interaction with U2AF65, indicating that Ser20 is essential for binding. SF1 is phosphorylated in vitro by PKG, but not by cAMP-dependent protein kinase A (PKA). Phosphorylation of SF1 also occurs in cultured neuronal cells and is increased on Ser20 in response to a cGMP analogue. These results suggest a new role for PKG in mammalian pre-mRNA splicing by regulating in a phosphorylation-dependent manner the association of SF1 with U2AF65 and spliceosome assembly.
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PMID:Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly. 1044 20

We tested the hypothesis that protein kinase (PK)G activation in response to nitric oxide ((*)NO) mediates tumor necrosis factor (TNF)-alpha-induced activation of the transcription factor activating protein-1 (AP-1) in pulmonary microvessel endothelial monolayers (PEM). The DNA-binding activity of AP-1 was assessed using the electrophoretic mobility shift assay. TNF treatment (1,000 U/ml) for 4 h induced a significant increase in DNA binding of AP-1. The effects of TNF were prevented by the superoxide radical scavenger superoxide dismutase (SOD) (100 U/ml), the (*)NO synthase inhibitor aminoguanidine (100 microM), the guanylate cyclase inhibitor ODQ (100 microM), and the PKG inhibitors KT5823 (1 microM) and 8-bromo-cyclic guanosine monophosphate (cGMP)-thioate (100 microM). Spermine-NO (1 microM) and L-arginine (400 microM) prevented the aminoguanidine-induced ablation of AP-1 activation in response to TNF. Phosphorylation of H-Arg-Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg-OH (BPDEtide), a specific substrate for PKG, measured the activity of cGMP-dependent protein kinase (PKG). TNF for 0.5 h induced an increase in PKG activity that was prevented by aminoguanidine, ODQ, KT5823, and 8-bromo-cGMP-thioate; however, SOD had no effect. The PKG agonist 8-bromo-cGMP (100 microM), when given alone, increased PKG activity but induced significant DNA-binding activity of AP-1 only when given in the ODQ + TNF Group. SIN-1 (1 mM, a peroxynitrite agonist) increased DNA-binding activity of AP-1. SOD prevented SIN-1-induced AP-1 activation, a response similar to that of the SOD + TNF Group. PEM were transfected with the chloramphenicol acetyltransferase (CAT) reporter plasmid pBLCAT2, which contains a regulation sequence responsive to AP-1. The pharmacologic profile of TNF-induced CAT activity was identical to TNF-induced DNA binding by AP-1. Thus, TNF-induced AP-1-dependent gene transcription is modulated by (*)NO-dependent mediated activation of PKG.
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PMID:Tumor necrosis factor-alpha-induced activating protein-1 activity is modulated by nitric oxide-mediated protein kinase G activation. 1061 72

cGMP-dependent protein kinase type I (cGK I), a major constituent of the atrial natriuretic peptide (ANP)/nitric oxide/cGMP signal transduction pathway, phosphorylates the vasodilator-stimulated phosphoprotein (VASP), a member of the Ena/VASP family of proteins involved in regulation of the actin cytoskeleton. Here we demonstrate that stimulation of human umbilical vein endothelial cells (HUVECs) by both ANP and 8-(4-chlorophenylthio)guanosine 3':5'-monophosphate (8-pCPT-cGMP) activates transfected cGK I and causes detachment of VASP and its known binding partner (zyxin) from focal adhesions in >60% of cells after 30 min. The ANP effects, but not the 8-pCPT-cGMP effects, reversed after 3 h of treatment. In contrast, a catalytically inactive cGK Ibeta mutant (cGK Ibeta-K405A) was incapable of mediating these effects. VASP mutated (Ser/Thr to Ala) at all three of its established phosphorylation sites (vesicular stomatitis virus-tagged VASP-AAA mutant) was not phosphorylated by cGK I and was resistant to detaching from HUVEC focal adhesions in response to 8-pCPT-cGMP. Furthermore, activation of cGK I, but not of mutant cGK Ibeta-K405A, caused a 1.5-2-fold inhibition of HUVEC migration, a dynamic process highly dependent on focal adhesion formation and disassembly. These results indicate that cGK I phosphorylation of VASP results in loss of VASP and zyxin from focal adhesions, a response that could contribute to cGK alteration of cytoskeleton-regulated processes such as cell migration.
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PMID:Regulation of human endothelial cell focal adhesion sites and migration by cGMP-dependent protein kinase I. 1085 Dec 46

For the type I cGMP-dependent protein kinases (cGKIalpha and cGKIbeta), a high affinity interaction exists between the C2 amino group of cGMP and the hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase activation in the type II isozyme of cGMP-dependent protein kinase (cGKII), alanine was substituted for the conserved threonine or serine. cGKII was found to require the C2 amino group of cGMP and its cognate serine or threonine hydroxyl for efficient cGMP activation. Of the two binding sites, disruption of cGMP-specific binding in the NH(2)-terminal binding site had the greatest effect on cGMP-dependent kinase activation, like cGKI. However, ligand dissociation studies showed that the location of the rapid and slow dissociation sites of cGKII was reversed relative to cGKI. Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH(2)-terminal, rapid dissociation binding site for cyclic nucleotide-dependent activation of cGKII. These findings suggest distinct mechanisms of activation for cGKII and cGKI isoforms. Because cGKII mediates the effects of heat-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl(-) channel, these findings define a structural target for drug design.
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PMID:The amino-terminal cyclic nucleotide binding site of the type II cGMP-dependent protein kinase is essential for full cyclic nucleotide-dependent activation. 1086 32

We have previously reported that nitric oxide-related cyclic guanosine-3',5'-monophosphate (GMP) protected spinal nonmotor neurons, but not motor neurons against chronic glutamate-induced toxicity, which is associated with selective motor neuronal death after glutamate stress. In this report, we investigated the effect of cyclic GMP against reactive oxygen species (ROS)-induced toxicity in cultured neurons from embryonic rat spinal cords. Pretreatment with a cGMP analogue, 8-bromoguanosine monophosphate (8br-cGMP), for 12-24 hours protected both spinal motor neurons and nonmotor neurons against injury induced by either hydrogen peroxide (H(2)O(2)), or a glutathione depletor, L-buthionine-[S,R]-sulfoximine (BSO). This protective effect was reversed by coadministration with the cGMP-dependent protein kinase (PKG) inhibitor Arg-Lys-Arg-Ala-Arg-Lys-Glu. Interestingly, when cultures were exposed to BSO for 24 hours to allow irreversible inhibition of glutathione synthesis, 8br-cGMP protected only nonmotor neurons. Our results indicate that cGMP attenuates oxidative injury to cultured spinal neurons, in a mechanism associated with glutathione synthesis.
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PMID:Neuroprotective effect of cyclic GMP against radical-induced toxicity in cultured spinal motor neurons. 1093 31

Small GTPase Rho and cGMP/cGMP-dependent protein kinase (cGK) pathways exert opposing effects in specific systems such as vascular contraction and growth. However, the direct interaction between these pathways has remained elusive. We demonstrate that cGK phosphorylates RhoA in vitro at Ser188, the same residue phosphorylated by cAMP-dependent protein kinase. In HeLa cells transfected with constitutively active cGK (C-cGK), stress fiber formation induced by lysophosphatidic acid or V14RhoA was blocked. By contrast, C-cGK failed to inhibit stress fiber formation in cells transfected with mutant RhoA with substitution of Ser188 to Ala. C-cGK did not affect actin reorganization induced by Rac1 or Rho-associated kinase, one of the effectors for RhoA. Furthermore, C-cGK expression inhibited the membrane translocation of RhoA. Collectively, our findings suggest that cGK phosphorylates RhoA at Ser188 and inactivates RhoA signaling. The physiological relevance of the direct interaction between RhoA and cGK awaits further investigation.
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PMID:cGMP-dependent protein kinase phosphorylates and inactivates RhoA. 1116 91

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

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

Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues.
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PMID:MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro. 1153 58

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


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