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)

cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I alpha and I beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI alpha and rI beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI alpha and rI beta had a molecular mass of about 75 kDa. rI alpha and rI beta had S20,W values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI alpha, and P-II and rI beta, were immunologically indistinguishable. P-I, P-II, rI alpha and rI beta had the same catalytic activity. However, rI alpha and rI beta were half-maximally activated at 0.1 microM and 1.3 microM cGMP, and 0.3 microM and 12 microM 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent KA values. P-I and rI alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI beta bound and rI beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I alpha amino-terminal domain to that of I beta increases the apparent KA value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.
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PMID:The activation of expressed cGMP-dependent protein kinase isozymes I alpha and I beta is determined by the different amino-termini. 166 12

Polymerase chain reaction (PCR) methodology and cDNA library screening were used to isolate a cDNA clone encoding a cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE) from bovine lung. Degenerate oligonucleotides based on cGB-PDE peptide sequences were used as primers for a PCR reaction with bovine lung cDNA as the template. An 824-base pair PCR product was recovered and used as a probe to screen a bovine lung cDNA library. A 4.5-kilobase pair cDNA clone encoding a full-length cGB-PDE was isolated. The open reading frame of this cDNA predicted an 875 amino acid (AA), 99,525-Da polypeptide. By Northern analysis, the cGB-PDE cDNA hybridized to a single lung 6.9-kilobase mRNA. The identity of the cGB-PDE cDNA was verified by comparison of the deduced AA sequence with several peptide sequences obtained from cGB-PDE. COS-7 cells transfected with cGB-PDE cDNA overexpressed cGMP-binding and cGMP-PDE activities characteristic of lung cGB-PDE. The sequence of cGB-PDE contained a segment (AA 578-812) that was homologous to the putative catalytic region conserved among all mammalian PDEs and a segment (AA 142-526) that was homologous to the putative cGMP binding region of the cGMP-stimulated PDE and the photoreceptor PDEs. As noted also for these PDEs, two internally homologous repeats were contained within the putative cGMP binding region of cGB-PDE. The amino-terminal 142 residues of cGB-PDE showed no significant homology to other PDEs and contained the serine (AA 92) which is phosphorylated by cGMP-dependent protein kinase.
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PMID:The structure of a bovine lung cGMP-binding, cGMP-specific phosphodiesterase deduced from a cDNA clone. 822 96

Synthetic oligonucleotides were used to amplify mouse brain cDNA sequences homologous to conserved regions of known cGMP-dependent protein kinases, and two classes of products were identified. The first class (CGKI) of amplification products contained approximately 1.0 kilobase (kb) of DNA sequence between the oligonucleotide primers, and this sequence showed a high degree of homology (90% identity) with the known bovine and human type I cDNA sequences for cGMP-dependent protein kinase. The second class (CGKII) of amplification products contained approximately 1.1 kb of DNA sequence between the oligonucleotide primers, and this sequence showed a much lower homology (65% identity) with the bovine and human type I cDNA sequences. Northern blot analysis showed that CGKI transcripts of 8.5 kb were abundant in brain and lung, whereas a 7-kb transcript could be detected in testis. CGKII transcripts of 6 kb were also abundant in brain and lung but could be detected at lower levels in kidney. The CGKII amplification product was used to screen a mouse brain cDNA library, and four overlapping cDNA clones were isolated which comprised the entire CGKII coding region. The predicted CGKII protein consists of 761 amino acids and has a molecular mass of 87 kDa. The CGKII protein shows highest homology to the catalytic (66% amino acid identity) and regulatory domains (45% identity) of bovine and human CGKI. Little homology is observed at the amino terminus or in the region linking the regulatory and catalytic domains. An expression vector for mouse CGKII was constructed and transfected into COS-1 cells where it directed the expression of a protein kinase which was activated by cGMP with an apparent K alpha of 300 nM cGMP.
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PMID:Cloning and expression of a novel cyclic GMP-dependent protein kinase from mouse brain. 851 91

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane.
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PMID:N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II. 863 33

6-Pyruvoyltetrahydropterin synthase (PTPS) participates in tetrahydrobiopterin cofactor biosynthesis. We previously identified in a PTPS-deficient patient an inactive PTPS allele with an Arg(16) to Cys codon mutation. Arg(16) is located in the protein surface exposed phosphorylation motif Arg(16)-Arg-Ile-Ser, with Ser(19) as the putative phosphorylation site for serine-threonine protein kinases. Purification of recombinant PTPS-S19A from bacterial cells resulted in an active enzyme (k(cat)/K(m) = 6.4 x 10(3) M(-1) s(-1)), which was similar to wild-type PTPS (k(cat)/K(m) = 4.1 x 10(3) M(-1) s(-1)). In assays with purified enzymes, wild-type but not PTPS-S19A was a specific substrate for the cGMP-dependent protein kinase (cGK) type I and II. Upon expression in COS-1 cells, PTPS-S19A was stable but not phosphorylated and had a reduced activity of approximately 33% in comparison to wild-type PTPS. Extracts from several human cell lines, including brain, contained a kinase that bound to and phosphorylated immobilized wild-type, but not mutant PTPS. Addition of cGMP stimulated phosphotransferase activity 2-fold. Extracts from transfected COS-1 cells overexpressing cGKII stimulated Ser(19) phosphorylation more than 100-fold, but only 4-fold from cGKI overexpressing cells. Moreover, fibroblast extracts from mice lacking cGKII exhibited significantly reduced phosphorylation of PTPS. These results suggest that Ser(19) of human PTPS may be a substrate for cGKII phosphorylation also in vivo, a modification that is essential for normal activity.
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PMID:Serine 19 of human 6-pyruvoyltetrahydropterin synthase is phosphorylated by cGMP protein kinase II. 1053 34

We previously showed that stimulation of cGMP-dependent protein kinase (PKG) stimulates L-type calcium current in newborn but not in adult rabbit ventricular myocytes. We have now isolated rabbit PKG type Ialpha cDNA (+1 to 2095), determined the sequence, and analyzed specific expression of PKG in adult and newborn rabbit heart by Western and Northern analyses to elucidate the developmental decline in the significance of PKG in cardiac function. We obtained full-length cDNA of PKG Ialpha from newborn rabbit heart mRNA with reverse transcription-polymerase chain reaction. The coding region of rabbit PKG Ialpha showed 94% homology to sequences of human and bovine PKG Ialpha. The deduced amino acid sequence of 671 amino acids showed seven substitutions between rabbit and either human or bovine PKG Ialpha. The major substitutions were found in the cGMP-binding domain. The cloned PKG 1alpha cDNA was expressed in COS cells. Expressed PKG showed cGMP stimulated PKG activity and immunoreactivity. Northern blot analysis of cardiac tissue demonstrated PKG Ialpha mRNA of 6.8 kb, with much higher levels in newborn than in adult cells. Western analysis in homogenates from ventricular tissues and isolated ventricular myocytes of rabbit heart showed much higher expression of PKG type I protein in newborn compared with adult cells. These findings suggest that PKG is developmentally regulated in rabbit heart and is expressed at a much higher level in newborn than in adult cells. The greater expression of PKG in newborn cells could be responsible for differences in the significance of cGMP in adult and newborn rabbit cells.
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PMID:Analysis of expression of cGMP-dependent protein kinase in rabbit heart cells. 1056 12

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

We have shown that cGMP-dependent protein kinase (PKG) mediates stimulation of L-type calcium current by cGMP in rabbit atrial myocytes. The human atrium may have similar PKG-dependent regulation of calcium current. To elucidate the significance of PKG in cardiac function, we have isolated human PKG type I alpha cDNA (+1 to 2016), determined the nucleotide sequence and analyzed specific expression of PKG in human atrium. We obtained full-length cDNA of PKG type I alpha from human atrial RNA using reverse transcriptase-polymerase chain reaction (RT-PCR). The coding region of human cardiac PKG I alpha showed 99.9% homology to previously published human PKG I alpha except for base No. 1983. At this position G was substituted for T and this resulted in an amino acid substitution from Leu649 to Phe649. The cloned PKG I alpha cDNA was expressed in COS cells and the expressed PKG showed cGMP-stimulated PKG enzyme activity and immunoreactivity. Ribonuclease protection assay, Western blot analysis, and PKG enzyme activity assays in homogenates from human atrial tissue demonstrated the presence of PKG mRNA and protein in human atrial tissue. Immunofluorescence staining confirmed that PKG is highly expressed in human atrial myocytes. These findings suggest that PKG is highly expressed in human atrium and that PKG-dependent phosphorylation may be important in regulation of calcium channel activity in human atrial myocytes.
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PMID:Expression of cGMP-dependent protein kinase in human atrium. 1144 35

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


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