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

Several of the hepatic microsomal cytochromes P-450 (CYP) including CYP3A are inducible by phenobarbital (PB). However, the intracellular pathways involved in the action of PB on CYP3A remain poorly known. With the aim to unravel some of the main aspects of PB signaling, we first devised a simple model of mouse cultured primary hepatocytes in which CYP3A mRNA and protein were strongly induced by PB in the absence of dexamethasone and were at maximum levels after a 48-h treatment with a 2-mM dose of PB. Under these culture conditions, we studied the effects of inhibitors and activators of different protein kinases or phosphatases on CYP3A mRNA and protein induction by PB. CYP3A-induced expression was inhibited by activators of cyclic AMP-dependent protein kinase (PKA) (dibutyryl-cyclic AMP and forskolin) whereas inhibition of PKA by PKA inhibitor enhanced induction. 8-br-cGMP produced effects similar to the activators of PKA, and so did the specific inhibitor of cGMP-dependent protein kinase, beta-phenyl-1, N(2)-etheno-8-bromoguanosine-3,5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS). Inhibition of Ca(2+)/calmodulin-dependent protein kinase by KN-62 or the intracellular Ca(2+) chelator BAPTA-AM produced an inhibition of CYP3A induction by PB. Specific inhibitors of protein kinase C, mitogen-activated protein kinase kinase, phosphatidylinositol-3-kinase, or serine/threonine phosphatase did not produce any effect. Taken together, our results suggest that CYP3A induction by PB is regulated positively by calmodulin-dependent protein kinase and cGMP-dependent protein kinase, and negatively by PKA in mouse hepatocytes in primary culture.
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PMID:Involvement of cyclic nucleotide- and calcium-regulated pathways in phenobarbital-induced cytochrome P-450 3A expression in mouse primary hepatocytes. 1045 3

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

GABA(A) receptors of rat cerebellar granule cells in culture have been studied by the whole cell patch clamp technique. The biphasic desensitization kinetic observed could be due either to different desensitization mechanisms of a single receptor population or to different receptor populations. The overall data indicate that the latter hypothesis is most probably the correct one. In fact, the fast desensitizing component was selectively potentiated by a benzodiazepine agonist and preferentially down-regulated by activation of the protein serine/threonine kinases A and G, as a consequence of the latter characteristic that receptor population was preferentially down-regulated by previous activation of N-methyl-d-aspartate glutamate receptors, via production of nitric oxide and PKG activation, most probably in dendrites. The other population is benzodiazepine insensitive and not influenced by activation of PKA or PKG. This slowly desensitizing population may correspond to the extrasynaptic delta subunit containing GABA(A) receptors described by other authors. Instead, the rapidly desensitizing population appears to represent dendritic synaptic GABA(A) receptors.
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PMID:Evidence of two populations of GABA(A) receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization. 1060 May 49

Phospholamban is a small integral membrane protein of cardiac, smooth, and slow-twitch skeletal muscle sarcoplasmic reticulum that interacts with the Ca2+ pump of these organelles and inhibits Ca(2+)-pump activity while in the dephosphorylated form. Three sites of Ser/Thr phosphorylation have been identified in the primary sequence of phospholamban, at Ser-10, Ser-16, and Thr-17. In vitro studies indicate that these residues are phosphorylated by PKC (Ser-10), PKA, PKG or PKC (Ser-16), and CaM kinase II (Thr-17). Phosphorylation of Ser-16 (or Thr-17) is accompanied by an increase in Ca2+ pump activity in direct proportion to the stoichiometry of phosphorylation. Dual phosphorylation of both Ser-16 and Thr-17 does not cause any further stimulation of pump function over that achieved by stoichiometric phosphorylation of a single site. Examination of the pattern of phosphorylation in vivo has been aided by the generation of polyclonal antibodies specific for the phosphorylated forms of phospholamban. beta-Adrenergic stimulation of cardiac muscle results in phosphorylation of both Ser-16 and Thr-17. The time course of Ser-16 phosphorylation precedes Thr-17. The spatial distribution of Ser-16 and Thr-17 phosphorylated forms of phospholamban is not identical; phospholamban located in the nuclear membrane of a cardiac myocyte is phosphorylated exclusively on Ser-16, whereas phospholamban molecules in the SR membrane of the same cell are phosphorylated on Ser-16 and/or Thr-17. Finally, we have identified a novel stimulus for the phosphorylation of phospholamban. Ca2+ store depletion, achieved by exposure of myocytes to SERCA inhibitors, prompts the phosphorylation of phospholamban on Ser-16. This would be expected to increase Ca2+ uptake by the SR in an attempt to achieve the refilling of the SR.
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PMID:Phosphorylation states of phospholamban. 1060 38

The role of each of the two different cGMP-binding sites (referred to as slow and fast sites) of type I cGMP-dependent protein kinase (PKG) in altering the rate of catalysis of phosphorylation of exogenous substrates (heterophosphorylation) or the rate of autophosphorylation has not been resolved. In the present study, the cGMP concentration required for half-maximal activation (A(50)) of wild-type PKG type Ibeta (WT) was 5-fold higher for heterophosphorylation than for autophosphorylation. cGMP occupation of the slow site was associated with an increase in the autophosphorylation rate, whereas occupation of the fast and slow site together was associated with a decrease in the autophosphorylation rate compared with the rate observed with occupation of the slow site alone. The contributions of each cGMP-binding site were investigated using PKG mutants containing substitutions of an invariant threonine residue that is critical for high affinity cGMP-binding in each site. Site-directed mutagenesis of Thr-317 of the fast site (T317A) increased the cGMP A(50) for heterophosphorylation 4-fold at 30 degrees C, with nominal effect on cGMP A(50) for autophosphorylation compared with WT. The analogous slow site mutation (T193A) increased the cGMP A(50) for heterophosphorylation and autophosphorylation 32- and 64-fold, respectively. Compared with WT, the cGMP A(50) of the double mutant (T193A/T317A) for heterophosphorylation was increased 300-fold, whereas the cGMP A(50) for autophosphorylation was similar to that of T193A. Thus, occupation of both cGMP-binding sites of PKG is required for maximal stimulation of heterophosphorylation, whereas occupation of the slow site alone is sufficient for stimulation of the rate of autophosphorylation, and additional occupation of the fast site reduces this rate.
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PMID:Distinguishing the roles of the two different cGMP-binding sites for modulating phosphorylation of exogenous substrate (heterophosphorylation) and autophosphorylation of cGMP-dependent protein kinase. 1061 99

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.
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PMID:Nitric oxide activation of p38 mitogen-activated protein kinase in 293T fibroblasts requires cGMP-dependent protein kinase. 1064 46

We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.
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PMID:Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta. 1064 36

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


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