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)

A purified bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) was rapidly phosphorylated by purified bovine lung cGMP-dependent protein kinase (cGK). Within a physiological concentration range, cGK catalyzed phosphorylation of cG-BPDE at a rate approximately 10 times greater than did equimolar concentrations of purified catalytic subunit of cAMP-dependent protein kinase (cAK). cG-BPDE was a poor substrate for either purified protein kinase C or Ca2+/calmodulin-dependent protein kinase II. Binding of cGMP to the cG-BPDE binding site was required for phosphorylation since (a) phosphorylation of cG-BPDE by the catalytic subunit of cAK was cGMP-dependent, (b) phosphorylation of cG-BPDE in the presence of a cGMP analog specific for activation of cGK was cGMP-dependent, and (c) occupation of the cG-BPDE hydrolytic site with competitive inhibitors did not produce the cGMP-dependent effect. cGMP-dependent phosphorylation of cG-BPDE by both cGK and cAK occurred at serine. Proteolytic digestion of cG-BPDE phosphorylated by either cGK or cAK revealed the same phosphopeptide pattern, suggesting that phosphorylation by the two kinases occurred at the same or adjacent site(s). Tryptic digestion of cG-BPDE phosphorylated by cGK and [gamma-32P]ATP produced a single major phosphopeptide of approximately 2 kDa with the following amino-terminal sequence: Lys-Ile-Ser-Ala-Ser-Glu-Phe-Asp-Arg-Pro-Leu-Arg- Radioactivity was released during the third cycle of Edman degradation. cG-BPDE is one of few specific in vitro cGK substrates of known function to be identified. Elevation of intracellular cGMP may cause phosphorylation of cG-BPDE by modulating the substrate site availability as well as by activating cGK. Such regulation would greatly increase the selectivity of the phosphorylation of cG-BPDE and would represent a unique mechanism of action of a cyclic nucleotide or other second messenger.
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PMID:Substrate- and kinase-directed regulation of phosphorylation of a cGMP-binding phosphodiesterase by cGMP. 216 96

Two murine monoclonal antibodies (H5 and B6) generated against bovine heart type II regulatory subunit of cAMP-dependent protein kinase were shown to cross-react equally well with the homologous subunit from porcine heart. The antibodies demonstrated specificity for only the type II regulatory subunit and showed negligible cross-reactivity with the type I regulatory subunit, the catalytic subunit, and cGMP-dependent protein kinase. Following limited proteolysis of type II regulatory subunit with chymotrypsin, the H5 monoclonal antibody was shown to cross-react with the Mr = 37,000 cAMP-binding domain corresponding to the COOH-terminal region of the polypeptide chain. To more specifically localize the antigenic sites, the porcine type II regulatory subunit was carboxymethylated and cleaved with cyanogen bromide. Both monoclonal antibodies cross-reacted with the NH2-terminal CNBr peptide, and this peptide demonstrated affinities similar to native bovine type II regulatory subunit in competitive displacement radioimmunoassays. Tryptic cleavage of this CNBr fragment destroyed all antigenicity for both monoclonal antibodies, whereas antigenicity was retained following chymotryptic digestion. A single major immunoreactive chymotryptic fragment that cross-reacted with H5 was isolated by gel filtration and reverse phase high performance liquid chromatography. this peptide retained the complete antigenic site and had the following sequence: Asn-Pro-Asp-Glu-Glu-Glu-Glu-Asp-Thr-Asp-Pro-Arg-Val-Ile-His-Pro-Lys-Thr-Asp-Gl n. This antigenic site was localized just beyond the major site of autophosphorylation, approximately a third of the distance from the NH2-terminal end of the polypeptide chain.
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PMID:Monoclonal antibodies as structural probes of surface residues in the regulatory subunit of cAMP-dependent protein kinase II from porcine heart. 618 75

The amino acid sequence around the site of the regulatory subunit of type I cAMP-dependent protein kinase (RI) that is phosphorylated by cGMP-dependent protein kinase has been determined. This site was found to be located near the site on RI previously shown to be very sensitive to hydrolysis by trypsin (Potter, R. L., and Taylor, S. S. (1979) J. Biol. Chem. 254, 2413-2418). The primary sequence surrounding the site is as follows: -Lys-Ala-Gly-Ser-Arg-Ala-Asp-Ser-Arg-Glu-Asp-Glu-Ile-Ser-Pro-Pro-Pro-Pro-Asn-Pro-Val-Val-Lys-Gly-Arg-Arg-Arg-Arg-Gly-Ala-Ile-Ser(P)-Ala-Glu-Val-Tyr-Thr-Glu-Glu-Asp-Ala-Ala-Ser-Tyr-Val-Arg-Lys-Val-Ile-Pro-Lys-Asp-Tyr-Lys-Thr-. As described previously (Geahlen, R. L., and Krebs, E. G. (1980) J. Biol. Chem. 255, 1164-1169), this site is specific for cGMP-dependent protein kinase and is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.
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PMID:Studies on the site in the regulatory subunit of type I cAMP-dependent protein kinase phosphorylated by cGMP-dependent protein kinase. 626 84

The amino acid sequence at the ATP-binding site on the cGMP-dependent protein kinase has been determined. For this determination the enzyme was labeled covalently by 5'-p-fluorosulfonyl[14C]benzoyladenosine and fragmented using cyanogen bromide or digested by trypsin after succinylation. The 14C-labeled peptides were purified by gel filtration and high performance liquid chromatography. The amino acid sequence around the site was found to be: -Val-Glu-Leu-Val-Gln-Leu-Lys-Ser-Glu-Glu-Ser-Lys-Thr-Phe-Ala-Met-*Lys-Ile-Leu-Lys--Lys-Arg-His-Ile-Val-Asp-Thr-Arg-Gln-Gln-Glu-His-Ile-Arg-Ser-Glu-Lys-, in which *Lys is the lysine residue that was modified by the affinity reagent. When this sequence was compared with that of the ATP-binding site of the catalytic subunit of cAMP-dependent protein kinase, a high degree of structural homology was observed for this site in the two proteins.
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PMID:Amino acid sequence at the ATP-binding site of cGMP-dependent protein kinase. 627 62

Achatin-I (Gly-D-Phe-Ala-Asp), a tetrapeptide having a D-phenylalanine residue and isolated from Achatina ganglia, has been proposed as an excitatory neurotransmitter of Achatina neurones. In the present study, it was demonstrated using Achatina giant neurones that achetin-I, perfused at alow concentration, enhanced an inward current (Iin) caused by 5-hydroxytryptamine (fast component) and an outward current (Iout) caused by FMRFamide (Phe-Met-Arg-Phe-NH2), and that this peptide suppressed an Iin caused by oxytocin, and Iout caused by acetylcholine and APGW-amide (Ala-Pro-Gly-Trp-NH2). These findings indicate that achatin-I acts not only as a neurotransmitter but also as a neuromodulator for these neurones. In the preliminary experiments, it was shown that an Iin caused by achatin-I on an Achatina giant neurone type, PON (periodically oscillating neurone), was suppressed by H-89 (a PKA inhibitor) and W-7 (calmodulin inhibitor), and that an Iin caused by achatin-I on v-RCON (ventral-right cerebral distinct neurone) was suppressed by KT5823 (PKG inhibitor), suggesting that achatin-I acts on PON via the cyclic AMP-PKA system and on v-RCON via the cyclic GMP-PKG system. Moreover, calmodulin would play a role to produce the Iin for achatin-I on PON via the system mentioned.
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PMID:Further study on the effects of achatin-I, an Achatina endogenous neuroexcitatory tetrapeptide having a D-phenylalanine residue, on Achatina neurones. 885 10

The ability of cGMP-dependent protein kinases (cGKs) to activate cAMP response element (CRE)-dependent gene transcription was compared with that of cAMP-dependent protein kinases (cAKs). Although both the type Ibeta cGMP-dependent protein kinase (cGKIbeta) and the type II cAMP-dependent protein kinase (cAKII) phosphorylated the cytoplasmic substrate VASP (vasodilator- and A kinase-stimulated phosphoprotein) to a similar extent, cyclic nucleotide regulation of CRE-dependent transcription was at least 10-fold higher in cAKII-transfected cells than in cGKIbeta-transfected cells. Overexpression of each kinase in mammalian cells resulted in a cytoplasmic localization of the unactivated enzyme. As reported previously, the catalytic (C) subunit of cAKII translocated to the nucleus following activation by 8-bromo-cyclic AMP. However, cGKIbeta did not translocate to the nucleus upon activation by 8-bromo-cyclic GMP. Replacement of an autophosphorylated serine (Ser79) of cGKIbeta with an aspartic acid resulted in a mutant kinase with constitutive kinase activity in vitro and in vivo. The cGKIbetaS79D mutant localized to the cytoplasm and was only a weak activator of CRE-dependent gene transcription. However, an amino-terminal deletion mutant of cGKIbeta was found in the nucleus as well as the cytoplasm and was a strong activator of CRE-dependent gene transcription. These data suggest that the inability of cGKs to translocate to the nucleus is responsible for the differential ability of cAKs and cGKs to activate CRE-dependent gene transcription and that nuclear redistribution of cGKs is not required for NO/cGMP regulation of gene transcription.
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PMID:Cyclic AMP- and cyclic GMP-dependent protein kinases differ in their regulation of cyclic AMP response element-dependent gene transcription. 1008 70

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

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.
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PMID:Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase. 1174 88

Outer hair cells (OHCs) in the mammalian organ of Corti display electromotility, which is thought to provide the local active mechanical amplification of the cochlear response. Prestin is the key molecule responsible for OHC electromotility. Several compounds, including cGMP, have been shown to influence OHC electromotility. There are two potential cAMP/cGMP-dependent protein kinase phosphorylation sites on prestin. Whether these sites are involved in cGMP-dependent reactions is as yet unknown. In this study, prestin cDNA was transiently transfected into TSA 201 cells. Cells that expressed prestin were selected to measure non-linear capacitance (NLC), a signature of outer hair cell motility. We applied cGMP and cAMP analogues and a protein kinase G (PKG) antagonist to the cells. Furthermore, nine mutations at putative phosphorylation sites of prestin were produced. The neutral amino acid alanine replaced serine/threonine at phosphorylation sites to change the conserved phosphorylation motif in order to mimic the dephosphorylated state of prestin, whereas replacement with the negatively charged aspartic acid mimicked the phosphorylated state. The properties of such modified prestin-expressing cells were examined, through measurement of NLC and with confocal microscopy. Our data demonstrate that cGMP is significantly more influential than cAMP in modifying the non-linear, voltage-dependent charge displacement in prestin-transfected cells. The electrical properties of the single and double mutations further indicate a possible interaction between the two PKG target sites. One of these sites may influence the membrane targeting process of prestin. Finally, a new topology map of prestin is proposed.
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PMID:Effects of cyclic nucleotides on the function of prestin. 1564 74

SUMMARY Colletotrichum acutatum causes Key lime anthracnose (KLA) and postbloom fruit drop (PFD) of citrus. We utilized restriction enzyme-mediated integration (REMI) mutagenesis to produce six non-pathogenic mutants from a KLA isolate after screening 1064 transformants on detached Key lime leaves. Subsequently, a gene designated KLAP1 (Key Lime Anthracnose Pathogenicity) was identified from one of the mutants and was demonstrated genetically to be required for pathogenicity to Key lime leaves. The predicted polypeptide encoded by KLAP1 contains a cAMP and cGMP-dependent protein kinase phosphorylation site, and two RGD (Arg-Gly-Asp) cell attachment sequences, a bipartite nuclear targeting sequence, a fungal G-protein alpha subunit signature, a putative metal-binding zinc finger (Cys(2)His(2)) and a putative HMG-I/Y ('high mobility group' non-histone chromatin protein encoding genes) DNA-binding domain (A+T hook), suggesting that KLAP1 may function as a transcription activator in C. acutatum. Sequences homologous to KLAP1 were detected in most C. acutatum isolates examined, and similarity was found in several classes of fungi, animals, plants and bacteria, indicating that KLAP1 is a putative, uncharacterized, conserved transcription activator in fungi. Targeted gene disruption of KLAP1 yielded mutants that were blocked in the penetration stage and were completely defective in pathogenicity on Key lime leaves, but remained pathogenic to flower petals. Complementation of a klap1-null mutant with a full-length KLAP1 gene clone restored complete ability to incite lesions on Key lime. The results indicate that KLAP1 is an important pathogenicity factor in C. acutatum.
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PMID:A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime anthracnose. 2056 76


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