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)

cGMP-dependent protein kinase (cGMP kinase) has been implicated in the regulation of the cytosolic calcium level ([Ca2+]i). In Chinese hamster ovary (CHO) cells stably transfected with the cGMP kinase I alpha (CHO-cGK cells), cGMP kinase suppressed the thrombin-induced increase in inositol 1,4,5-trisphosphate and [Ca2+]i (Ruth, P., Wang, G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M., Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular calcium release via a pertussis toxin (PTX)-insensitive pathway in CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated [Ca2+]i. In contrast, cGMP kinase suppressed calcium influx stimulated by insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The effects of PTX and cGMP kinase on [Ca2+]i were not additive. 8-Bromo-cGMP had no effect on [Ca2+]i stimulated by IGF-1 or IGF-2 in wild type CHO cells. These results suggested that cGMP kinase inhibited the different signaling pathways by the phosphorylation of a PTX-sensitive G protein. cGMP kinase phosphorylated the alpha subunits of Gi1, Gi2, and Gi3 in vitro. Phosphorylation stoichiometry was 0.4 mol of phosphate/mol of G alpha i1 after reconstitution of heterotrimeric Gi1 in phospholipid vesicles. The alpha subunit of Gi was also phosphorylated in vivo. These results show that cGMP kinase blocks transduction of distinct hormone pathways that signal via PTX-sensitive Gi proteins.
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PMID:Cyclic GMP-dependent protein kinase blocks pertussis toxin-sensitive hormone receptor signaling pathways in Chinese hamster ovary cells. 772 18

Ejaculated ram sperm were demembranated with Triton X-100, separated from the detergent-soluble matrix, and reactivated [San Agustin and Witman (1993): Cell Motil. Cytoskeleton 24:264-273]. The percent motility of models prepared from freshly washed sperm was comparable to that of the washed sample before demembranation, regardless of whether cAMP was included in the reactivation medium. However, demembranated models derived from aging or metabolically inhibited sperm exhibited a lower percent reactivation and required cAMP to attain the level of motility of freshly washed sperm. Cyclic AMP was approximately 100 times more effective than cGMP. The requirement for cAMP could be bypassed by addition of porcine heart cAMP-dependent protein kinase (PKA) catalytic subunit to the reactivation medium, demonstrating that cAMP was acting via PKA. The cAMP stimulation of reactivation was not affected by inclusion of the PKA inhibitor PKI(5-24) in the reactivation medium, but was decreased when the models were preincubated with PKI(5-24) prior to reactivation. The cytosol-free models retained > 90% of the sperm PKA activity; therefore, the PKA appears to be anchored to internal sperm structures. This PKA could not be extracted by cAMP or Triton X-100 alone, but only by cAMP and Triton X-100 in combination. We conclude that cAMP-dependent protein phosphorylation is critical for sperm motility, but that the essential protein phosphate sites turn over slowly under our reactivation conditions, so that the cAMP requirement is apparent only in models prepared from sperm having a low internal ATP or cAMP content. Interestingly, reactivation was rapidly blocked by the peptide arg-lys-arg-ala-arg-lys-glu, which has been reported to be a selective inhibitor of cGMP-dependent protein kinase.
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PMID:Role of cAMP in the reactivation of demembranated ram spermatozoa. 802 Jan 7

Two size forms of the class B N-type calcium channel alpha 1 subunit were recently identified with CNB1, an antipeptide antibody directed against an intracellular loop of this channel (Westenbroek, R.E., Hell, J.W., Warner, C., Dubel, S.J., Snutch, T.P., and Catterall, W.A. (1992) Neuron 9, 1099-1115). To investigate the biochemical differences between these two size forms, the antibodies CNB3 and CNB4 were raised against peptides with sequences corresponding to the COOH-terminal end of the full-length form. Immunoblot experiments demonstrated that both antibodies specifically recognize the longer form of 250 kDa, indicating that the COOH-terminal regions of the two size forms of the class B N-type channel alpha 1 subunit are different. Phosphorylation experiments with immunopurified calcium channels and different second messenger-activated protein kinases revealed that both the 220- and 250-kDa forms of the class B N-type calcium channel alpha 1 subunit are substrates for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. These three kinases incorporated approximately 1 mol of phosphate/mol of binding sites for omega-conotoxin (omega-CgTx) GVIA, a ligand specific for the N-type calcium channel, and may regulate the activity of both forms in vivo. In contrast, calcium- and calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated only the long form of the class B N-type calcium channel alpha 1 subunit, with a stoichiometry of 0.5 mol of phosphate/mol of total omega-CgTx GVIA binding sites. Specific phosphorylation of the long form of the class B alpha 1 subunit by CaM kinase II may differentially regulate the function of N-type calcium channels containing different size forms of their alpha 1 subunits in vivo.
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PMID:Differential phosphorylation of two size forms of the N-type calcium channel alpha 1 subunit which have different COOH termini. 812 57

Paramecium dyneins were tested as substrates for phosphorylation by cAMP-dependent protein kinase, cGMP-dependent protein kinase, and two Ca(2+)-dependent protein kinases that were partially purified from Paramecium extracts. Only cAMP-dependent protein kinase caused significant phosphorylation. The major phosphorylated species was a 29 kDa protein that was present in both 22 S and 12 S dyneins; its phosphate-accepting activity peaked with 22 S dynein. In vitro phosphorylation was maximal at five minutes, then decreased. This decrease in phosphorylation was inhibited by the addition of vanadate or NaF. The 29 kDa protein was not phosphorylated by a heterologous cAMP-dependent protein kinase, the bovine catalytic subunit. Phosphorylation of dynein did not change its ATPase activity. In sucrose gradient fractions from the last step of dynein purification, phosphorylation by an endogenous kinase occurred. This phosphorylation could not be attributed to the small amounts of cAMP- and cGMP-dependent protein kinases known to be present, nor was it Ca(2+)-dependent. This previously uncharacterized ciliary protein kinase used casein as an in vitro substrate.
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PMID:In vitro phosphorylation of ciliary dyneins by protein kinases from Paramecium. 812 14

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.
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PMID:Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase. 838 Mar 42

The large number of covalently bound phosphates on the extracellular phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP) have been implicated in biological functions such as mineral deposition and osteoclast binding. In the present study the state of phosphorylation of BSP and OPN was evaluated by in vitro 32P labeling using a series of protein kinases and quantification. Both the purified bovine BSP and OPN were radiolabeled by [32P]ATP and factor-independent protein kinase. Quantification of 32P radioactivity incorporated on dephosphorylated BSP and OPN provided 6.6 and 8.9 mol of phosphate incorporated/mol, respectively. Native OPN incorporated 1.07 and BSP 2.46 mol of phosphate/mol by factor-independent protein kinase. These data led to calculations that OPN and BSP, respectively, contain 7.83 and 4.14 mol of phosphate/mol in their natural state. Thrombin digests of 32P-labeled BSP showed radioactivity to be associated with fragment of approximately molecular mass values 30 kDa (N-terminal half), with no observable radioactivity associated with the 40-kDa fragment (C-terminal half). Similar experiments with 32P-labeled OPN provided two radiolabeled thrombin fragments, with molecular mass 30 kDa (N-terminal half) and 20 kDa (C-terminal half), both were radioactive. The major phosphorylation was associated with the N-terminal half containing 7.0 mol of phosphate, and 1.9 mol of phosphate were associated with the C-terminal half. Additional experiments of in vitro phosphorylation of OPN and BSP by several other known protein kinases were carried out. cAMP-dependent protein kinase showed no phosphorylation of OPN or BSP, while protein kinase C and cGMP-dependent protein kinase led to minor phosphorylation, each of the latter introduced about 1 mol of phosphate/mol of OPN and BSP molecule.
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PMID:Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases. 866 67

In Paramecium tetraurelia cells synchronous exocytosis induced by aminoethyldextran (AED) is accompanied by an equally rapid dephosphorylation of a 63 kDa phosphoprotein (PP63) within 80 ms. In vivo, rephosphorylation occurs within a few seconds after AED triggering. In homogenates (P)P63 can be solubilized in all three phosphorylation states (phosphorylated, dephosphorylated and rephosphorylated) and thus tested in vitro. By using chelators of different divalent cations, de- and rephosphorylation of PP63 and P63 respectively can be achieved by an endogenous protein phosphatase/kinase system. Dephosphorylation occurs in the presence of EDTA, whereas in the presence of EGTA this was concealed by phosphorylation by endogenous kinase(s), thus indicating that phosphorylation of P63 is calcium-independent. Results obtained with protein phosphatase inhibitors (okadaic acid, calyculin A) allowed us to exclude a protein serine/threonine phosphatase of type I (with selective sensitivity in Paramecium). Protein phosphatase 2C is also less likely to be a candidate because of its requirement for high Mg2+ concentrations. According to previous evidence a protein serine/threonine phosphatase of type 2B (calcineurin; CaN) is possibly involved. We have now found that bovine brain CaN dephosphorylates PP63 in vitro. Taking into account the specific requirements of this phosphatase in vitro, with p-nitrophenyl phosphate as a substrate, we have isolated a cytosolic phosphatase of similar characteristics by combined preparative gel electrophoresis and affinity-column chromatography. In Paramecium this phosphatase also dephosphorylates PP63 in vitro (after 32P labelling in vivo). Using various combinations of ion exchange, affinity and hydrophobic interaction chromatography we have also isolated three different protein kinases from the soluble fraction, i.e. a cAMP-dependent protein kinase (PKA), a cGMP-dependent protein kinase (PKG) and a casein kinase. Among the kinases tested, PKA cannot phosphorylate P63, whereas either PKG or the casein kinase phosphorylate P63 in vitro. On the basis of these findings we propose that a protein phosphatase/kinase system is involved in the regulation of exocytosis in P. tetraurelia cells.
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PMID:Protein phosphatase and kinase activities possibly involved in exocytosis regulation in Paramecium tetraurelia. 869 88

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Ibeta cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (approximately 0.8 phosphate/cGK monomer) approximately 3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (approximately 67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Ibeta cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.
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PMID:Arginine 75 in the pseudosubstrate sequence of type Ibeta cGMP-dependent protein kinase is critical for autoinhibition, although autophosphorylated serine 63 is outside this sequence. 870 27

The effects of cyclic GMP (cGMP) and activation of cGMP-dependent protein kinase (PKG) on the phosphorylation of the inositol 1,4, 5-trisphosphate (IP3) receptor were examined in intact rat aorta using the technique of back phosphorylation. Aorta treated with the nitric oxide donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside, or the selective PKG activator, 8-(4-para-chlorophenylthio)-cGMP (8-CPT-cGMP), demonstrated increased IP3 receptor phosphorylation in situ, which was both time- and concentration-dependent with a stoichiometry of 0.5 mol of phosphate/mol of receptor above control. Treatment of aorta with the adenyl cyclase activator, forskolin, also demonstrated increased phosphorylation of the IP3 receptor on the PKG site, although the selective cAMP-dependent protein kinase activator, 8-(4-para-chlorophenylthio)-cAMP (8-CPT-cAMP), did not increase the phosphorylation of the IP3 receptor. Moreover, the PKG selective inhibitor, KT 5823, inhibited both sodium nitroprusside and forskolin-induced IP3 receptor phosphorylation more potently than the selective cAMP-dependent protein kinase inhibitor, KT 5720, suggesting that PKG mediates the increase in IP3 receptor phosphorylation by both cyclic nucleotides in intact aorta. These results provide further support for the notion that PKG is activated by both cAMP and cGMP in intact vascular smooth muscle and that PKG performs a critical role in cyclic nucleotide-dependent relaxation of blood vessels.
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PMID:Phosphorylation of the inositol 1,4,5-trisphosphate receptor. Cyclic GMP-dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta. 870 97

cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experimental studies were undertaken using four synthetic cGMP analogues, PET-cGMP, 8-Br-PET-cGMP, Rp-8-Br-PET-cGMPS, and Sp-8-Br-PET-cGMPS. With excised patches from either Xenopus oocytes expressing a cloned rat rod channel alpha-subunit or from native Xenopus rod photoreceptors, Rp-8-Br-PET-cGMPS competitively suppressed the cGMP-induced current with an IC50 of 25 microM and Sp-8-Br-PET-cGMPS inhibited this current with an IC50 of 105 microM. On the expressed rat rod channel, 8-Br-PET-cGMP behaved as a very weak partial agonist at high concentrations and an antagonist (IC50 = 64 microM) at lower concentrations when coapplied with cGMP. PET-cGMP did not activate channel currents alone but showed a synergism when coapplied with subsaturating concentrations of cGMP. Because Sp-8-Br-PET-cGMPS is a potent activator of type I cGMP-dependent protein kinase, but a competitive antagonist of channel activation, it will be a useful reagent for discriminating between those effects of cGMP that are mediated by a protein kinase and those mediated by channel activation. Because the PET derivatives all contain a phenyl-substituted 5-membered ring system fused to the amino group in position 2 and the nitrogen in position 1 of the guanine ring, the results support the idea that N1 and N2 are important for channel activation. They also suggest a minor role for the cyclic phosphate group in binding or activation.
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PMID:Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: beta-phenyl-1,N2-etheno-substituted cGMP analogues as probes of the cGMP-binding site. 898 20


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