Cyclic Nucleotide-dependent Protein Kinase Activity Research Articles

Construction and functional enrichment analysis of the competitive endogenous RNA regulatory network for nonarteritic anterior ischemic optic neuropathy based on high-throughput sequencing.

Based on the transcriptome high-throughput sequencing of nonarteritic anterior ischemic optic neuropathy (NAION), this study constructed a competitive endogenous RNA (ceRNA) network, enriched and analyzed it, screened long noncoding (lnc)RNAs and circular (circ)RNAs that may participate in the competitive endogenous mechanism in NAION, and inferred its function. Four milliliters of peripheral blood from NAION patients and the control group was extracted from clinical samples, transcriptome high-throughput sequencing was performed, and the sequencing data were visualized. Based on the principle of the ceRNA network, the lncRNA-micro (mi)RNA-messenger (m)RNA interaction axis and circRNA-miRNA‒mRNA interaction axes were constructed. The differentially expressed genes (DEGs) in the interaction axis were enriched and analyzed by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the functions and signalling pathways of lncRNAs and circRNAs in the interaction network were speculated. Fifty-one circRNAs were differentially expressed in the sequencing data: 25 were upregulated, and 26 were downregulated. For 996 differentially expressed lncRNAs, 317 were upregulated and 679 were downregulated, and for 1161 differentially expressed mRNAs, 698 were upregulated and 463 were downregulated. Thirty-three differentially expressed miRNAs, upregulated miRNA 18 and downregulated miRNA 15 were identified. After screening, 13 coexpressed mRNAs, 15 lncRNAs, and 3 miRNAs were finally constructed in the lncRNA-miRNA-mRNA network, and the circRNA-miRNA-mRNA network was constructed by 159 mRNAs, 26 miRNAs, and 34 circRNAs. In the lncRNA network, GO enrichment analysis obtained 182 biological processes, 12 cell components and 38 molecular functions, which are related mainly to the regulation of the activity of proteins and enzymes such as cyclic nucleotide-dependent protein kinase activity and magnesium ion-dependent protein serine/threonine phosphatase activity. KEGG analysis involved mainly the forkhead box O (FoxO) signalling pathway, apelin signalling pathway, etc. In the circRNA enrichment results, 353 biological processes, 52 cell components, and 45 molecular functions were obtained, involving mainly calcium channel regulation, neutrophil activation, mRNA transport, and metabolism. KEGG mainly involved the Wnt signalling pathway, apelin signalling pathway, Hippo signalling pathway, oxytocin signalling pathway, etc. This paper provides a new idea for lncRNAs and circRNAs to mediate the occurrence and development of NAION through the ceRNA mechanism. This study lays a foundation for the in-depth study of the pathogenesis of NAION.

New perspectives in cyclic nucleotide-mediated functions in the CNS: the emerging role of cyclic nucleotide-gated (CNG) channels

Cyclic nucleotides play fundamental roles in the central nervous system (CNS) under both physiological and pathological conditions. The impact of cAMP and cGMP signaling on neuronal and glial cell functions has been thoroughly characterized. Most of their effects have been related to cyclic nucleotide-dependent protein kinase activity. However, cyclic nucleotide-gated (CNG) channels, first described as key mediators of sensory transduction in retinal and olfactory receptors, have been receiving increasing attention as possible targets of cyclic nucleotides in the CNS. In the last 15 years, consistent evidence has emerged for their expression in neurons and astrocytes of the rodent brain. Far less is known, however, about the functional role of CNG channels in these cells, although several of their features, such as Ca(2+) permeability and prolonged activation in the presence of cyclic nucleotides, make them ideal candidates for mediators of physiological functions in the CNS. Here, we review literature suggesting the involvement of CNG channels in a number of CNS cellular functions (e.g., regulation of membrane potential, neuronal excitability, and neurotransmitter release) as well as in more complex phenomena, like brain plasticity, adult neurogenesis, and pain sensitivity. The emerging picture is that functional and dysfunctional cyclic nucleotide signaling in the CNS has to be reconsidered including CNG channels among possible targets. However, concerted efforts and multidisciplinary approaches are still needed to get more in-depth knowledge in this field.

Effects of spinal versus supraspinal administration of cyclic nucleotide-dependent protein kinase inhibitors on morphine tolerance in mice

The consequences of becoming tolerant to the analgesic effects of morphine include increased risk of unwanted side effects, such as respiratory depression, because the patient is required to take larger doses of the opioid to get the same relief from pain. Many studies suggest that phosphorylation plays a role in the neuroplasticity associated with opioid tolerance. This study examines the effect of inhibiting cyclic nucleotide-dependent protein kinase activity in the brain or spinal cord of morphine-tolerant mice. KT5720, a cyclic adenosine monophosphate (cAMP)-dependent protein kinase inhibitor, or KT5823, a cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, was centrally administered in morphine-tolerant and placebo-treated mice prior to a systemically administered challenge dose of morphine. KT5720 completely reversed morphine tolerance in the tail-flick assay when the pretreatment was administered intracerebroventricularly (i.c.v.); KT5823 had no effect on morphine via this route. When either of these drugs was administered intrathecally (i.t.), the activity of morphine was greatly diminished in the tolerant animals, with no effect on morphine antinociception in the placebo group. These data suggest that cAMP-dependent protein kinase activity may be upregulated in the brain with morphine tolerance, and that this upregulation is critical to the expression of tolerance to the antinociceptive effects of morphine. In the spinal cord, however, the activity of cyclic nucleotide-dependent protein kinases, and possibly their substrate proteins, may be affected by chronic morphine exposure such that inhibition of these kinases produces hyperalgesia.

Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium

Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K = 10(3) Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation. To identify ciliary targets of cyclic nucleotide-dependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[gamma-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the deciliation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22S dynein, and a high molecular weight protein (greater than 300K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.

Protein kinase activity in canine basilar arteries after subarachnoid hemorrhage.

To investigate the intracellular physiology of cerebral arterial vasospasm after subarachnoid hemorrhage (SAH), we measured the activity of three protein kinases related to vascular smooth muscle function. Myosin light chain kinase (MLCK), cyclic adenosine monophosphate (cAMP)-dependent protein kinase and cyclic guanosine monophosphate (cGMP)-dependent protein kinase activity was determined in canine basilar arteries using the SAH model created by two intracisternal injections of autologous blood. Moderate chronic cerebral vasospasm was confirmed angiographically 7 days after the first injection of blood, with a mean decrease in the diameter of the basilar artery to 61.2 +/- 2.3% (SE) of preinjection controls. Enzymatic analysis of basilar arteries excised from control dogs and from SAH model dogs 2 to 10 days after an intracisternal injection of blood demonstrated detectable MLCK and cyclic nucleotide-dependent protein kinase activity. The MLCK activity was approximately 3 times higher than the cyclic nucleotide-dependent protein kinase activity. There was no significant difference in basilar artery protein kinase activity between control animals and SAH model animals at any time for up to 10 days after the intracisternal injection of blood. As MLCK is involved with smooth muscle contraction and cAMP- and cGMP-dependent protein kinases are involved in smooth muscle relaxation, our results suggest that the metabolic machinery of vascular smooth muscle in the cerebral arterial wall is intact after SAH and that prolonged vasospasm represents an appropriate physiological response by the vascular smooth muscle to external stimuli. If this is correct, the successful pharmacological manipulation of vasospasm after SAH is more likely than if profound disruption had occurred, and further investigative efforts in this area should be encouraged.

Variations in epidermal cyclic Nucleotide-dependent protein kinase activity during moult cycle of the crayfish Orconectes limosus and hormonal control of kinase activity by 20-hydroxyecdysone

1. 1. In correlation to changes in ecdysone hemolymph titers during moulting of the crayfish high kinase activity is observed in the moult stages D 0− D 2 3 and in postmoult stage B. 2. 2. Both basal (measured in the absence of cyclic nucleotides) and total (in the presence of cyclic nucleotides) protein kinase activity were enhanced, probably caused by an increase in tissue Levels of cAMP and cGMP and furthermore by the disapperance of a kinase inhibirory protein. 3. 3. Injection of 20-hydroxyecdysone into intermoult animals increased integumental kinase activity in a time- and dose-dependent manner, maximal activity occurring after 5 hr at a dose of 1 μg/g fresh weight. 4. 4. In vitro incubation of an integumental extract with 20-hydroxyecdysone showed no enhancement of cAMP and cGMP levels in the tissues.

Regulation of cyclic nucleotide-dependent protein kinase activity by histones and poly(L-arginine).

Interaction of cGMP-dependent protein kinase with histones H2A, H2B, H3, and H4, or poly(L-arginine) resulted in changes in enzyme conformation such that inactivation of cGMP binding and activation of basal catalytic activity (assayed without cGMP) occurred. Total kinase activity as determined by phosphorylation of exogenous substrates subsequently decreased, but autophosphorylation of the enzyme was enhanced. The reaction was specific for nucleosome core histones and poly(L-arginine); H1, troponin, and poly(L-lysine) had no effect. Inactivation of cyclic nucleotide binding sites followed pseudo-first order kinetics and, at various histone concentrations, exhibited saturation kinetics at low ionic strength (2 mM potassium phosphate, pH 6.8), but non-saturation kinetics at higher ionic strength (37.5 mM potassium phosphate, pH 6.8, 12.5 mM MgCl2). Saturation kinetics was observed with poly(L-arginine) at both low and high ionic strength. Kinetic parameters measured under saturation conditions were determined for each core histone and poly(L-arginine). Core histones and poly(L-arginine) were noncompetitive inhibitors of cGMP binding; core histones and poly(L-arginine) interacted competitively at an enzyme site designated as the poly(L-arginine) binding site. Regulatory subunits of cAMP-dependent protein kinase contain a similar poly(L-arginine) binding site. Modulator proteins bind to poly(L-arginine) or arginyl residues in histone to prevent interaction with the poly(L-arginine) binding site on the enzymes. Through this mechanism, modulator proteins maintain cyclic nucleotide dependency and full enzyme activity.

Cyclic nucleotide-dependent protein kinase activity in acinar cells from guinea pig pancreas

Acinar cells from guinea pig pancreas possess two distinct protein kinase activities. Cyclic GMP-dependent kinase elutes as a single peak on diethylaminoethyl (DEAE)-cellulose chromatography, is not inhibited by protein kinase inhibitor, and has a greater affinity for cyclic GMP (half-maximal activation at 20 nM) than for cyclic AMP (half-maximal activation at 100 nM). Cyclic AMP-dependent kinase elutes as two peaks on DEAE-cellulose chromatography, is inhibited by protein kinase inhibitor, and has a greater affinity for cyclic AMP (half-maximal activation at 20 nM) than for cyclic GMP (half-maximal activation at 7 μm). Binding of cyclic 3H-nucleotides to the enzyme preparation was rapid, specific, temperature-dependent, and reversible, and there was a close correlation between the ability of a particular cyclic nucleotide to inhibit binding of cyclic 3H-nucleotide and its ability to activate protein kinase. Binding of cyclic 3H-nucleotide could not be described as a simple bimolecular reaction and native cyclic nucleotides accelerated the dissociation of bound, labeled cyclic nucleotide. Vasoactive intestinal peptide or secretin, each of which increases cellular cyclic AMP, caused endogenous activation of protein kinase and inhibition of cyclic [3H]AMP binding but did not alter binding of cyclic [3H]GMP. In contrast, cholecystokinin which increases cellular cyclic GMP did not alter protein kinase activity and did not alter binding of cyclic [3H]GMP or cyclic [3H]AMP.

Cyclic nucleotide-dependent protein kinase activity of adult female locust fat body

Locust fat body protein kinase is dependent on, and maximally stimulated by 5 × 10 −6 M cyclic GMP and to a lesser extent by cyclic AMP. It exhibits a pH optimum of 6.9, an unusual requirement for high Mg 2+ (maximum at 100 mM) or an alternative requirement for Mn 2+ (maximum at 5 mM). Maximal stimulation by Mg 2+ is twice that by Mn 2+. Rabbit muscle protein kinase inhibitor uncharacteristically inhibits both cyclic AMP and cyclic GMP stimulated protein kinase activity of locust fat body, the former 5-fold more than the latter, with histone IIA as acceptor. The degree of phosphorylation differs for different acceptor proteins: histone IIA > locust vitellin > arginine-rich histone. Casein is barely phosphorylated and bovine serum albumin (fraction V) not at all. The high threshold for Mg 2+ activation may be related to the high levels of Mg 2+ in locust haemolymph, to the preferential phosphorylation of vitellin, and to the fact that the fat body is the site of vitellogenin synthesis.

Cyclic nucleotide-dependent protein kinase activity in malignant and cyclic AMP-induced “differentiated” neuroblastoma cells in culture

The activity of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase was demonstrated in the supernatant (S) fraction (100,000 × g) of mouse and human neuroblastoma (NB) cells. In cAMP-induced “differentiated” mouse NB cells, the cAMP-dependent protein kinase (cAMP-PK) activity did not significantly change. Cyclic GMP did not stimulate the PK activity in S-fraction. The cAMP-PK or cGMP-PK activity was not detected in the membrane (M) fraction. The present results in combination with previous data support the concept that the major portion of binding proteins is distinct from the regulatory subunits of cAMP-PK. For example, the level of binding proteins markedly increases in S-fraction of “differentiated” NB cell, but cAMP-PK activity does not change. cAMP binding proteins are present in the M-fraction, but cAMP-PK activity is not demonstrable. Cyclic GMP binds with the soluble proteins with about 10-fold less binding affinity than cAMP; however, cGMP does not stimulate PK activity.

CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES FROM SILKWORM EGGS: OCCURRENCE OF TWO CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES, CHANGES IN THEIR ACTIVITIES AND LEVELS OF CYCLIC NUCLEOTIDES DURING DEVELOPMENT OF THE EGGS.

Two cyclic nucleotide-dependent protein kinases, designated as protein kinase-I and -II, were obtained from the eggs of the silkworm, Bombyx mori. Protein kinase-I is highly dependent on cGMP, whereas protein kinase-II is dependent on cAMP. In developing non-diapause eggs, the level of cyclic nucleotide-dependent protein kinase activity is quite high but that in the diapause eggs is not. The developmental changes in the two protein kinases and the level of cyclic nucleotides were also studied during the development of the eggs.