Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The generation of reactive oxygen species (ROS) has been implicated in the regulation of sperm capacitation and acrosome reaction; however, the mechanisms underlying this regulation remain unclear. To examine the cellular processes involved, we studied the effect of different concentrations of hydrogen peroxide (H(2)O(2)) on protein tyrosine phosphorylation under various conditions. Treatment of spermatozoa with H(2)O(2) in medium without heparin caused a time- and dose-dependent increase in protein tyrosine phosphorylation of at least six proteins in which maximal effect was seen after 2 h of incubation with 50 microM H(2)O(2). At much higher concentrations of H(2)O(2) (0.5 mM), there is significant reduction in the phosphorylation level, and no protein tyrosine phosphorylation is observed at 5 mM H(2)O(2) after 4 h of incubation. Exogenous NADPH enhanced protein tyrosine phosphorylation similarly to H(2)O(2). These two agents, but not heparin, induced Ca(2+)-dependent tyrosine phosphorylation of an 80-kDa protein. Treatment with H(2)O(2) (50 microM) caused approximately a twofold increase in cAMP, which is comparable to the effect of bicarbonate, a known activator of soluble adenylyl cyclase in sperm. This report suggests that relatively low concentrations of H(2)O(2) are beneficial for sperm capacitation, but that too high a concentration inhibits this process. We also conclude that H(2)O(2) activates adenylyl cyclase to produce cAMP, leading to protein kinase A-dependent protein tyrosine phosphorylation.
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PMID:Role of hydrogen peroxide in sperm capacitation and acrosome reaction. 1456 55

CFTR channels conduct HCO(3)(-) in addition to Cl(-) in airway epithelial cells. A defective HCO(3)(-)-transporting function of CFTR may underlie the pathogenesis of cystic fibrosis. In the present study, we have investigated whether a HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) is functionally coupled with CFTR and thus forms an autoregulatory mechanism for HCO(3)(-) transport in human airway epithelial Calu-3 cells. A reverse transcriptase-polymerase chain reaction showed that transcripts of both full-length and truncated sACs are present in Calu-3 cells. Truncated sAC protein is the predominant, if not the only, isoform expressed in Calu-3 cells. HCO(3)(-) stimulated a modest increase in cAMP production, and the increase was sensitive to 2-hydroxyestradiol (2-HE), a sAC inhibitor, but not to SQ22,536, a blocker of conventional transmembrane adenylyl cyclases. These results suggest that sAC is functional in Calu-3 cells. Adding 25 mM HCO(3)(-) to the bath stimulated CFTR-mediated whole cell currents in the absence, but not in the presence, of 2-HE. In cell-attached membrane patches, 25 or 50 mM HCO(3)(-) in the bath markedly increased the product of channel number and open probability of CFTR, and this activation was attenuated by 2-HE. These findings demonstrate that sAC signaling pathway is involved in the regulation of CFTR function in human airway epithelium and thereby provides a link between the level of intracellular HCO(3)(-)/CO(2) and the modulation of HCO(3)(-)-conductive CFTR function by cAMP/PKA.
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PMID:Regulation of CFTR channels by HCO(3)--sensitive soluble adenylyl cyclase in human airway epithelial cells. 1595 23

A previously identified, calmodulin-binding, sea urchin sperm flagellar adenylyl cyclase (AC) was cloned and sequenced and found to be a homologue of mammalian sperm soluble adenylyl cyclase (sAC). Compared to the mammalian sAC, the sea urchin sAC (susAC) has several long amino acid insertions, some of which contain protein kinase A phosphorylation sites. The enzymatic activity of susAC shows a steep pH dependency curve, the specific activity doubling when the pH is increased from 7.0 to 7.5. This suggests that like sperm dynein ATPase, the susAC is probably activated by increases in intracellular pH occurring upon spawning into seawater and also when sperm respond to contact with the egg jelly layer. The susAC is strongly activated by manganese, but has low activity in magnesium. Gene database searches identified sAC homologues in species known to have cyclic AMP-dependent sperm motility. This implies (as shown in mouse) that susAC has a role in sperm motility, most probably through axonemal protein phosphorylation or ion channel regulation.
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PMID:A soluble adenylyl cyclase from sea urchin spermatozoa. 1597 50

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.
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PMID:Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation. 1612 82

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.
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PMID:Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP. 1759 88

In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H(+)-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endosomes. The specific PKA activator N(6)-monobutyryl-3'-5'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI (mPKI, 10 microM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chlorophenylthio)-2-O-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 microM), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 microM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with mPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.
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PMID:Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells. 1816 Apr 85

Acidic luminal pH and low [HCO(3)(-)] maintain sperm quiescent during maturation in the epididymis. The vacuolar H(+)-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO(3)(-), induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N(6)-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [(32)P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.
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PMID:AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells. 1921 18

Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signaling pathways involved. We show here that protein kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP), which activates mitochondrial PKA, does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO(2)-HCO(3)(-)-sAC-cAMP-PKA (mito-sAC) signaling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability.
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PMID:Cyclic AMP produced inside mitochondria regulates oxidative phosphorylation. 1925 71

Ion channels in mammal sperm, including Ca2+, Na+, K+, Cl- and HCO3- channels, each play a key role in the process of sperm capacitation. Ca2+, HCO3- and ROS, as signal molecules, activate soluble adenylyl cyclase (sAC) with the cooperation of cyclic adenosine monophosphate (cAMP), Ca2+ and intracellular pH and, via a cross talk between the cAMP/protein kinase A (PKA) and tyrosine phosphatase signaling pathways, promote the biological effect of sperm capacitation.
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PMID:[Regulation of ion and ion channels in sperm capacitation]. 1932 81

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.
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PMID:Soluble adenylyl cyclase controls mitochondria-dependent apoptosis in coronary endothelial cells. 1933 6


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