Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ATP is released at the neuromuscular junction to regulate development and proliferation. The sequential expression of P2X and P2Y receptors has been correlated to these effects in many species and cell lines. We have therefore investigated ATP mediated signalling in differentiated primary human skeletal muscle cells. ATP was capable to trigger Ca2+ transients in these cells via P2Y receptors which were not attributable to Ca2+ influx via P2X receptors. Instead, ATP propagated the formation of inositol phosphate (IP) with an EC50 of 21.3 microM. The Ca2+ transient provoked by ATP was abrogated roughly 75% by the phospholipase C (PLC) inhibitor, U73122. Interestingly, the ryanodine sensitive Ca2+ pool was not involved in ATP triggered Ca2+ release. On mRNA level and by a pharmacological approach we confirmed the presence of the P2Y1, P2Y2, P2Y4 and P2Y6 receptors. Substantially, ATP activated IP formation via a P2Y1 receptor. In addition, ATP elicited extracellular signal regulated kinase (ERK)1/2 phosphorylation in a time and concentration dependent manner, again mainly via P2Y1 receptors. The ATP mediated ERK1/2 phosphorylation was strictly dependent on phospholipase C and PI3 kinase activity. Importantly, ATP mediated ERK1/2 phosphorylation was Ca2+ independent. This observation was corroborated by the finding that conventional protein kinase C inhibitors did not suppress ATP triggered ERK1/2 phosphorylation. Taken together, these observations highlight the importance of ATP as a co-neurotransmitter at the neuromuscular junction via dual signalling, i.e. IP3 receptor mediated Ca2+ transients and Ca2+ insensitive phosphorylation of ERK1/2.
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PMID:Extracellular ATP activates ERK1/ERK2 via a metabotropic P2Y1 receptor in a Ca2+ independent manner in differentiated human skeletal muscle cells. 1653 96

Glycogen synthase kinase (GSK)-3 was identified initially as an enzyme that regulates glycogen synthesis in response to insulin, but more recent studies indicate that it is also involved in numerous cellular processes, including cell survival, cell cycle regulation, proliferation, and differentiation. Because extracellular ATP exerts trophic actions on astrocytes, we investigated a possible signaling linkage from P2 purinergic receptors to GSK3beta. Addition of ATP to primary cultures of rat cortical astrocytes resulted in phosphorylation of Ser9 on GSK3beta and a concomitant decrease in GSK3 activity. UTP and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) increased phosphorylation of Ser9 on GSK3beta indicating that metabotropic P2Y and ionotropic P2X receptors are coupled to GSK3beta. Signaling studies showed that phosphorylation of Ser9-GSK3beta in response to ATP was inhibited by downregulation of protein kinase C (PKC) but not by blockade of Akt or p70 S6 kinase pathways. PKC also links P2 receptors to ERK in astrocytes, but inhibition of ERK signaling did not block phosphorylation of Ser9-GSK3beta stimulated by P2 receptors. Mechanical strain, which releases ATP, also stimulated Ser9 phosphorylation and this was attenuated by hydrolysis of extracellular ATP with apyrase or by blockade of P2 receptors. We conclude that P2 receptors are coupled to GSK3beta by a PKC-dependent pathway that is independent of Akt, p70 S6 kinase, and ERK pathways. These findings suggest that purinergic signaling contributes to the regulation of GSK3beta functions, one of which may be the response of astrocytes to CNS injury on release of ATP.
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PMID:P2 purinergic receptors signal to glycogen synthase kinase-3beta in astrocytes. 1681 Jun 87

Loss-of-function mutations of the parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease (AR-JP). Parkin was shown to function as a RING-type E3 ubiquitin protein ligase. However, the function of parkin in neuronal cells remains elusive. Here, we show that expression of parkin-potentiated adenosine triphosphate (ATP)-induced currents that result from activation of the P2X receptors which are widely distributed in the brain and involved in neurotransmission. ATP-induced inward currents were measured in mock-, wild-type or mutant (T415N)-parkin-transfected PC12 cells under the conventional whole-cell patch clamp configuration. The amplitude of ATP-induced currents was significantly greater in wild-type parkin-transfected cells. However, the immunocytochemical study showed no apparent increase in the number of P2X receptors or in ubiquitin levels. The increased currents were attenuated by inhibition of cAMP-dependent protein kinase (PKA) but not protein kinase C (PKC) or Ca2+ and calmodulin-dependent protein kinase (CaMKII). ATP-induced currents were also regulated by phosphatases and cyclin-dependent protein kinase 5 (CDK5) via dopamine and cyclic AMP-regulated phosphoprotein (DARPP-32), though the phosphorylation at Thr-34 and Thr-75 were unchanged or rather attenuated. We also tried to investigate the effect of alpha-synuclein, a substrate of parkin and also forming Lysine 63-linked multiubiquitin chains. Expression of alpha-synuclein did not affect the amplitude of ATP-induced currents. Our finding provides the evidence for a relationship between parkin and a neurotransmitter receptor, suggesting that parkin may play an important role in synaptic activity.
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PMID:Parkin potentiates ATP-induced currents due to activation of P2X receptors in PC12 cells. 1682 4

At the neuromuscular junction, ATP is co-released with the neurotransmitter acetylcholine (ACh) and once in the synaptic space, it is degraded to the presynaptically active metabolite adenosine. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of extracellular ATP (100 muM) and the slowly hydrolysable ATP analog 5'-adenylylimidodiphosphate lithium (betagamma-imido ATP) (30 muM) on miniature end-plate potential (MEPP) frequency. We found that application of ATP and betagamma-imido ATP decreased spontaneous secretion by 45.3% and 55.9% respectively. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A(1) adenosine receptor antagonist and alpha,beta-methylene ADP sodium salt (alphabeta-MeADP), which is an inhibitor of ecto-5'-nucleotidase, did not prevent the inhibitory effect of ATP, demonstrating that the nucleotide is able to modulate spontaneous ACh release through a mechanism independent of the action of adenosine. Blockade of Ca(2+) channels by both, Cd(2+) or the combined application of nitrendipine and omega-conotoxin GVIA (omega-CgTx) (L-type and N-type Ca(2+) channel antagonists, respectively) prevented the effect of betagamma-imido ATP, indicating that the nucleotide modulates Ca(2+) influx through the voltage-dependent Ca(2+) channels related to spontaneous secretion. betagamma-Imido ATP-induced modulation was antagonized by the non-specific P2 receptor antagonist suramin and the P2Y receptor antagonist 1-amino-4-[[4-[[4-chloro-6-[[3(or4)-sulfophenyl] amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl] amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid (reactive blue-2), but not by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt (PPADS), which has a preferential antagonist effect on P2X receptors. Pertussis toxin and N-ethylmaleimide (NEM), which are blockers of G(i/o) proteins, prevented the action of the nucleotide, suggesting that the effect is mediated by P2Y receptors coupled to G(i/o) proteins. The protein kinase C (PKC) antagonist chelerythrine and the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) occluded the effect of betagamma-imido ATP, while the protein kinase A (PKA) antagonist KT-5720 and the inhibitor of the calcium/calmodulin-dependent protein kinase II (CAMKII) KN-62 failed to do so. betagamma-Imido ATP did not affect 10, 15 and 20 mM K(+)-evoked release and application of reactive blue-2 before incubation in high K(+) induced a higher asynchronous secretion. Thus, our results show that at mammalian neuromuscular junctions, ATP induces presynaptic inhibition of spontaneous ACh release due to the modulation of Ca(2+) channels related to tonic secretion through the activation of P2Y receptors coupled to G(i/o) proteins. We also demonstrated that at increasing degrees of membrane depolarization evoked by K(+), endogenously released ATP induces presynaptic inhibition as a means of preventing excessive neurotransmitter secretion.
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PMID:Presynaptic inhibition of spontaneous acetylcholine release mediated by P2Y receptors at the mouse neuromuscular junction. 1684 2

This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of [(3)H]thymidine/5-bromo-2'-deoxyuridine incorporation and the number of cells in both a time- and dose-dependent manner. AMP-CPP (a P2X(1) and P2X(3) agonist), ATP-gammaS (a P2Y agonist), and 2-methylthio-ATP (a P2X and P2Y agonist) stimulated [(3)H]thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. Reverse transcription-polymerase chain reaction analysis revealed P2X(3), P2X(4), P2Y(1), and P2Y(2) expression in mouse ESCs. Adenylate cyclase inhibitor (SQ 22536), phospholipase C inhibitors (neomycin or U 73122), and protein kinase C (PKC) inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [(3)H]thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC alpha, delta, and zeta from the cytosol to the membrane compartment. ATP and its agonists increased [Ca(2+)](i). In addition, the ATP-induced increase in [(3)H]thymidine incorporation was completely inhibited by a combination of EGTA (extracellular Ca(2+) chelator) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (intracellular Ca(2+) chelator). ATP phosphorylated Akt and p44/42 mitogen-activated protein kinases (MAPKs) in a time-dependent manner, and either suramin or reactive blue 2 (RB2) blocked the ATP-induced phosphorylation of Akt. Suramin, RB2, the phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [(3)H]thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and the MAPK kinase inhibitor (PD 98059). Suramin, RB2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 levels. In conclusion, ATP stimulates mouse ESC proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors.
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PMID:ATP stimulates mouse embryonic stem cell proliferation via protein kinase C, phosphatidylinositol 3-kinase/Akt, and mitogen-activated protein kinase signaling pathways. 1691 26

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.
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PMID:Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages. 1698 Feb 98

Differentiation of skeletal muscle cells both in vivo and in vitro is accompanied by the development of voltage-dependent processes and alterations in purinergic signalling. To date at least two independent methods have been used to induce differentiation in primary cultures, namely, appropriate modification of culturing conditions and overexpression of specific protein kinase C (PKC) isoenzymes. Here we characterize and compare the development of purinergic and depolarization-dependent alterations using these two methods to induce differentiation in C2C12 cells. We demonstrate that depolarization- and ATP-evoked Ca(2+) responses underwent functional development during differentiation, and the characteristics of this progress were dependent on the actual differentiation-promoting stimulus. Overexpression of PKCalpha anticipated the appearance of robust increases in the intracellular calcium concentration upon ATP administration but failed to do so after depolarizing stimuli. Moreover, the first phase of the biphasic ATP-induced response observed in differentiated myotubes induced by culturing was not present in differentiated PKCalpha-overexpressing cells, suggesting that although purinergic signalling developed very early, purinergic stimuli failed to activate the voltage-dependent mechanisms of these cells even at subsequent stages of differentiation. Disruption of the coupling of purinergic signalling to depolarization-activated mechanisms may be explained by our observations that PKCalpha-overexpression changed the purinergic receptor pattern of immature myoblasts differently from what was seen in the course of culturing-induced differentiation. PKCalpha-specific alterations were characterized by the lack of increase in the expression of P2X(7) receptors and the failure of P2Y(4) receptors to appear and P2Y(2) receptors to disappear. The effects of PKCalpha-overexpression were proven to be specific since the overexpression of the hyperproliferative isoenzyme PKCdelta failed to induce any of the changes promoted by PKCalpha. Our data suggest that the method of inducing differentiation in skeletal muscle cells modifies not only the course of development but also the interaction of depolarization-dependent and purinergic pathways.
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PMID:Differences in purinergic and voltage-dependent signalling during protein kinase Calpha overexpression- and culturing-induced differentiation of C2C12 myoblasts. 1705 44

P2X receptors (P2XR) act as ligand-gated, cation-selective ion channels. A common characteristic of all seven P2X family members is a conserved consensus sequence for protein kinase C (PKC)-mediated phosphorylation in the intracellular N-terminus of the receptor. Activation of PKC has been shown to enhance currents through P2X(3)R, however the molecular mechanism of this potentiation has not been elucidated. In the present study we show that activation of PKC can enhance adenosine triphosphate (ATP)-mediated Ca(2+) signals approximately 2.5-fold in a DT-40 3KO cell culture system (P2 receptor null) transiently overexpressing P2X(3)R. ATP-activated cation currents were also directly studied using whole cell patch clamp techniques in HEK-293 cells, a null background for ionotropic P2XR. PKC activation resulted in a approximately 8.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X(3)R cDNA, but had no effect on currents through either P2X(4)R- or P2X(7)R-transfected cells. P2X(3)R-transfected HEK-293 cells were metabolically labeled with (32)PO(4)(-) and following treatment with phorbol-12-myristate-13-acetate (PMA) and subsequent immunoprecipitation, there was no incorporation of (32)PO(4)(-) in bands corresponding to P2X(3)R. Similarly, in vitro phosphorylation experiments, utilizing purified PKC catalytic subunits failed to establish phosphorylation of either P2X(3)R or P2X(3)R-EGFP. These data indicate that PKC activation can enhance both the Ca(2+) signal as well as the cation current through P2X(3)R, however it appears that the regulation is unlikely to be a result of direct phosphorylation of the receptor.
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PMID:Protein kinase C regulation of P2X3 receptors is unlikely to involve direct receptor phosphorylation. 1705 68

Phospholipase D (PLD) is a ubiquitous enzyme that can be activated by extracellular adenosine 5'-triphosphate (ATP) or phorbol 12-myristate 13-acetate (PMA) in B-lymphocytes from subjects with chronic lymphocytic leukaemia (CLL). In this study, ATP- but not PMA-induced PLD stimulation in CLL B-lymphocytes was abolished in the presence of an anti-P2X(7) receptor monoclonal antibody, as well as in B-lymphocytes from CLL subjects homozygous for the Glu(496) to Ala loss-of-function P2X(7) polymorphism. Rottlerin, an inhibitor of novel protein kinase C (PKC) isoforms, but not GF 109203X, an inhibitor of conventional PKC isoforms, impaired the ATP-stimulated PLD activity in CLL B-lymphocytes. In contrast, both inhibitors impaired PLD activity stimulated by PMA, a known mediator of PKC activation. The inhibition of P2X(7)-stimulated PLD activity by rottlerin was attributed to a target downstream of P2X(7) activation, as the ATP-mediated (86)Rb(+) efflux from CLL B-lymphocytes was not altered in the presence of rottlerin. Our results indicate a possible role for novel PKC isoforms in the regulation of P2X(7)-mediated PLD activity.
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PMID:Rottlerin inhibits P2X(7) receptor-stimulated phospholipase D activity in chronic lymphocytic leukaemia B-lymphocytes. 1713 Sep 1

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X(7) ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X(7) receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y(2) in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y(2) expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y(2) sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y(2) mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X(7)-antagonists oATP and BBG. The BzATP-evoked P2Y(2) receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y(2)siRNA, thus supporting evidence of increased P2Y(2) activity. To further investigate the mechanisms by which P2X(7) receptors mediated the P2Y(2) mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y(2) mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X(7)-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X(7) receptors in astrocytes enhances P2Y(2) mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways.
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PMID:Activation of P2X(7) receptors stimulates the expression of P2Y(2) receptor mRNA in astrocytes cultured from rat brain. 1762 42


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