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)

We determined the effect of nucleotides and protein kinase A (PKA) on the Ca(2+)-dependent gating of the cloned intermediate conductance, Ca(2+)-dependent K(+) channel, hIK1. In Xenopus oocytes, during two-electrode voltage-clamp, forskolin plus isobutylmethylxanthine induced a Ca(2+)-dependent increase in hIK1 activity. In excised inside-out patches, addition of ATP induced a Ca(2+)-dependent increase in hIK1 activity (NP(o)). In contrast, neither nonhydrolyzable (AMP-PNP, AMP-PCP) nor hydrolyzable ATP analogs (GTP, CTP, UTP, and ITP) activated hIK1. The ATP-dependent activation of hIK1 required Mg(2+) and was reversed by either exogenous alkaline phosphatase or the PKA inhibitor PKI(5-24). The Ca(2+) dependence of hIK1 activation was best fit with a stimulatory constant (K(s)) of 350 nM and a Hill coefficient (n) of 2.3. ATP increased NP(o) at [Ca(2+)] >100 nM while having no effect on K(s) or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg(2+)-dependent fashion, being reversed by alkaline phosphatase. Neither PKI(5-24) nor CaMKII(281-309) or PKC(19-31) affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg(2+)- and PKI(5-24)-dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII(281-309) and PKC(19-31) had no effect on the ATP-dependent activation. The Ca(2+)-dependent activation (K(s) and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca(2+)-dependent and occurs via an increase in NP(o) without an effect on either Ca(2+) affinity or apparent cooperativity.
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PMID:Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. 1061 55

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.
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PMID:P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes. 1069 92

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.
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PMID:Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway. 1076 82

Isolated segments of porcine tracheal epithelium were mounted in Ussing chambers, current required to maintain transepithelial potential difference at 0 mV (short circuit current, I(SC)) was monitored and effects of nucleotides upon I(SC) were studied. Mucosal UTP (100 microM) evoked a transient rise in I(SC) that was followed by a sustained fall below basal I(SC) maintained for 30 min. Mucosal ATP (100 microM) also stimulated a transient rise in I(SC) but in contrast to UTP did not inhibit basal I(SC). Submucosal UTP and ATP both transiently increased I(SC). UTP-prestimulated epithelia were refractory to ATP but prestimulation with ATP did not abolish the response to UTP. The epithelia thus appear to express two populations of apical receptors allowing nucleotides to modulate I(SC). The UTP-induced rise was reduced by pretreatment with either bumetanide (100 microM), diphenylamin-2-carboxylic acid (DPC, 1 mM), or Cl(-) and HCO(3)(-)-free solution whilst the fall was abolished by amiloride pretreatment. Thapsigargin (0.3 microM) abolished the UTP-induced increase in I(SC) but not the subsequent decrease. Staurosporine (0.1 microM) inhibited basal I(SC) and blocked UTP-induced inhibition of I(SC). Inhibitors of either protein kinase C (PKC) (D-erythro sphingosine) or PKA (H89) had no effect. This study suggests that UTP stimulates Cl(-) secretion and inhibits basal Na(+) absorption. ATP has a similar stimulatory effect, which may be mediated by activation of P2Y(2) receptors and an increase in [Ca(2+)](in), but no inhibitory effect, which is likely mediated by activation of a pyrimidine receptor and possible inhibition of a protein kinase other than PKC or PKA.
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PMID:Differential effects of UTP and ATP on ion transport in porcine tracheal epithelium. 1080 75

We have previously demonstrated that Ca(2+)/calmodulin-dependent protein kinase (CaMK) mediates pyrimidinoceptor potentiation of LPS-elicited inducible nitric oxide synthase (iNOS) induction in murine J774 macrophages. In the present paper, we have explored the role of cyclo-oxygenase (COX)-dependent prostaglandin E(2) (PGE(2)) formation in this event. In J774 macrophages predominantly expressing P2Y(6) receptors, the simultaneous addition of UTP and lipopolysaccharide (LPS) resulted in potentiated increase in PGE(2) release. UTP-induced increased PGE(2) release was demonstrated by a concomitant increase in COX-2 protein expression, and was decreased by inhibitors specific for phosphatidylinositide-phospholipase C (PI-PLC), CaMK, protein kinase C (PKC), nuclear factor-kappa B (NF-kappaB) or COX-2. NS-398 (a selective COX-2 inhibitor) reduced LPS plus UTP-elicited iNOS induction and nitrite accumulation, supporting for the positive regulation of iNOS gene expression by endogenous PGE(2). Moreover, the cyclic AMP/PKA-dependent up-regulation of iNOS expression mediated by PGE(2) was drawn from the inhibitory effects of 2',5'-dideoxyadenosine, KT5720 and H-89. Exogenous PGE(2) induced NF-kappaB activation and potentiated nitrite accumulation in response to LPS. In addition to COX-2 induction, arachidonic acid (AA) release and steady-state mRNA levels of type V secretory phospholipase A(2) (sPLA(2)) and Ca(2+)-independent PLA(2) (iPLA(2)) were also increased in the presence of LPS and UTP; the LPS-induced increase in iPLA(2) activity was also potentiated by UTP. Taken together, we conclude that UTP-mediated COX-2 and iPLA(2) potentiation and PGE(2) formation contribute to the iNOS induction, and that CaMK activation is the primary step in the UTP enhancement of COX-2 induction.
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PMID:Pyrimidinoceptor potentiation of macrophage PGE(2) release involved in the induction of nitric oxide synthase. 1086 83

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.
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PMID:Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney. 1091 72

In apical membrane vesicles from beef tracheal epithelia expressing up to 30% of the proteins as functional cystic fibrosis transmembrane conductance regulator (CFTR)-- i.e. a voltage-independent and PKA-sensitive 36Cl- flux--an ATPase activity, different from P, F0F1 and V types, was reproducibly detected. Its specific activity averaged 20 micromol Pi h(-1) mg(-1) with an apparent affinity for ATP of 530 +/- 30 microM. Its possible involvement in CFTR functions was supported by (1) the linear relationship between the ATPase activity and the magnitude of 36Cl- fluxes (turnover rate: 3 ATP hydrolyzed per CFTR per second), (2) the same rank of potency of ATP, ITP, GTP, UTP and CTP to be hydrolyzed and to open CFTR chloride channels, (3) the similar and parallel inhibition of the ATPase and CFTR Cl- fluxes by NS004 (IC50: 60 microM) and (4) the potency of anti-R domain antibodies to increase by 18% the ATPase activity.
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PMID:Biochemical evidence for ATPase activity in CFTR-enriched apical membrane vesicles from tracheal epithelium. 1093 May 4

The properties of the inward-rectifying Cl- conductance in rat choroid plexus epithelial cells were investigated to allow comparisons to be made with ClC-2. All experiments were performed using the whole-cell configuration of the patch-clamp method. The conductance was transiently activated using an electrode solution which contained 375 nM catalytic subunit of protein kinase A (PKA). PKA failed to activate the conductance, however, when cells were pre-incubated with phorbol esters, which activate protein kinase C [1 microM phorbol 12-myristate 13-acetate (PMA) and 1 microM phorbol 12,13-dibutyrate (PDBu)]. Sustained activation of the conductance by PKA was observed in Ca2+-free conditions (5 mM BAPTA in the electrode solution), or when 100 nM calphostin C, a PKC inhibitor, was added to the electrode solution. The inward-rectifying Cl- conductance in choroid plexus is therefore similar to ClC-2 in that it is inhibited by PKC. The inward-rectifying conductance was blocked when Cd2+ (30 and 300 microM) and Zn2+ (1, 30 and 300 microM) were added to the bath solution. ClC-2 channels are also blocked by Zn2+ and Cd2+. The magnitude of the inward conductance was dependent on the concentration of ATP in the electrode solution. The conductance was not observed when ATP in the electrode was replaced with non-hydrolysable ATP analogues [adenosine 5'-O-(3-thiotriphosphate) (ATP[gamma-S]) and 5'-adenylylimidodiphosphate (AMP-PNP)), but it was supported by UTP and GTP. These data contrast with those of previous studies in which ClC-2 channels were activated in the absence of ATP. In conclusion, the inward-rectifying Cl- channel in rat choroid plexus shares some properties with ClC-2 (inhibition by PKC and block by divalent cations), but differs in that it depends on intracellular ATP.
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PMID:Properties of the inward-rectifying Cl- channel in rat choroid plexus: regulation by intracellular messengers and inhibition by divalent cations. 1104 61

Priming of NB4 promyelocytic cells with all-trans retinoic acid, followed by extracellular ATP in the presence of a phosphodiesterase inhibitor, elevated cAMP and activated protein kinase A. The order of potency for cAMP production was ATP (EC50 = 95 +/- 13 micromol/L) > ADP > AMP = adenosine. The order of potency of ATP analogues was 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (EC50 = 54 +/- 15 micromol/L) = adenosine 5'-O-(3-thio) triphosphate (EC50 = 66 +/- 4 micromol/L) > ATP > beta,gamma-methylene ATP (EC50 = 200 +/- 55 micromol/L). Adenosine 5'-O-thiomonophosphate and adenosine 5'-O-(2-thio) diphosphate inhibited ATP-induced cAMP production. Differentiation also occurred as measured by increased expression of CD11b and N-formyl peptide receptor and changes in cell morphology. UTP did not elevate cAMP or induce differentiation, indicating that P2Y2, P2Y4, and P2Y6 receptors were not involved. The P2Y11 receptor, a cAMP-linked receptor on promyelocytic HL-60 cells, was detected in NB4 cells by reverse transcription-polymerase chain reaction and northern blotting. This receptor has the same order of potency with respect to cAMP production as that observed in HL-60 cells.
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PMID:Extracellular ATP couples to cAMP generation and granulocytic differentiation in human NB4 promyelocytic leukaemia cells. 1105 May 28

1. In rat mesangial cells extracellular nucleotides were found to increase arachidonic acid release by a cytosolic phospholipase A(2) through the P2Y(2) purinergic receptor. 2. In this study we investigated the effects of ATP and UTP on interleukin-1ss (IL-1ss)-induced mRNA expression and activity of group IIA phospholipase A(2) (sPLA(2)-IIA) in rat mesangial cells. 3. Treatment of cells for 24 h with extracellular ATP potentiated IL-1ss-stimulated sPLA(2)-IIA induction, whereas UTP had no effect. 4. We obtained the following evidence that the P2Y(2) receptor is not involved in the potentiation of sPLA(2)-IIA induction: (i) ATP-gamma-S had no enhancing effect; (ii) suramin, a P(2) receptor antagonist, did not inhibit ATP-mediated potentiation; (iii) inhibition of degradation of extracellular nucleotides by the 5'-ectonucleotidase inhibitor AOPCP did not enhance sPLA(2)-IIA induction and (iv) adenosine deaminase treatment completely abolished the ATP-mediated potentiation of sPLA(2)-IIA induction. 5. In contrast, treatment of mesangial cells with adenosine or the A2A receptor agonist CGS 21680 mimicked the effects of ATP in enhancing IL-1ss-stimulated sPLA(2)-IIA induction, whereas the specific A2A receptor antagonist ZM 241385 completely abolished the potentiating effect of ATP or adenosine. 6. The protein kinase A inhibitor Rp-8-Br-cyclic AMPS dose-dependently inhibited the enhancing effect of ATP or adenosine indicating the participation of an adenosine receptor-mediated cyclic AMP-dependent signalling pathway. 7. These data indicate that ATP mediates proinflammatory long-term effects in rat mesangial cells via its degradation product adenosine through the A2A receptor resulting in potentiation of sPLA(2)-IIA induction.
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PMID:Potentiation of cytokine induction of group IIA phospholipase A(2) in rat mesangial cells by ATP and adenosine via the A2A adenosine receptor. 1115 59


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