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)

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.
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PMID:Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. 164 31

We recently reported that cultured gland serous cells release chondroitin sulfate proteoglycans (CSPGs) in response to beta-adrenergic agonists. In this study, we analyzed this regulatory pathway and other cellular mechanisms responsible for CSPG secretion. We show the following. 1) Isoproterenol increased CSPG secretion in a concentration-dependent manner, with maximal stimulation (50%) obtained at 10(-5) M; at this concentration, the beta-agonist also stimulated protein kinase A (PKA) by 50%, whereas it increased cellular adenosine 3',5'-cyclic monophosphate (cAMP) content by 300%. 2) Phenylephrine (10(-5) M), 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (1.6 x 10(-7) M), and A23187 (10(-6) M) also stimulated CSPG secretion; this stimulation was concomitant with protein kinase C (PKC) translocation from cytosol to membrane, was blocked by sphingosine (2 x 10(-5) M), and was additive with that elicited by isoproterenol. 3) All PKC activators potentiated the isoproterenol-induced increased in cAMP accumulation without modifying the activation of PKA elicited by the beta-agonist. Our results indicate that although the signaling pathways triggered by alpha- and beta-adrenergic agonists converge at the level of adenylate cyclase in tracheal serous cells, PKA and PKC independently regulate CSPG secretion.
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PMID:Regulation of secretion in cultured tracheal serous cells by protein kinases A and C. 165 65

Brief incubation of a mammary epithelial cellular preparation from lactating rats with isoprenaline is shown to result in major re-distribution of the activity of cyclic AMP-dependent protein kinase (measured in the presence of saturating cyclic AMP) within the cell. Activity in the soluble fraction was halved and a corresponding increase in the sedimentable activity occurred. Similar effects were observed when cell-free extracts were treated with cyclic AMP in the presence of inhibitors of phosphodiesterase and subsequently fractionated by a simple one-step centrifugation procedure. The concentration of the catalytic subunit of cyclic AMP-dependent protein kinase, assessed by quantitative Western blot analysis, did not reflect these activity changes. Quantitation of the regulatory subunits (R-I plus R-II) of A-kinase enabled independent assessment of the possible total A-kinase holoenzyme in mammary epithelial cells and was in reasonable agreement with the measured total A-kinase activity. Isoprenaline selectively increased the apparent mean specific catalytic activity of the C-subunit in the particulate fraction.
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PMID:Cyclic AMP-dependent protein kinase in mammary epithelial cells; activity and subcellular distribution are acutely modulated by isoprenaline. 165 69

The regulation of the plasma membrane Ca2+ pump by hormones via phosphorylation in intact cells has not been clearly established. We now present evidence that the Ca2+ pump is phosphorylated on both serine and threonine residues in unstimulated and stimulated cultured rat aortic endothelial cells. Among the stimuli tested, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was most potent and increased the level of phosphorylation threefold, while the cAMP-dependent protein kinase activator 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated the phosphorylation 1.6-fold. Two-dimensional tryptic phosphopeptide maps of the Ca2+ pump from unstimulated and CPT-cAMP-stimulated cells have identical patterns (five phosphopeptides) while PMA-stimulated cells have three additional phosphopeptides. Isoproterenol-, ATP-, angiotensin II-, and bradykinin-stimulated cells also have increased levels of Ca2+ pump phosphorylation. Stimuli-induced phosphorylation of the Ca2+ pump was rapid (5-10 min) and was concomitant with stimulated calcium efflux from the same cells. This is the first direct evidence that the plasma membrane Ca2+ pump in intact cells is regulated by various hormones or agonists via cAMP-dependent protein kinase or protein kinase C phosphorylation.
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PMID:Hormone-induced phosphorylation of the plasma membrane calcium pump in cultured aortic endothelial cells. 165 40

1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca(2+)-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)-dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca(2+)-dependent inactivation is entirely sensitive to beta-adrenergic stimulation. These facts suggest that either Ca(2+)-dependent inactivation results from Ca(2+)-dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)-dependent inactivation is modulated by protein kinase A.
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PMID:Ca2+ and voltage inactivate Ca2+ channels in guinea-pig ventricular myocytes through independent mechanisms. 166 48

The effect of the beta-adrenergic receptor agonist isoproterenol on guanine nucleotide-dependent phospholipase C (PLC) activity was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes. In the presence of guanosine 5'-(gamma-thiotriphosphate) (GTP[S]) isoproterenol caused a dose-dependent stimulation of [3H]inositol phosphate ([3H]InsP) formation. The activation of PLC by GTP[S] occurred after an initial lag period of 1-2 min and was followed by a sustained rate of [3H]InsP formation which remained linear for 4-5 min. Isoproterenol decreased the lag period for GTP[S]-induced [3H]InsP formation and increased PLC activity at all time points following this lag. Consequently, isoproterenol shifted the dose-response curve for GTP[S] to the left (10-fold) and increased the maximal response. The EC50 value for isoproterenol-induced activation of PLC was 104 +/- 17 nM. Isoproterenol also potentiated GTP-dependent PLC activity but was ineffective in stimulating the enzyme in the presence of AIF4-. The PLC activation by isoproterenol was completely inhibited by propanolol and atenolol but was unaffected by prazosin or yohimbine. Although GTP[S] and isoproterenol could increase cAMP formation in this membrane preparation, the isoproterenol-induced stimulation of PLC occurred in the absence of ATP and was independent of cAMP formation. Furthermore, addition of cAMP, 8-bromo-cAMP, forskolin, or either the regulatory or catalytic subunits of cAMP-dependent protein kinase failed to stimulate [3H]InsP formation and had no effect on the responses elicited by GTP[S] and isoproterenol. Isoproterenol also stimulated [3H]InsP2 and [3H]InsP3 production in intact erythrocytes. Cholera toxin had no effect on [3H]InsP formation in the intact cells under conditions where it stimulated cAMP accumulation. In addition, the activation of PLC by GTP[S] and isoproterenol was unaffected in membranes prepared from cholera toxin-treated erythrocytes. These data demonstrate that stimulation of turkey erythrocyte beta-adrenergic receptors by isoproterenol results in a direct activation of guanine nucleotide-dependent PLC.
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PMID:Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes. 167 88

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.
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PMID:Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol. 176 64

1. In the adipocyte, phosphorylation/dephosphorylation of regulatory proteins is a common mechanism of metabolic regulation. We have observed a very prominent phosphoprotein doublet of 61 kDa and 63 kDa in rat adipocytes that is markedly responsive to hormones. The 63 kDa band was the predominant phosphoprotein in the cell in response to 0.1 microM-isoprenaline, whereas the 61 kDa band was nearly absent. Insulin alone did not alter 32P incorporation into the doublet, but partially counteracted the effects of isoprenaline, decreasing label in the 63 kDa band by as much as 50% and resulting in the reappearance of the 61 kDa band. 2. Subcellular fractionation demonstrated that both phosphoprotein bands were fat-associated. Neither insulin nor isoprenaline altered this localization. Peptide maps (one-dimensional) of the 61/63 kDa bands demonstrated close sequence similarity. Amino acid analysis revealed the presence of phosphoserine and phosphothreonine. The latter was more prominent in the 61 kDa band. Isoprenaline caused an absolute increase in both phosphoamino acids. 3. Permeabilization of 32P-labelled isoprenaline-treated cells with digitonin initiated rapid dephosphorylation of the 63 kDa band, with reappearance of the 61 kDa band. Insulin increased the rate of dephosphorylation by 2-3-fold when present with isoprenaline before permeabilization. 4. In permeabilized adipocytes, cyclic AMP (1 microM-1 mM) increased phosphorylation of the 61/63 kDa doublet by 4-10-fold in the presence of [gamma-32P]ATP, but insulin had no effect. 5. We conclude that this prominent phosphoprotein, migrating as a 61/63 kDa doublet, is coupled to the cyclic AMP-dependent protein kinase and is associated with an insulin-stimulated phosphoprotein phosphatase activity. This fat-associated phosphoprotein, which is under counter-regulatory hormonal control, may play a role in hormone-dependent lipid metabolism.
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PMID:Counter-regulation by insulin and isoprenaline of a prominent fat-associated phosphoprotein doublet in rat adipocytes. 184 60

MEPP frequency (f) was measured in mouse phrenic nerve hemidiaphragm preparations during exposure to adrenoceptor agonist and antagonist drugs. Epinephrine, norepinephrine (NE), and phenylephrine caused a concentration-dependent increase in frequency that was blocked by prazosin but not by yohimbine or nadolol. Isoproterenol had no effect on MEPP(f). The response to NE was not affected by prior incubation of the tissues with pertussis toxin. The response was, however, reduced or abolished by prior exposure to drugs, the actions of which include protein kinase inhibition, and also to a calmodulin inhibitory concentration of W-7. H-7, an inhibitor of protein kinase C and of cyclic nucleotide-dependent kinases, was ineffective. The response to NE was enhanced by 10 mM Li+. The data indicate the existence of a presynaptic alpha 1-adrenoceptor in the motor neuron terminal and suggest that modulation of transmitter release might be mediated by inositol triphosphate liberation, Ca2+ release into the cytosol and activation of a calmodulin-dependent system.
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PMID:Transduction of the modulatory effect of catecholamines at the mammalian motor neuron terminal. 184 23

1. Receptor-mediated modulation of the delayed outward potassium current (IK) was investigated in guinea-pig single ventricular cells by using whole-cell voltage clamp and intracellular dialysis. 2. Isoprenaline increased IK in a dose-dependent manner with a half-maximum dose of 1.8 X 10(-8) M. Isoprenaline (10(-6) M) maximally increased IK by a factor of 2.85. This effect did not depend on the concentration of intracellular Ca2+ [( Ca2+]i). 3. External application of 10(-5) M-forskolin and internal application of 5 X 10(-5) M-cyclic AMP or 5 X 10(-6) M of the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) also increased IK about 3-fold. The effect of isoprenaline on IK was masked by previous application of cyclic AMP. 4. All the above phosphorylating agents increased the amplitude of IK without a significant change in the current kinetics. 5. In the presence of 10(-5) M-forskolin, an additional application of 10(-8) M-12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C (PKC), produced a further increase in IK, suggesting that the active sites of PKA and PKC on the IK channel are different. 6. Acetylcholine (10(-6) M) suppressed IK when the current was previously enhanced by 2 X 10(-8) M-isoprenaline, but had little effect in the absence of isoprenaline. 7. We conclude that beta-adrenergic modulation of IK is mediated by cyclic AMP-dependent phosphorylation but not by an increase in [Ca2+]i, that PKA and PKC enhance IK independently, and that acetylcholine antagonizes beta-adrenergic stimulation of IK most probably by inhibiting adenylate cyclase.
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PMID:Mechanism of receptor-mediated modulation of the delayed outward potassium current in guinea-pig ventricular myocytes. 216 57


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