Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antibodies that recognize the alpha 2 delta and alpha 1 subunits of skeletal muscle L-type calcium channels have been used to investigate the subunit components and phosphorylation of omega-conotoxin (omega-CgTx)-sensitive N-type calcium channels from rabbit brain. Photolabeling of the N-type channel with a photoreactive derivative of 125I-omega-CgTx results in the identification of a single polypeptide of 240 kDa. MANC-1, a monoclonal antibody recognizing alpha 2 delta subunits of L-type calcium channels from skeletal muscle, immunoprecipitates the omega-CgTx-labeled 240-kDa polypeptide and approximately 6% of the digitonin-solubilized 125I-omega-CgTx-labeled N-type channels. MANC-1 also immunoprecipitates a phosphoprotein of 240 kDa that comigrates with 125I-omega-CgTx-labeled N-type calcium channels, but not with L-type calcium channels, in sucrose gradients. Both cAMP-dependent protein kinase and protein kinase C are effective in the phosphorylation of this polypeptide. Similar to the alpha 1 subunits of skeletal muscle L-type calcium channels, the immunoprecipitation of the 240-kDa phosphoprotein by MANC-1 is prevented by the detergent Triton X-100. Anti-CP-(1382-1400), an antipeptide antibody against a highly conserved segment of the alpha 1 subunits of calcium channels, immunoprecipitates the 240-kDa phosphopeptide in Triton X-100. The 240-kDa protein is phosphorylated to a stoichiometry of approximately 1 mol of phosphate/mol of omega-CgTx-binding N-type calcium channels by both cAMP-dependent protein kinase and protein kinase C. Our results show that the 240-kDa polypeptide is an alpha 1-like subunit of an omega-CgTx-sensitive N-type calcium channel. The N-type calcium channels containing this subunit are phosphorylated by cAMP-dependent protein kinase and protein kinase C and contain noncovalently associated alpha 1-like and alpha 2 delta-like subunits as part of their oligomeric structure.
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PMID:Phosphorylation of an alpha 1-like subunit of an omega-conotoxin-sensitive brain calcium channel by cAMP-dependent protein kinase and protein kinase. 165 16

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.
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PMID:The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells. 166 7

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.
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PMID:Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells. 166 12

A monoclonal antibody against phospholamban has been reported to increase Ca2+ uptake by cardiac sarcoplasmic reticulum. We compared the effect of this antibody on Ca2+ pump ATPase activity of cardiac sarcoplasmic reticulum vesicles to the effect of cAMP-dependent phosphorylation of phospholamban. The antibody markedly stimulated the Ca(2+)-dependent ATPase activity in parallel to the increase in Ca2+ uptake by cardiac sarcoplasmic reticulum. When the Ca(2+)-dependent profile of the ATPase activity was compared, the KCa was shifted from 1.24 to 0.62 microM by the antibody, whereas cAMP-dependent phosphorylation of phospholamban shifted the KCa to 0.84 microM. When cardiac sarcoplasmic reticulum vesicles were treated with both cAMP-dependent protein kinase and the antibody, the stimulation was the same as that with the antibody alone. Thus, the Ca2+ pump ATPase seems to be fully activated by the antibody. The stoichiometry between Ca2+ uptake and ATPase rate was around 1 and no significant change was observed by the treatment with the antibody. Therefore, the stimulation of Ca2+ uptake of cardiac sarcoplasmic reticulum by the antibody occurred by the stimulation of Ca2+ pump ATPase, not by other mechanisms such as channel activity of phospholamban. These results indicate that the binding of the antibody to phospholamban produces essentially the same mode of action on Ca2+ pump ATPase as that of phospholamban phosphorylation. The antibody and phospholamban phosphorylation appear to release the inhibitory action of phospholamban on Ca2+ pump ATPase, resulting in the stimulation of Ca2+ pump.
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PMID:Effects of monoclonal antibody against phospholamban on calcium pump ATPase of cardiac sarcoplasmic reticulum. 166 13

1. The intracellular mechanism of heterosynaptic facilitation (HSF) formation in identified neurons from the snail Planorbis corneus has been studied. 2. Facilitation of excitatory postsynaptic currents (EPSC) were induced by (a) stimulation of pallial nerve, and (b) addition to extracellular saline of serotonin, NaF, papaverine, theophylline, caffeine or dibutril-cAMP. 3. A depression of EPSC in solutions containing tolbutamide, a cAMP-dependent protein kinase inhibitor was observed. 4. In some cases the similar facilitation or depression of the current induced by acetylcholine application (ACh-current) was found in the same neuron. 5. The effects on ACh-current were distorted in solutions containing caffeine, a well-known activator of calcium ions release from the intracellular depot. 6. According to our findings, we suggest that adenylate cyclase activity of postsynaptic cells could underlie the formation of HSF and it is likely that this activity was modulated by intracellular concentration of calcium ions.
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PMID:Analysis of heterosynaptic facilitation in identified giant neurons from cerebral ganglion of the pond snail Planorbis corneus. 167 48

1. The activation process of Ca(2+)-dependent potassium channel was studied electrophysiologically and pharmacologically using identified neurons of the land snail, Euhadra peliomphala. 2. Ca(2+)-mediated delayed outward K current (IKD) was dose-dependently reduced by the calmodulin inhibitors, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, week) and N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7, potent). These antagonists also caused a slight membrane depolarization and increase in impulse discharge frequency with decrease in the amplitude of both action potential and after hyperpolarization. 3. The cAMP-dependent protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide (H-8) did not produce any significant effect on IKD and membrane potential. 4. Calmodulin, when injected into the neuron which had been treated with either W-5 or W-7, transiently restored the suppressed IKD nearly to the pretreatment level, and caused hyperpolarization of the cell. In contrast, calcium chloride, intracellularly injected in the same way, had little effect on both the IKD and the membrane potential shifted by these antagonists. 5. Intracellular injection of kinase II, a Ca2+/calmodulin-dependent protein kinase, caused an increase in the IKD and membrane hyperpolarization. Similar but weak effects were produced when a catalytic subunit (CS) of cAMP-dependent protein kinase was intracellularly injected. However, the neurons pretreated with W-7 no longer had any detectable increase in the IKD and hyperpolarization of the membrane. 6. These results suggest the possibility that Ca2+/camodulin-dependent protein phosphorylation may finally mediate the activation of a certain number of potassium channels.
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PMID:Activation process of calcium-dependent potassium channel in Euhadra neurons: involvement of calcium/calmodulin and subsequent protein phosphorylation. 167 36

A model that summarizes some of the neural and molecular mechanisms contributing to short- and long-term sensitization is shown in Figure 14. Sensitizing stimuli lead to the release of a modulatory transmitter such as 5-HT. Both serotonin and sensitizing stimuli lead to an increase in the synthesis of cAMP and the modulation of a number of K+ currents through protein phosphorylation. Closure of these K+ channels leads to membrane depolarization and the enhancement of excitability. An additional consequence of the modulation of the K+ currents is a reduction of current during the repolarization of the action potential, which leads to an increase in its duration. As a result, Ca2+ flows into the cell for a correspondingly longer period of time, and additional transmitter is released from the cell. Modulation of the pool of transmitter available for release (mobilization) also appears to occur as a result of sensitizing stimuli. Recent evidence indicates that the mobilization process can be activated by both cAMP-dependent protein kinase and protein kinase C. Thus, release of transmitter is enhanced not only because of the greater influx of Ca2+ but also because more transmitter is made available for release by mobilization. The enhanced release of transmitter leads to enhanced activation of motor neurons and an enhanced behavioral response. Just as the regulation of membrane currents is used as a read out of the memory for short-term sensitization, it also is used as a read out of the memory for long-term sensitization. But long-term sensitization differs from short-term sensitization in that morphological changes are associated with it, and long-term sensitization requires new protein synthesis. The mechanisms that induce and maintain the long-term changes are not yet fully understood (see the dashed lines in Fig. 14) although they are likely to be due to direct interactions with the translation apparatus and perhaps also to events occurring in the cell nucleus. Nevertheless, it appears that the same intracellular messenger, cAMP, that contributes to the expression of the short-term changes, also triggers cellular processes that lead to the long-term changes. One possible mechanism for the action of cAMP is through its regulation of the synthesis of membrane modulatory proteins or key effector proteins (for example, membrane channels). It is also possible that long-term changes in membrane currents could be due in part to enhanced activity of the cAMP-dependent protein kinase so that there is a persistent phosphorylation of target proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Neural and molecular bases of nonassociative and associative learning in Aplysia. 167 7

1. The involvement of protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity (BA) was evaluated in identified neurons of the snail. Euhadra peliomphala by examining the effect of various protein kinases and their inhibitors on the membrane properties induced by PTZ. 2. In neurons which normally exhibited spontaneous regular firing, PTZ elicited BA, the negative slope resistance (NSR) in the steady-state current (I)-voltage (V) relationship and a reduction of the delayed potassium current (IKD) in a dose-dependent manner. These were inhibited by the cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide. 3. Intracellular injection of catalytic subunit (CS) of cAMP-dependent protein kinase enhanced PTZ-induced NSR and reduction of IKD, as well as a conversion of the BA to a long-lasting depolarization of the membrane, whereas a saturating dose of the CS occluded PTZ action on the NSR and IKD. 4. Ca2+/calmodulin-dependent protein kinase II (CaMKII), when intracellularly injected during the depolarizing phase of PTZ-induced bursting cycle, changed to a prolonged hyperpolarization of the membrane. This kinase also restored the PTZ-suppressed IKD nearly to the pre-PTZ level. However, when intracellular injection of CaMKII and application of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin inhibitor, to the inside and outside of the neuron were simultaneously carried out, neither post-burst hyperpolarization nor restoration of the IKD was observed. 5. Intracellular injection of calmodulin, together with calcium chloride, had little effect on both the BA and reduction of IKD induced by PTZ. 6. Simultaneous application of 40 microM 1-(5-isoquinolinsulfonyl)-2-methylpiperazine, which selectively suppressed the phosphatidylserine-dependent protein phosphorylation in extracts from Euhadra ganglia, to both the inside and outside of the neuron, did not produce any significant change in the membrane properties induced by PTX. Intracellular injection of protein kinase C also brought about no effect. 7. These findings suggest that PTZ stimulates cAMP-dependent protein phosphorylation which, in turn, is involved in the development of NSR and reduction of IKD, leading to the depolarization of the membrane. In addition, we propose that the Ca2+ ions, increased during the depolarizing phase of the BA cycle, form a Ca2+/calmodulin complex and subsequent protein phosphorylation, coupled with the opening of potassium channels, leading to the membrane hyperpolarization.
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PMID:The molecular mechanism underlying pentylenetetrazole-induced bursting activity in Euhadra neurons: involvement of protein phosphorylation. 168 38

Cytosol of the guinea pig adrenals was found to contain a protein kinase which was dependent on the presence of both calcium and phospholipids (phosphatidylserine and diolein), i.e., calcium/phospholipid-dependent protein kinase (protein kinase C). The peak of protein kinase C was separated from type II cAMP-dependent protein kinase by DE-52 chromatography. 12-0-Tetradecanoylphorbol-13-acetate (TPA) caused dose-dependent increments of cortisol formation without affecting cAMP formation by guinea pig adrenocortical cells as well as angiotensin II did. TPA-activated cortisol production was blocked by the addition of aminoglutethimide and cycloheximide, suggesting that the site of action of TPA might be located at a point before the production of pregnenolone in the mitochondria. Since TPA showed an increase in the cortisol production, protein kinase C may be involved in modulating steroidogenesis in the guinea pig adrenals in addition to the classical cAMP-dependent protein kinase pathway.
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PMID:Involvement of protein kinase C in the regulation of cortisol production by guinea pig adrenocortical cells. 168 88

Exposing primary cultures of cerebellar granule neurons to 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 hr decreases the Ca2+/phosphatidylserine/diolein-dependent protein kinase C (PKC; ATP:protein phosphotransferase, EC 2.7.1.37) by approximately 90% in the 100,000 x g supernatant and pellet fractions of neuronal culture homogenates. Immunoblot analysis of the homogenates with polyclonal antibodies raised against either the beta-type PKC peptide or total rat brain PKC reveals a virtual loss of 78-kDa PKC immunoreactivity in the supernatant and a marked decrease of PKC immunoreactivity in the pellet. Exposure of the cultures to 50 microM glutamate for 15 min (no Mg2+) induces the translocation of supernatant PKC immunoreactivity to the pellet. Such translocation persists after glutamate withdrawal and is followed by a progressive increase in neuronal death, which begins 2 hr later. Neuronal death approaches completion in about 24 hr. PMA-induced down-regulation of PKC decreases glutamate-elicited neurotoxicity. Yet, the culture exposure to 100 nM PMA fails to decrease the high-affinity binding of [3H]glutamate to neuronal membranes and does not reduce glutamate-induced activation of ionotropic or metabolotropic receptors (assayed as total membrane current measured in whole-cell voltage-clamped neurons, 45Ca2+ uptake in intact monolayers, inositolphospholipid hydrolysis, and transcriptional activation and translation of c-fos mRNA). Moreover, the immediate cell-body swelling and activation of spectrin proteolysis elicited by glutamate remain unchanged. On the other hand, PMA-induced PKC down-regulation reduces any increase in 45Ca2+ uptake or Ca2(+)-dependent proteolysis (measured as spectrin degradation) after glutamate withdrawal. These results support the view that PKC translocation is operative in glutamate-induced destabilization of cytosolic ionized Ca2+ homeostasis and neuronal death.
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PMID:Down-regulation of protein kinase C protects cerebellar granule neurons in primary culture from glutamate-induced neuronal death. 168 50


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