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 mechanisms by which an activator of cyclic AMP-dependent protein kinase, corticotropin releasing factor (CRF) and the protein kinase C stimulant, phorbol myristate acetate (PMA) regulate the level of intracellular free calcium in the mouse anterior pituitary cell line AtT-20 were examined using the fluorescence probe Quin 2. The increase in cytosolic calcium in AtT-20 cells induced by CRF and PMA was blocked by calcium channel antagonists indicating that both agents stimulate calcium influx. The ability of PMA to raise cytosolic calcium levels was prevented by the sodium channel antagonist tetrodotoxin, suggesting that phorbol esters depolarize the cell membrane or increase action potential frequency to enhance calcium influx. The K+ channel antagonists, tetraethylammonium, cesium and 4-aminopyridine, inhibited PMA-stimulated calcium influx in AtT-20 cells. Thus, one mechanism by which protein kinase C activation may lead to a depolarization of the cell membrane is through a reduction in K+ currents. In contrast, neither tetraethylammonium or cesium reduced CRF-stimulated calcium influx into AtT-20 cells. The stimulation of calcium influx by CRF, therefore, appears to not involve changes in K+ currents in AtT-20 cells. CRF activates cyclic AMP-dependent protein kinase to stimulate calcium influx either by facilitating calcium conductance directly or by modifying the membrane potential or firing activity of AtT-20 cells.
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PMID:Phorbol esters and corticotropin releasing factor stimulate calcium influx in the anterior pituitary tumor cell line, AtT-20, through different intracellular sites of action. 253 67

The effects of pyrethroids and DDT on the alpha-subunit protein of the rat brain sodium channel were studied by using both native and exogenously added cAMP-dependent protein kinases. For this purpose, the sodium channel was partially purified, using the method of Hartshorne and Catterall [J Biol Chem 259: 1667-1675, 1984], and 32P-phosphorylated using [gamma-32P]ATP and exogenously added catalytic subunit of cAMP-dependent protein kinase. By comparing the phosphorylation patterns of the isolated sodium channel to those of the partially purified or unpurified (i.e. intact synaptosomes) preparations, it was concluded that the alpha-subunit of the voltage-sensitive sodium channel protein is the only phosphorylatable protein present at the 260 kD molecular weight range on the sodium dodecyl sulfate-polyacrylamide gel electrophoretogram. Phosphorylation of the alpha-subunit was induced by depolarization, and this process was inhibited by 10(-6) to 10(-10) M 1R-deltamethrin, but not by 1S-deltamethrin, the latter being an inactive enantiomer of the former. DDT produced a similar effect, but only at a higher concentration range. By using lysed synaptosomal membranes, it was possible to study the direct effects of these compounds on the alpha-subunit, which were similar to those produced by depolarization of intact synaptosomes.
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PMID:Modification by pyrethroids and DDT of phosphorylation activities of rat brain sodium channel. 254 81

The alpha subunit of the rat brain sodium channel is phosphorylated by cAMP-dependent protein kinase in vitro and in situ at multiple sites which yield seven tryptic phosphopeptides. Phosphopeptides 1-4 and 7 are derived from phosphorylation sites between residues 554 and 623 in a single large CNBr fragment from the cytoplasmic segment connecting homologous domains I and II of the alpha subunit (Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535). In the present work, antibodies were prepared against a synthetic peptide corresponding to residues 676-692 (AbSP15), which contain one additional potential phosphorylation site at Ser686-Ser687 in a different predicted CNBr fragment of this same intracellular segment. AbSP15 recognizes native and denatured sodium channels specifically and immunoprecipitates phosphorylated CNBr fragments of low molecular mass that contain a new site phosphorylated by cAMP-dependent protein kinase. Comparison of tryptic phosphopeptides derived from intact alpha subunits with those derived from the phosphorylated CNBr fragments isolated by immunoprecipitation with AbSP15 indicates that the two previously unidentified phosphopeptides 5 and 6 derived from the intact alpha subunit arise from phosphorylation of the site containing Ser686-Ser687. These results identify a new cAMP-dependent phosphorylation site and show that the major cAMP-dependent phosphorylation sites of the rat brain sodium channel, which are phosphorylated both in vitro and in intact neurons, are all located in a cluster between residues 554 and 687 in the intracellular segment between domains I and II.
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PMID:Phosphorylation of the alpha subunit of rat brain sodium channels by cAMP-dependent protein kinase at a new site containing Ser686 and Ser687. 254 90

The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.
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PMID:Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus. 255 2

Studies in the past several years have provided direct evidence that protein phosphorylation is involved in the regulation of neuronal function. Electrophysiological experiments have demonstrated that three distinct classes of protein kinases, i.e., cyclic AMP-dependent protein kinase, protein kinase C, and CaM kinase II, modulate physiological processes in neurons. Cyclic AMP-dependent protein kinase and kinase C have been shown to modify potassium and calcium channels, and CaM kinase II has been shown to enhance neurotransmitter release. A large number of substrates for these protein kinases have been found in neurons. In some cases (e.g., tyrosine hydroxylase, acetylcholine receptor, sodium channel) these proteins have a known function, whereas most of these proteins (e.g., synapsin I) had no known function when they were first identified as phosphoproteins. In the case of synapsin I, evidence now suggests that it regulates neurotransmitter release. These studies of synapsin I suggest that the characterization of previously unknown neuronal phosphoproteins will lead to the elucidation of previously unknown regulatory processes in neurons.
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PMID:Protein phosphorylation and neuronal function. 258 86

Antibodies against a peptide (SP19) corresponding to a highly conserved, predicted intracellular region of the sodium channel alpha subunit bind rat brain sodium channels with a similar affinity as the peptide antigen, indicating that the corresponding segment of the alpha subunit is fully accessible in the intact channel structure. These antibodies recognize sodium channel alpha subunits from rat or eel brain, rat skeletal muscle, rat heart, eel electroplax, and locust nervous system. alpha subunits from all these tissues except rat skeletal muscle are substrates for phosphorylation by cAMP-dependent protein kinase. Disulfide linkage of alpha and beta 2 subunits was observed for both the RI and RII subtypes of rat brain sodium channels and for sodium channels from eel brain but not for sodium channels from rat heart, eel electroplax, or locust nerve cord. Treatment with neuraminidase reduced the apparent molecular weight of sodium channel alpha subunits from rat and eel brain and eel electroplax by 22,000-58,000, those from heart by 8000, and those from locust nerve cord by less than 4000. Our results provide the first identification of sodium channel alpha subunits from rat heart and locust brain and nerve cord and show that sodium channel alpha subunits are expressed with different subunit associations and posttranslational modifications in different excitable tissues.
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PMID:Biochemical properties of sodium channels in a wide range of excitable tissues studied with site-directed antibodies. 284 76

The alpha subunit of the sodium channel purified from rat brain is rapidly and selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 3 to 4 mol of 32P/mol of saxitoxin-binding activity. The rate of phosphorylation is comparable to that of the synthetic peptide analog of the phosphorylation site of pyruvate kinase, one of the best substrates for cAMP-dependent protein kinase. An endogenous cAMP-dependent protein kinase that is present in the partially purified sodium channel preparations also selectively phosphorylates the alpha subunit. The specificity and rapidity of the phosphorylation reaction are consistent with the hypothesis that the alpha subunit is phosphorylated by cAMP-dependent protein kinase in vivo.
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PMID:Selective phosphorylation of the alpha subunit of the sodium channel by cAMP-dependent protein kinase. 628 61

In purified preparations of voltage-sensitive sodium channels, the alpha subunit is selectively phosphorylated by cAMP-dependent protein kinase (Costa, M. R. C., Casnellie, J. E., and Catterall, W. A. (1982) J. Biol. Chem., 7918-7921). We have developed methods to measure sodium channel phosphorylation in both lysed synaptosomal membranes and intact synaptosomes. Incubation of lysed synaptosomal membranes with exogenously added catalytic subunit of cAMP-dependent kinase and [gamma-32P]ATP resulted in rapid phosphorylation of the alpha subunit as detected by specific immuno-precipitation, sodium dodecyl sulfate-gel electrophoresis, and autoradiography. Analysis of tryptic phosphopeptides revealed five major sites of reaction. The level of phosphorylation of these sites on the sodium channel in intact synaptosomes was monitored using a rephosphorylation method in which those sites not phosphorylated in situ were labeled with [gamma-32P]ATP and exogenously added protein kinase after lysis of the synaptosomes. Incubation of synaptosomes with 8-Br-cAMP completely blocked labeling of the alpha subunit in rephosphorylation indicating marked stimulation of phosphorylation of the sites on the sodium channel in situ. Phosphorylation was complete in 15 s and all four of the tryptic phosphopeptides detected under these conditions could be phosphorylated in situ. These results show that the sodium channel can be rapidly phosphorylated by endogenous cAMP-dependent protein kinase in intact synaptosomes. In addition, since ATP and protein kinase are only available inside the synaptosomes, they also show that the alpha subunit is a transmembrane polypeptide exposed on both sides of the synaptosomal membrane. The functional consequences of 8-Br-cAMP-stimulated phosphorylation were examined using ion flux and neurotoxin-binding methods. Binding of saxitoxin and scorpion toxin were unaffected, but neurotoxin-activated 22Na+ influx mediated by the sodium channel was reduced 16 to 26% (P less than 0.01) under various experimental conditions. The potential physiological significance of this action is considered.
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PMID:Cyclic AMP-dependent phosphorylation of the alpha subunit of the sodium channel in synaptic nerve ending particles. 633 Jan 3

Both the neuronal IIA as well as the cardiac SkM2 isoform of the pore forming alpha-subunit of voltage dependent sodium channels are modulated by Protein Kinase A. While alphaIIA becomes attenuated upon PKA stimulation, alphaSkM2 becomes upregulated. PKC dependent phosphorylation of a serine, located in the highly conserved cytoplasmatic region between the third and the fourth transmembraneous domain has been found to be a prerequisite for PKA modulation of the alphaIIA isoform. We used site-directed mutagenesis, expression in Xenopus laevis oocytes and the two-electrode voltage clamp technique to test, whether phosphorylation of the corresponding serine in alphaSkM2 is required for the PKA modulation of also the cardiac isoform. The results clearly indicate that serine 1504 does not play a significant role in the PKA modulation of the cardiac sodium channel isoform, further underlining the differential modulation of the two isoforms by identical signal transduction cascades.
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PMID:Modulation of cardiac sodium channel isoform by cyclic AMP dependent protein kinase does not depend on phosphorylation of serine 1504 in the cytosolic loop interconnecting transmembrane domains III and IV. 747 28

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. We have identified soluble rat brain phosphatases that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity were observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 nM okadaic acid and contained a 36-kDa polypeptide recognized by a monoclonal antibody directed against the catalytic subunit of phosphatase 2A. Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcinerurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin increased apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites, as assayed by back phosphorylation. These results indicate that phosphatase 2A and calcineurin dephosphorylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.
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PMID:Identification of soluble protein phosphatases that dephosphorylate voltage-sensitive sodium channels in rat brain. 770 24


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