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)

Most neurotransmitter receptors examined to date are either regulated by phosphorylation or contain consensus sequences for phosphorylation by protein kinases. The nicotinic acetylcholine receptor (AChR), which mediates depolarization at the neuromuscular junction, has served as a model for the study of the structure, function, and regulation of ligand-gated ion channels. The AChR is phosphorylated by protein kinase A, protein kinase C, and an unidentified protein tyrosine kinase. Tyrosine phosphorylation of the AChR is correlated with a modulation of the rate of receptor desensitization and is associated with AChR clustering. We showed that agrin, a neuronally derived extracellular matrix protein, induces AChR clustering and tyrosine phosphorylation. In addition, we identified two protein tyrosine kinases, Fyn and Fyk, that appear to be involved in the regulation of synaptic transmission at the neuromuscular junction by phosphorylating the AChR. The two kinases are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. As demonstrated by coimmunoprecipitation, Fyn and Fyk associate with the AChR. Furthermore, the AChR is phosphorylated in Fyn and Fyk immunoprecipitates. We investigated the molecular basis for the association of the AChR with Fyn and Fyk using fusion proteins derived from the kinases. The AChR bound specifically to the SH2 domain fusion proteins of Fyn and Fyk. The association of the AChR with the SH2 domains is dependent on the state of AChR tyrosine phosphorylation and is mediated by the delta subunit of the receptor. These data provide evidence that the protein tyrosine kinases Fyn and Fyk may act to phosphorylate the AChR in vivo.
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PMID:Phosphorylation of the nicotinic acetylcholine receptor by protein tyrosine kinases. 754 72

Agrin induces the accumulation of nicotinic acetylcholine receptors (AChRs) in the myofiber membrane at synaptic sites in vertebrate skeletal muscle and causes an increase in tyrosine phosphorylation of the AChR beta subunit. To examine further the mechanism of agrin-induced AChR phosphorylation and the relationship between changes in protein phosphorylation and AChR aggregation, the effect of the protein tyrosine phosphatase inhibitor sodium pervanadate was tested on chick myotubes in culture. Pervanadate caused an increase in the phosphotyrosine content of a variety of proteins, including the AChR. Pervanadate also prevented agrin-induced AChR aggregation and slowed the rate at which AChRs were extracted from intact myotubes by mild detergent treatment. The rate at which phosphorylation of the AChR beta subunit and receptor detergent extractability changed following pervanadate-induced phosphatase inhibition was increased by agrin, indicating that agrin activates a protein tyrosine kinase rather than inhibiting a protein tyrosine phosphatase. The present results, taken together with previous findings on the inhibition of agrin-induced AChR aggregation by protein kinase inhibitors, demonstrate that protein tyrosine phosphorylation regulates the formation and stability of AChR aggregates, apparently by strengthening the interaction between AChRs and the cytoskelton.
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PMID:Regulation of the interaction of nicotinic acetylcholine receptors with the cytoskeleton by agrin-activated protein tyrosine kinase. 789 76

Forskolin treatment of cells expressing Torpedo acetylcholine receptors leads to enhanced assembly efficiency of subunits, which correlates with increased phosphorylation of the gamma subunit. To determine the role of the two potential protein kinase A sites of the gamma subunit in receptor assembly, cell lines expressing different mutant receptors were established. Mouse fibroblast cell lines stably expressing wild-type Torpedo acetylcholine receptor alpha, beta, delta subunits plus one of three gamma subunit mutations (S353A, S354A, or S353,354A) were established to identify the protein kinase A phosphorylation sites of gamma in vivo, and to determine if increased phosphorylation of the gamma subunit leads to enhanced expression of receptors. We found that both serines (353, 354) in gamma are phosphorylated in vivo by protein kinase A, however, phosphorylation of either or both of these sites does not lead to increased assembly efficiency. We established a cell line expressing alpha, beta, and gamma(S353,354A) subunits only (no delta), and found that the presence of delta (or its phosphorylation) is also not necessary for the observed stimulation by forskolin. alpha beta gamma, alpha gamma, and beta gamma associations were stimulated by forskolin but alpha beta and alpha delta interactions were not. These data imply that the presence of gamma is necessary for forskolin action. We postulate that forskolin may stimulate acetylcholine receptor expression through a cellular protein that is involved in the folding and/or assembly of protein complexes, and that forskolin may regulate the action of such a protein through phosphorylation.
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PMID:Cyclic AMP-regulated AChR assembly is independent of AChR subunit phosphorylation by PKA. 796 4

The nicotinic acetylcholine receptor (AChR) is a pentameric complex made up of four types of subunits in the stoichiometry alpha 2 beta gamma delta. These subunits have been shown to be differentially phosphorylated by cAMP-dependent protein kinase (PKA) protein kinase C, and a protein tyrosine kinase. A variety of studies have suggested that phosphorylation of the AChR in vitro and in vivo regulates the rate of desensitization of the receptor. In this study we have used site-specific mutagenesis and patch-clamp techniques to examine the role of phosphorylation in the regulation of desensitization of the AChR expressed in Xenopus oocytes Expression of wild-type AChR in Xenopus oocytes results in the constitutive phosphorylation of the AChR on the gamma and delta subunits. This phosphorylation is apparently due to the high basal level of PKA in oocytes since a specific peptide inhibitor of PKA completely eliminated phosphorylation of the AChR by oocyte extracts in vitro. The phosphorylation of the AChR in oocytes was not significantly enhanced by forskolin or cAMP analogs or by coexpression with the catalytic subunit of PKA, suggesting that the basal activity of PKA in oocytes is sufficient to phosphorylate the receptor to a high stoichiometry. Using site-specific mutagenesis, the sites of phosphorylation were determined to be serines 353 and 354 on the gamma subunit and serines 361 and 362 on the delta subunit. To examine the functional properties of wild-type and mutant receptors lacking phosphorylation sites, we used patch-clamp techniques to measure the responses of out-side-out patches to repetitive pulses of ACh using a rapid perfusion system. Wild-type and mutant receptors showed rapid concentration-dependent activation and desensitization to applied agonist. The time constant of desensitization of ensemble mean currents ranged from several hundred milliseconds at low ACh concentrations to 100-200 msec at saturating concentrations. The desensitization time constants for mutant receptors lacking all phosphorylation sites were significantly slower than wild-type phosphorylated receptors at all concentrations of ACh tested. In addition, mutant receptors that had the serine residues changed to glutamate residues in order to mimic the negative charge of the phosphorylated serine residue produced receptors that had desensitization rates approaching those of the wild-type phosphorylated receptor. These results provide further support that phosphorylation of the nicotinic ACh receptor regulates rate of desensitization.
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PMID:Role of phosphorylation in desensitization of acetylcholine receptors expressed in Xenopus oocytes. 802 70

The predicted protein kinase activity of the cloned gene product of the human myotonic dystrophy locus has been experimentally verified. Affinity-purified recombinant DM protein kinase became phosphorylated itself and transphosphorylated histone H1. These activities were not present in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhibit distinct sizes, 43 and 66 kDa, respectively. DMPKH was further purified by velocity sedimentation on sucrose gradients; both activities migrated with the recombinant protein at 41 S, consistent with discrete multimeric particles. Phosphoamino acid analysis showed that threonine (predominantly) and serine were phosphorylated in both DMPKH and histone H1. Although PKA and PKC are the known types of protein kinase with closest sequence homology to the DM protein kinase domain, purified DMPKH was inhibited by 4 mM but not 0.04-0.4 mM H7 and H8, which inhibit PKA and PKC with Ki's of 0.4-15 microM. Specific inhibitors of other classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-dependent protein kinase II (KN-62) did not inhibit DMPKH. DMPKH did not phosphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, and CKI and -II, respectively. These experimental results suggest that the active center of the recombinant human myotonic dystrophy protein kinase may have properties distinct from the well-studied classes of serine/threonine protein kinases, in contrast to predictions based upon primary structure alone.
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PMID:Phosphorylation reactions of recombinant human myotonic dystrophy protein kinase and their inhibition. 807 83

1. Nicotinic acetylcholine receptor (AChR)-operated non-contractile Ca2+ mobilization (unaccompanied by muscle contraction) depressed contractile Ca2+ mobilization (accompanied by muscle contraction) in mouse diaphragm muscles. In the process of nicotinic AChR desensitization, the enhancing role of calcitonin gene-related peptide (CGRP) on the non-contractile Ca2(+)-induced depression of contractile Ca2+ mobilization was investigated by measurement of Ca2(+)-aequorin luminescence in the presence of neostigmine (0.1 microM). 2. When the phrenic nerve was stimulated with paired pulses at intervals of 150, 300, 600, 1000 and 2000 ms, contractile Ca2+ transients were elicited during the generation of non-contractile Ca2+ mobilization. The amplitude of the contractile Ca2 transients elicited by the second pulse (S2) was depressed at the shorter pulse intervals, but not at the longer pulse intervals. 3. The extent of depression of S2 was enhanced when the duration of non-contractile Ca2+ mobilization was prolonged by CGRP (10 nM). However, CGRP failed to enhance the depression of S2 when non-contractile Ca2+ mobilization was not observed at the low external Ca2+ concentration (1.3 mM). 4. The enhancing effect by CGRP on the depression of S2 was counteracted by staurosporine (3 nM), a protein kinase-C inhibitor, despite prolongation of the duration of non-contractile Ca2+ mobilization. 5. When H-89 (1 microM), a protein kinase-A inhibitor, completely blocked non-contractile Ca2+ mobilization, the depression of S2 was diminished. The prolongation of the duration of non-contractile Ca2+ mobilization by AA373 (300 microM), a protein kinase-A activator, enhanced the depression of S2. The enhancing effect was observed neither with CGRP nor with AA373, in the presence of H-89 (0.1 microM). 6. These findings suggest that the CGRP mobilizes non-contractile Ca2+ through activation of protein kinase-A, which in turn may activate protein kinase-C, then enhance the desensitization of postsynaptic nicotinic AChRs at the neuromuscular junction.
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PMID:Enhancement by calcitonin gene-related peptide of non-contractile Ca2(+)-induced nicotinic receptor desensitization at the mouse neuromuscular junction. 886 31

This study examined the distribution of muscarinic acetylcholine receptor-immunoreactive neurons in the amygdaloid complex of the rat, with emphasis on the central nucleus. The monoclonal antibody M35 raised against purified muscarinic acetylcholine receptor protein was used to visualize muscarinic acetylcholine receptor-immunoreactive cells. Muscarinic acetylcholine receptor immuno-reactivity was high in the central nucleus and low to moderate in all other regions of the amygdaloid complex. Within the central nucleus, the muscarinic acetylcholine receptor-immunoreactive neurons were found predominantly in the lateral subdivision. This region contained medium-sized neurons (largest diameter ranging from 10 to 15 microns), with a round or slightly ovoid cell shape. At the subcellular level, however, the labeled neurons revealed relatively few muscarinic acetylcholine receptor-immunoreactive postsynaptic densities. Immunofluorescent double-labeling demonstrated that nearly all of the muscarinic acetylcholine receptor-immunoreactive neurons (98.6%) in the central nucleus expressed abundant amounts of nicotinic acetylcholine receptors, further substantiating the cholinoceptive character of these cells. In addition, the vast majority of these muscarinic acetylcholine receptor-immunoreactive neurons (94.3%) were GABAergic neurons. The muscarinic acetylcholine receptor-immunoreactive neurons expressed moderate levels of protein kinase gamma, one of the likely intracellular mediators between muscarinic acetylcholine receptors and their elicited physiological response. The number and staining intensity of muscarinic acetylcholine receptor-immunoreactive neurons in the central nucleus varied dramatically among rats. This individual variation correlated positively with the rat's expression of conditioned immobility and correlated negatively with active shock avoidance performance. These results suggest that the GABAergic/cholinoceptive neuronal elements in the central nucleus are involved in the expression of fear-induced behaviors. This interpretation is further elaborated in a forthcoming paper.
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PMID:Muscarinic acetylcholine receptor immunoreactivity in the amygdala--I. Cellular distribution correlated with fear-induced behavior. 897 59

The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors.
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PMID:Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells. 977 56

Electrical activity of myotubes represses nicotinic acetylcholine receptor (AChR) gene expression. This effect is mimicked by okadaic acid and blocked by tetrodotoxin (TTX) or staurosporine in cultured myocytes [Altiok et al., EMBO J. 16 (1997) 717-725]. In this study, we investigated the mechanism of this repression. We show that addition of exogenous phospholipase D (PLD) and C inhibits AChR expression in a manner which parallels that of okadaic acid. Furthermore, okadaic acid caused an increase of the threonine phosphorylation of protein kinase Czeta (PKCzeta) and activator of transcription factor (ATF2) and a decrease of the phosphorylation of Sp1. All these effects were reversed by staurosporine, and TTX also abolished ATF2 phosphorylation. These data reveal a possible involvement of PLD, c-jun N-terminal kinase, PKCzeta and Sp1 in the repression of AChR genes by electrical activity.
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PMID:Electrical activity regulates AChR gene expression via JNK, PKCzeta and Sp1 in skeletal chick muscle. 1116 54

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) alpha4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3eta, known to interact directly with neuronal AChRs. 14-3-3eta is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3eta interacted with the recombinant alpha4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3eta with recombinant alpha4 subunits was abolished when serine 441 of the alpha4 subunit was mutated to alanine (alpha4(S441A)). The surface levels of recombinant wild-type alpha4beta2 AChRs were approximately 2-fold higher than those of mutant alpha4(S441A)beta2 AChRs. The interaction significantly increased the steady state levels of the alpha4 subunit and alpha4beta2 AChRs but not that of the mutant alpha4(S441A) subunit or mutant alpha4(S441A)beta2 AChRs. The EC50 values for activation by acetylcholine were not significantly different for alpha4beta2 AChRs and alpha4(S441A)beta2 AChRs coexpressed with 14-3-3eta in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native alpha4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of alpha4beta2 AChRs through its interaction with the alpha4 subunit.
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PMID:The chaperone protein 14-3-3eta interacts with the nicotinic acetylcholine receptor alpha 4 subunit. Evidence for a dynamic role in subunit stabilization. 1135 1


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