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 have synthesized a tetradecapeptide corresponding to residues 354-367 of the delta-subunit of Torpedo acetylcholine receptor. This peptide contains the sequence Arg-Arg-Ser-Ser which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMP-dependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMP-dependent protein kinase. Antibodies elicited against peptide 354-367 were shown to cross-react with native AChR and to bind specifically to the delta- and gamma-subunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMP-dependent phosphorylation of both the delta- and gamma-subunits. This suggests that the phosphorylation sites in the delta- and gamma-subunits are highly cross-reactive, and is in agreement with the demonstration that an endogenous cAMP-dependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the delta-subunit isolated from phosphorylated AChR yields a single 25-kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354-367 within this phosphorylated fragment.
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PMID:Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor. 370 19

Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms.
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PMID:Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies. 381 58

Calmodulin was previously found to inhibit the Ca2+/phospholipid-dependent phosphorylation of an endogenous substrate, called the 87-kilodalton protein, in a crude extract prepared from rat brain synaptosomal cytosol. We investigated the mechanism of this inhibition, using Ca2+/phospholipid-dependent protein kinase and the 87-kilodalton protein, both of which had been purified to homogeneity from bovine brain. Rabbit brain calmodulin and some other Ca2+-binding proteins inhibited the phosphorylation of the 87-kilodalton protein by this kinase in the purified system. Calmodulin also inhibited the Ca2+/phospholipid-dependent phosphorylation of H1 histone, synapsin I, and the delta subunit of the acetylcholine receptor, with use of purified components. These results suggest that calmodulin may be a physiological regulator of Ca2+/phospholipid-dependent protein kinase.
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PMID:Inhibition by calmodulin of calcium/phospholipid-dependent protein phosphorylation. 623 11

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.
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PMID:Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes. 625 May 98

We have found that the acetylcholine receptor (AChR) of Torpedo californica is phosphorylated and dephosphorylated in situ by a membrane-bound protein kinase and phosphatase [1]. There is increasing evidence that other neurotransmitters [2], light [3-6], polypeptide hormones [7], and growth factors [8-12] also may act by regulating the level of phosphorylation of membrane proteins. These observations suggest that membrane protein phosphorylation may be a general regulatory mechanism affecting the response of cells to exogenous metabolic and physical signals. To understand the role of membrane protein phosphorylation, we have chosen to study acetylcholine receptor-enriched membranes purified from the electric organ of T californica. This organ is an ideal model system for such studies since it is a rich source of the acetylcholine receptor. Membranes can be purified from the electric organ that are enriched in the AChR[13] and that show cholinergic agonist-dependent changes in cation flux [14]. Moreover, the AChR from T californica has been purified [15[, biochemically characterized [16], and used to generate specific antibodies [17]. Receptor, enriched membranes contain only a few other proteins that are closely associated with the receptor in the postsynaptic membrane. Such associated proteins may play a critical role in regulating the function of the AChR in the postsynaptic membrane. We have taken advantage of these conditions to study phosphorylation of the membrane-bound AChR in this well-defined, homogeneous system.
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PMID:Reversible phosphorylation of the membrane-bound acetylcholine receptor. 626 75

Postsynaptic membranes, rich in the nicotinic acetylcholine receptor, were isolated from the electric organ of Torpedo californica and shown to contain a cAMP-dependent protein kinase and a calcium/calmodulin-dependent protein kinase. The cAMP-dependent protein kinase phosphorylated the gamma and delta subunits of the acetylcholine receptor. The phosphorylated subunits were identified after purification of the acetylcholine receptor by affinity chromatography on a choline carboxymethyl affinity gel. In contrast, the calcium/calmodulin-dependent protein kinase phosphorylated proteins that were separated from the acetylcholine receptor by affinity chromatography. Protein kinase inhibitor, a specific inhibitor of the catalytic subunit of cAMP-dependent protein kinase, abolished the basal endogenous phosphorylation of the gamma and delta subunits of the receptor. cAMP activation of the endogenous phosphorylation of the gamma and delta subunits was dose dependent with a half-maximal response at 25 nM. Studies were also carried out with acetylcholine receptor purified from T. californica and catalytic subunit of cAMP-dependent protein kinase purified from bovine heart. The purified acetylcholine receptor was rapidly and specifically phosphorylated on the gamma and delta subunits by the purified catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 1.0 and 0.89 mol of (32)P per mol of receptor, respectively. The initial rates of phosphorylation of the gamma and delta subunits of the receptor were comparable to those of histone f2B and synapsin I (protein I), two of the most effective substrates for the catalytic subunit. Under the conditions used, the gamma and delta subunits had K(m) values of 4.0 and 3.3 muM and V(max) values of 2.7 and 2.1 mumol/min per mg, respectively. The results are consistent with the idea that the acetylcholine receptor is phosphorylated in vivo by a cAMP-dependent protein kinase.
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PMID:cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. 630 72

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca2+/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca2+/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca2+/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed alpha, beta, gamma and delta, only the gamma- and delta-subunits were phosphorylated by the cAMP-dependent protein kinase (+ cAMP), or by its purified catalytic subunits.
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PMID:cAMP, not Ca2+/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax. 632 Aug 88

A photoaffinity ATP ligand is used to identify the protein kinase present in acetylcholine receptor-enriched membranes from Torpedo californica. Incubation of these membranes with 8-azido-[alpha-32P]ATP and subsequent irradiation with UV light resulted in covalent labeling of a major band of Mr 43,000. Alkali-stripped membranes that show a selective reduction in the Mr 43,000 polypeptide also show a corresponding reduction in incorporation of photoaffinity label. In addition, the neutralized alkaline extract also showed one band at Mr 43,000 when labeled with the photoaffinity ligand. After alkali extraction, endogenous protein kinase activity decreased in the membranes in proportion to the loss of Mr 43,000 peptide. Moreover, the alkaline extract was able to phosphorylate casein in an exogenous assay system. These results suggest that a Mr 43,000 polypeptide in acetylcholine receptor-enriched membranes is the acetylcholine receptor kinase.
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PMID:Identification of a molecular weight 43,000 protein kinase in acetylcholine receptor-enriched membranes. 657 58

Calcium plus calmodulin (Ca2+/CaM)-dependent protein kinase activity was demonstrated in subcellular fractions from Torpedo californica electric organ. A protein kinase activity dependent on Ca2+/CaM was purified about 200-fold from electric organ cytosol using DEAE-cellulose and CaM-affinity chromatography. The most effective exogenous substrates for this enzyme were the synapse-specific protein Synapsin I (Protein I) and histone f3. Phosphorylase b, skeletal muscle myosin light chains, casein, phosvitin, histone f2b, and G-substrate were relatively poorly phosphorylated by Torpedo CaM-protein kinase. Thus, the enzyme differs in its substrate specificity from known cyclic nucleotide-dependent protein kinases, myosin light chain kinase and phosphorylase kinase. The Km for ATP was 15-20 microM; for Synapsin I, 0.8 microM; and for CaM, 85 nM. Two major endogenous substrates (Mr = 62,000 and 54,000) for CaM-protein kinase co-purified with the enzyme through the CaM-affinity column step. These two substrates, as well as the enzyme, were present in other subcellular fractions in addition to the cytosol, including crude membranes and purified synaptic vesicles. A third major substrate (Mr = 39,000) could be separated from the enzyme during purification and appeared to be localized primarily in the cytosol. CaM-protein kinase increased the phosphorylation of both serine and threonine residues in endogenous substrates. In contrast to previous reports, no evidence for Ca2+/CaM-dependent phosphorylation of any subunit of the acetylcholine receptor was obtained.
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PMID:Calmodulin-dependent protein kinase and associated substrates in Torpedo electric organ. 687 98

1. The effect of colchicine treatment on acetylcholine receptor function was examined in potassium depolarized, voltage-clamped snake twitch fibre endplates. Receptor function was assessed by analysis of miniature endplate currents (m.e.p.c.) as well as acetylcholine (ACh)-induced single channel currents. 2. Pretreatment of snake muscle fibres with colchicine (10 microM to 100 microM) for 16-18 h had no effect on m.e.p.c. amplitude or decay rates. At higher concentrations (1 mM), there was a slight decrease in the average m.e.p.c. amplitude. 3. Colchicine produced a concentration-dependent decrease in the extent of m.e.p.c. amplitude recovery following a 10 min exposure to 540 microM carbachol. Exposure of 100 microM colchicine-treated preparations to 0.5 microM staurosporine further reduced the extent of m.e.p.c. amplitude recovery following carbachol exposure. 4. The decrease in m.e.p.c. amplitude following carbachol exposure was not due to a shift in the m.e.p.c. reversal potential. In addition, the distribution of m.e.p.c. amplitudes remained unimodal in both control and colchicine (100 microM)-treated preparations following carbachol exposure. 5. In addition to the normal, large conductance (approximately 48 pS) ACh-activated channels, a population of small conductance (approximately 29 pS) channels was observed in colchicine-treated preparations following exposure to carbachol. In preparations treated with both colchicine and staurosporine and then exposed to carbachol, the conductance of these small channels was identical to that of colchicine or staurosporine alone. 6. We suggest that prolonged exposure of snake twitch fibre endplates to agonist results in the activation and desensitization of ACh receptors. Furthermore, we propose that for a subpopulation of the inactivated receptors, restoration of function requires both the integrity of a subsynaptic cytoskeletal component and phosphorylation by a staurosporine-sensitive protein kinase. One plausible mechanism is that some receptors become destabilized in the membrane and phosphorylation of a cytoskeletal component, whose distribution may depend on an intact microtubular system, is required to re-anchor these receptors. If this anchoring process is inhibited either by disruption of the cytoskeleton with colchicine, or inhibition of the kinase by staurosporine, these receptors remain activatable, but have a reduced conductance.
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PMID:Requirement of a colchicine-sensitive component of the cytoskeleton for acetylcholine receptor recovery. 753 19


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