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

---

Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of phosphorylation on muscarinic acetylcholine receptors (mAChRs) were studied in vitro and in vivo using rat brain plasma membrane and receptors partially purified at least 2500-fold. Purified mAChRs were phosphorylated in vitro by cAMP-dependent protein kinase and dephosphorylated by calcineurin. Phosphorylation of purified mAChRs was enhanced by carbachol and blocked by atropine. The filtrate which passed through glass fiber filters and high speed supernates were assayed for mAChRs by an ammonium sulfate precipitation method. Following incubation of the plasma membrane under phosphorylating conditions and ultracentrifugation at 300,000 g, the mAChRs appeared in the high speed supernate. This release was stimulated by adding carbachol to the incubation medium. In rats treated with carbachol, brain mAChRs redistributed from the heavy into the light membrane fractions. Ultrastructural examination of the light membrane fractions and the 300,000 g supernatant fractions after in vivo and in vitro carbachol treatment calcineurin increased the reincorporation of added partially purified receptors into the plasma membrane. The release and reincorporation of mAChRs strongly imply that there is a translocation and recycling of mAChRs between plasma membrane and cytosol in vivo. The significance and the function of the translocation of mAChRs remain to be investigated.
...
PMID:Phosphorylation-dephosphorylation of muscarinic acetylcholine receptors: evidence for the in vivo and in vitro release of receptors from rat brain plasma membrane. 217 18

The present studies examine the effects of in vivo and in situ progesterone treatment in the regulation of site-specific phosphorylation of the chicken oviduct progesterone receptor (PR). By gas-phase protein sequencing we have identified three hormonally regulated phosphorylation sites: Ser-211, Ser-260, and Ser-530. We determined phosphorylation stoichiometries by analyzing the amounts of phosphorylated and dephosphorylated serine at each site. Stoichiometries of sites 211 and 260 were about 20% under basal conditions and increased 1.5-2-fold by in situ progesterone treatment. Site 530 was virtually absent under basal conditions and induced to greater than 33% by in situ progesterone treatment. We tested several protein kinases for phosphorylation of the PR in vitro on these sites or peptides containing these sites. We found that the catalytic subunit of cAMP-dependent protein kinase mimicked the in vivo, hormone-induced altered mobility of PRs in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the in vivo and in vitro alterations were reversed by alkaline phosphatase. Finally, we showed that cAMP-dependent protein kinase phosphorylated Ser-528.
...
PMID:Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites. 239 63

Isolated triads from rabbit skeletal muscle were shown to contain an intrinsic protein kinase which was neither Ca2+/calmodulin-dependent nor cAMP-dependent. The protein substrates phosphorylated by this protein kinase exhibited apparent molecular weights of 300,000, 170,000, 90,000, 80,000, 65,000, 56,000, 52,000, 51,000, 40,000, 25,000, 22,000, and 15,000. Purification of the 1,4-dihydropyridine receptor from phosphorylated triads has demonstrated that the 170,000- and 52,000-Da subunits of the 1,4-dihydropyridine receptor are phosphorylated by this intrinsic protein kinase in isolated triads. Monoclonal antibodies to the 170,000-Da subunit of the dihydropyridine receptor immunoprecipitated the 170,000-Da phosphoprotein from detergent extracts of phosphorylated triads. The mobility of the 170,000-Da phosphoprotein in sodium dodecyl sulfate-polyacrylamide gels was not changed with or without reduction, demonstrating that the 170,000-Da phosphoprotein is not the glycoprotein subunit of the receptor. Our results demonstrate that the 170,000- and 52,000-Da subunits of the dihydropyridine receptor are phosphorylated by an intrinsic protein kinase in isolated triads. In addition, our results also demonstrate that the 175,000-Da glycoprotein subunit of the dihydropyridine receptor is not phosphorylated in isolated triads by the intrinsic protein kinase, cAMP-dependent protein kinase, or endogenous Ca2+/calmodulin-dependent protein kinase.
...
PMID:Phosphorylation of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel by an intrinsic protein kinase in isolated triads from rabbit skeletal muscle. 243 99

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.
...
PMID:Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells. 245 53

Following incubation of HPV 1-induced warts in the presence of [32P] phosphate several of the E4-encoded proteins were found to be radiolabeled. Two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 17K E4 polypeptides had incorporated [32P]phosphate whereas those of 16K were unlabeled. Purified E4 gene products were separated by ion exchange chromatography into a large number of different species, which were of similar size but of different charge due to varying extents of phosphorylated peptides have been isolated and identified. Phosphoserine and phosphothreonine were identified in all 16/17K E4 fractions but not phosphotyrosine. Both HPV 1 E4 16K and 17K fractions were phosphorylated in vitro by cAMP-dependent protein kinase but not by myosin light chain kinase or by phosphorylase kinase. Incubation with cAMP PK gave incorporation of approx. 0.5 mole phosphate/mol of protein indicating that the cAMP-dependent protein kinase site(s) was partially phosphorylated in vivo. This view was supported by the fact that species which were more heavily phosphorylated in vivo incorporated less phosphate after cAMP-dependent protein kinase phosphorylation. HPV 1 E4 was also phosphorylated at serine and threonine residues by a crude cytoplasmic extract prepared from cultured human keratinocytes and cultured human retinoblasts. These results are discussed in the light of the known effects of phosphorylation on the interactions of other keratinocyte-specific proteins.
...
PMID:Phosphorylation of the human papillomavirus type 1 E4 proteins in vivo and in vitro. 247 Jan 93

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein.
...
PMID:In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases. 249 51

A protein consisting of human (Hu)-IFN-alpha A to which the COOH-terminal 16 amino acids of Hu-IFN-gamma were fused was prepared by constructing an expression vector by oligonucleotide-directed mutagenesis. The hybrid protein Hu-IFN-alpha A/gamma was expressed under the control of phage lambda PL promoter. The protein was purified with the use of a monoclonal antibody against Hu-IFN-alpha or the COOH-terminal amino acid sequence of Hu-IFN-gamma. The purified protein exhibited a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has antiviral activity on human and bovine cells. Unlike Hu-IFN-alpha A, but similar to Hu-IFN-gamma, the hybrid Hu-IFN-alpha A/gamma can be phosphorylated by [gamma 32P]ATP and cAMP-dependent protein kinase. The phosphorylated molecule binds to the IFN-alpha/beta receptor. The introduction of a phosphorylation site into Hu-IFN-alpha A by fusion of the region of Hu-IFN-gamma which contains the phosphorylation site provides a new reagent for studies of receptor binding, pharmacokinetics, and other studies where labeled interferons are useful. Furthermore, the introduction of phosphorylation sites into proteins provides a new principle for the preparation of a wide variety of reagents for many purposes.
...
PMID:Construction and phosphorylation of a fusion protein Hu-IFN-alpha A/gamma. 250 45

The structure of phospholamban, a 30-kDa oligomeric protein integral to cardiac sarcoplasmic reticulum, was probed using ultraviolet absorbance and circular dichroism spectroscopy. Purified phospholamban was examined in three detergents: octyl glucoside, n-dodecyloctaethylene glycol monoether (C12E8) and sodium dodecyl sulfate (SDS). Ultraviolet absorption spectra of phospholamban reflected its aromatic amino acid content: absorption peaks at 275-277 nm and 253, 259, 265 and 268 nm were attributed to phospholamban's one tyrosine and two phenylalanines, respectively. Phospholamban phosphorylated at serine 16 by the catalytic subunit of cAMP-dependent protein kinase exhibited no absorbance changes when examined in C12E8 or SDS. Circular dichroism spectroscopy at 250-190 nm demonstrated that phospholamban possesses a very high content of alpha-helix in all three detergents and is unusually resistant to denaturation. Dissociation of phospholamban subunits by boiling in SDS increased the helical content, suggesting that the highly ordered structure is not dependent upon oligomeric interactions. The purified COOH-terminal tryptic fragment of phospholamban, containing residues 26-52 and comprising the hydrophobic, putative membrane-spanning domain, also exhibited a circular dichroism spectrum characteristic of alpha-helix. Circular dichroism spectra of phosphorylated and dephosphorylated phospholamban were very similar, indicating that phosphorylation does not alter phospholamban secondary structure significantly. The results are consistent with a two-domain model of phospholamban in which each domain contains a helix and phosphorylation may act to rotate one domain relative to the other.
...
PMID:Secondary structure of detergent-solubilized phospholamban, a phosphorylatable, oligomeric protein of cardiac sarcoplasmic reticulum. 252 65

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 as determined by SDS/PAGE) is a major cytosolic substrate for cAMP-stimulated protein phosphorylation in dopamine-innervated regions of rat CNS (Walaas et al., 1983c). This acidic phosphoprotein has now been identified in bovine caudate nucleus cytosol and purified to homogeneity from this source. The purification procedure involved diethylaminoethyl-cellulose chromatography, ammonium sulfate fractionation, phenyl-Sepharose CL-4B chromatography, and fast protein liquid chromatography using Mono Q anion-exchange resin. Two isoforms of ARPP-21 (ARPP-21A and ARPP-21B) were obtained, which were present in approximately equal amounts in the starting material. ARPP-21A was purified 2610-fold with a final yield of 20% and ARPP-21B was purified 2940-fold with a final yield of 21%. The purified preparations of both isoforms were judged to be homogenous by SDS/PAGE. ARPP-21A and ARPP-21B yielded identical 2-dimensional thin-layer tryptic phosphopeptide maps, identical amino acid compositions and closely related, but distinct, reverse-phase high-pressure liquid chromatograms of tryptic digests. The amino acid composition of ARPP-21 showed a high content of glutamic acid/glutamine, and no methionine, tryptophan, tyrosine, phenylalanine, or histidine. ARPP-21 was stable to heat denaturation and to 50% (vol/vol) ethanol treatment and was partially soluble at pH 2. The Mr determined for ARPP-21 by SDS/PAGE was 21,000. The Stokes radius of ARPP-21 was 26.3 A, and the sedimentation coefficient of ARPP-21 was 1.3 S; these values yield a calculated molecular mass of 13,700 Da and a frictional ratio of 1.7, indicative of an elongated tertiary structure. ARPP-21 was an excellent substrate for cAMP-dependent protein kinase and was either not phosphorylated or only poorly phosphorylated by cGMP-dependent protein kinase, calcium/calmodulin-dependent protein kinase I, calcium/calmodulin-dependent protein kinase II, casein kinase II, or protein kinase C. The purified catalytic subunit of cAMP-dependent protein kinase catalyzed the incorporation of 1.2 mol phosphate/mol purified ARPP-21. Phosphorylation occurred exclusively on seryl residues. Phospho-ARPP-21 was dephosphorylated effectively by protein phosphatase-1 or -2A, but not by protein phosphatase-2B or -2C. Rabbit polyclonal and mouse monoclonal antibodies were prepared to purified ARPP-21. These antibodies specifically immunoprecipitated ARPP-21, which was found to be highly enriched in the caudate nucleus and putamen of monkey brain.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:ARPP-21, a cyclic AMP-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Purification and characterization of the protein from bovine caudate nucleus. 253 84

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.
...
PMID:Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells. 254 36


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---