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)

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are multisubunit proteins and are regulated by protein phosphorylation. The purpose of this study was to determine: 1) which subunits are the preferential targets of various protein kinases when the channels are phosphorylated in vitro in their native membrane-bound state and 2) the consequences of these phosphorylations in functional assays. Using as substrates channels present in purified transverse (T) tubule membranes, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a multifunctional Ca2+/calmodulin-dependent protein kinase (CaM protein kinase) preferentially phosphorylated the 165-kDa alpha 1 subunit to an extent that was 2-5-fold greater than the 52-kDa beta subunit. A protein kinase endogenous to the skeletal muscle membranes preferentially phosphorylated the beta peptide and showed little activity toward the alpha 1 subunit; however, the extent of phosphorylation was low. Reconstitution of partially purified channels into liposomes was used to determine the functional consequences of phosphorylation by these kinases. Phosphorylation of channels by PKA or PKC resulted in an activation of the channels that was observed as increases in both the rate and extent of Ca2+ influx. However, phosphorylation of channels by either the CaM protein kinase or the endogenous kinase in T-tubule membranes was without effect. Phosphorylation did not affect the sensitivities of the channels toward the dihydropyridines. Taken together, the results demonstrate that the alpha 1 subunit is the preferred substrate of PKA, PKC, and CaM protein kinase when the channels are phosphorylated in the membrane-bound state and that phosphorylation of the channels by PKA and PKC, but not by CaM protein kinase or an endogenous T-tubule membrane protein kinase, results in activation of the dihydropyridine-sensitive Ca2+ channels from skeletal muscle.
J Biol Chem 1991 Sep 05
PMID:Dihydropyridine-sensitive calcium channels from skeletal muscle. II. Functional effects of differential phosphorylation of channel subunits. 165 34

Hearts isolated from 1-yr-old non-insulin-dependent diabetic rats exhibited reduced responsiveness to the beta-adrenergic agonist isoproterenol. Over a concentration range of 3 x 10(-9) to 10(-7) M, isoproterenol-mediated stimulation in the rate of left ventricular pressure decline, a measure of myocardial relaxation, and the rate of left ventricular pressure rise, a measure of myocardial contractility, were significantly depressed in the diabetic hearts. To clarify the basis for this defect, individual steps involved in the actions of the beta-adrenergic agonists were examined. Dihydroalprenolol binding assays revealed that neither beta-adrenergic receptor number nor binding affinity was affected by the diabetic condition. Also unaffected by diabetes was isoproterenol-mediated stimulation of adenylate cyclase activity, myocyte accumulation of adenosine 3',5'-cyclic monophosphate (cAMP), or the increase in cAMP-dependent protein kinase activity ratio. However, it was found that both in the presence and absence of cAMP-dependent protein kinase, activity of the sarcolemmal calcium transporter was significantly depressed in the diabetic heart. Also attenuated was protein kinase-induced enhancement of sarcoplasmic reticular calcium transport. The likelihood that these abnormalities contribute to alterations in calcium homeostasis and myocardial contractile function is discussed.
Am J Physiol 1991 Sep
PMID:Defective response to cAMP-dependent protein kinase in non-insulin-dependent diabetic heart. 165 26

Previous investigations revealed that under physiological conditions in the presence of MgATP the phosphorothioate analogue of cAMP, (Rp)-cAMPS, is a competitive inhibitor and antagonist for cAMP for cAMP-dependent protein kinases I and II [DeWit et al., (1984) Eur. J. Biochem. 142, 255-260]. For the type I holoenzyme, the antagonist properties of (Rp)-cAMPS are shown here to be absolutely dependent on MgATP. In the absence of MgATP, (Rp)-cAMPS serves as a weak agonist with a Ka of 7.9 microM. The high-affinity binding of MgATP imposes a barrier on cAMP-induced activation of the homoenzyme--a barrier that both cAMP and (Sp)-cAMPS, but not (Rp)-cAMPS, can overcome. In the absence of MgATP, this barrier no longer exists, and (Rp)-cAMPS functions as an agonist. The holoenzyme also was formed with mutant regulatory subunits. Replacing the essential arginine, predicted to bind the exocyclic oxygens of cAMP, in site A with lysine abolishes high-affinity binding of cAMP to site A. The holoenzyme formed with this mutant R-subunit is activated by (Rp)-cAMPS in both the presence and absence of MgATP. These results suggest that the stereospecific requirements for holoenzyme activation involve this guanidinium side chain. Mutations that eliminate the high-affinity binding of MgATP, such as the introduction of an autophosphorylation site in the autoinhibitory domain, also generate a holoenzyme that can be activated by (Rp)-cAMPS. In the case of the type II holoenzyme, (Rp)-cAMPS is an antagonist in both the presence and absence of MgATP, emphasizing distinct roles for MgATP in these two forms of cAMP-dependent protein kinase.
Biochemistry 1991 Sep 03
PMID:Identifying the molecular switches that determine whether (Rp)-cAMPS functions as an antagonist or an agonist in the activation of cAMP-dependent protein kinase I. 165 6

The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.
J Biol Chem 1991 Sep 25
PMID:Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro. 165 36

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.
J Biochem 1991 Sep
PMID:Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells. 166 12

The single human tyrosine hydroxylase (TH) gene generates four different mRNA species through alternative splicing events. TH-1 and TH-2 mRNAs are expressed mostly in the brain. We have produced large amounts of the corresponding proteins in Escherichia coli to analyze their respective molecular characteristics. The polypeptides have molecular weights similar to those of TH expressed in Xenopus oocytes and react with antibodies to TH. The two isoforms were purified with a purity of 90% using a three-step procedure. The phosphorylation sites have been determined in the two isoforms after labeling with [gamma-32P]ATP in the presence of cAMP-dependent protein kinase (PKA) or calmodulin-dependent protein kinase II (CaM-PK II). In both isoforms, Ser-40 was found to be phosphorylated by PKA, and Ser-19 and Ser-40 were found to be phosphorylated by CaM-PK II. The putative phosphorylation site generated by alternative splicing (Ser-31) was phosphorylated specifically by CaM-PK II in TH-2 only. The kinetic properties of the two isoforms in the presence of various concentrations of the substrate (tyrosine) and of the natural cofactor [6R)-tetrahydrobiopterin) were also analyzed. TH produced in E. coli is unphosphorylated but nevertheless active. At 50 microM tyrosine and 300 microM (6R)-tetrahydrobiopterin, the specific activities of TH-1 and TH-2 are 1300 and 620 nmol of dihydroxyphenylalanine/min/mg, respectively. Phosphorylation of TH-1 and TH-2 by PKA activates both isoenzymes as shown by the increase in the affinity for the cofactor. No changes in kinetic parameters of the isoenzymes were observed after phosphorylation by CaM-PK II. Dopamine was found to inhibit both TH isoenzymes to the same extent as shown by their similar Ki values for dopamine. These values were increased after phosphorylation of each enzyme by PKA. Unlike TH-1, phosphorylation of TH-2 by CaM-PK II resulted in an increase of the Ki value for dopamine. This property may be related to the presence of the additional phosphorylated residue in TH-2 isoform.
J Biol Chem 1991 Sep 15
PMID:Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing. 168 Jan 28

The properties of stretch-activated K+ channels in the membrane of loach (Misgurnus fossilis) embryos were studied using the patch-clamp technique. It was found that in the early stages of embryogenesis (2-256 cells) the stretch sensitivity of stretch-activated (SA) channels changes dramatically during the cell cleavage cycle. At the beginning of interphase the stretch sensitivity of SA channels and the probability of being in the open state (P0) were minimal, whereas at prometaphase they were increased 10-100-fold. Application of ATP to the cytoplasmic surface of excised inside-out patches induced a reversible increase in resting P0 and of stretch sensitivity of the SA channels in 50% of the patches, but the non-hydrolysable analogue of ATP, 5'-adenylylimidodiphosphate (AMP-PNP), was not effective. Phosphatase inhibitors (orthovanadate and para-nitrophenyl phosphate) prolonged the effect of ATP. Combined application of ATP, cAMP and cAMP-dependent protein kinase (PK) induced a reversible increase in the SA channel activity in 70% of those excised patches which did not respond to ATP. Inhibitors of PK prevented its activating effect. Dibutyryl-cAMP (dB cAMP) transiently increased activity of SA channels in intact cells. These results suggest that activity of SA channels may be regulated through cAMP-dependent phosphorylation and thus provide the basis for explanation of stretch sensitivity modulation during the cell cycle.
Proc Biol Sci 1991 Sep 23
PMID:Sensitivity of stretch-activated K+ channels changes during cell-cleavage cycle and may be regulated by cAMP-dependent protein kinase. 168 40

Apical membrane Cl- channels control the rate of transepithelial Cl- secretion in airway epithelia. cAMP-dependent protein kinase and protein kinase C regulate Cl- channels by phosphorylation; in cystic fibrosis cells, phosphorylation-dependent activation of Cl- channels is defective. Another important signaling system involves arachidonic acid, which is released from cell membranes during receptor-mediated stimulation. Here we report that arachidonic acid reversibly inhibited apical membrane Cl- channels in cell-free patches of membrane. Arachidonic acid itself inhibited the channel and not a cyclooxygenase or lipoxygenase metabolite because (i) inhibitors of these enzymes did not block the response, (ii) fatty acids that are not substrates for the enzymes had the same effect as arachidonic acid, and (iii) metabolites of arachidonic acid did not inhibit the channel. Inhibition occurred only when fatty acids were added to the cytosolic surface of the membrane patch. Unsaturated fatty acids were more potent than saturated fatty acids. Arachidonic acid inhibited Cl- channels from both normal and cystic fibrosis cells. These results suggest that fatty acids directly inhibit apical membrane Cl- channels in airway epithelial cells.
Proc Natl Acad Sci U S A 1990 Sep
PMID:Fatty acids inhibit apical membrane chloride channels in airway epithelia. 169 96

Cystic fibrosis (CF) is a common lethal genetic disease that manifests itself in airway and other epithelial cells as defective chloride ion absorption and secretion, resulting at least in part from a defect in a cyclic AMP-regulated, outwardly-rectifying Cl- channel in the apical surface. The gene responsible for CF has been identified and predicted to encode a membrane protein termed the CF transmembrane conductance regulator (CFTR). Identification of a cryptic bacterial promoter within the CFTR coding sequence led us to construct a complementary DNA in a low-copy-number plasmid, thereby avoiding the deleterious effects of CFTR expression on Escherischia coli. We have used this cDNA to express CFTR in vitro and in vivo. Here we demonstrate that CFTR is a membrane-associated glycoprotein that can be phosporylated in vitro by cAMP-dependent protein kinase. Polyclonal and monoclonal antibodies directed against distinct domains of the protein immunoprecipitated recombinant CFTR as well as the endogenous CFTR in nonrecombinant T84 cells. Partial proteolysis fingerprinting showed that the recombinant and non-recombinant proteins are indistinguishable. These data, which establish several characteristics of the protein responsible for CF, will now enable CFTR function to be studied and will provide a basis for diagnosis and therapy.
Nature 1990 Sep 27
PMID:Expression and characterization of the cystic fibrosis transmembrane conductance regulator. 169 61

Human cDNA clones for a heat-stable protein kinase inhibitor (PKI) protein of the cAMP-dependent protein kinase (PKA) were isolated using a mouse PKI cDNA fragment. Two human cDNA clones of 1.7 and 2.0 kb were sequenced and shown to encode the entire open reading frame of 228 nucleotides. Together these clones comprised 2147 nucleotides of the mRNA. The deduced amino acid sequence of the human clones showed 100% identity to the rabbit skeletal muscle PKI protein and 97% identity to the mouse brain PKI. The mouse and human PKI cDNAs shared nucleotide homology in their 3' untranslated regions as well as in the 32 nucleotides immediately 5' of the translation initiation site. Northern blot analysis of human skeletal muscle RNA with a human cRNA probe detected a major mRNA of approximately 4.0 kb. Transient overexpression in COS cells verified that a heat-stable inhibitor of protein kinase was produced by he human PKI cDNA, and protein extracts from the transfected COS cells inhibited both the C alpha and C beta isoforms of the PKA catalytic subunit with equal efficacy. Functional expression of the human PKI protein was further studied by assaying the ability of PKI expression vectors to inhibit PKA catalytic subunit stimulation of transcription from the human enkephalin promoter. In these studies, elimination of a conserved alternative translation start site in the 5' untranslated region of PKI was shown to potentiate the inhibitory activity of the PKI expression vector.
Mol Endocrinol 1991 Sep
PMID:Inhibition of protein kinase-A by overexpression of the cloned human protein kinase inhibitor. 177 Sep 51


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