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)

Synthetic peptides based on the threonine phosphorylation site and proposed inhibitory site of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were prepared and analyzed as substrates for cAMP-dependent protein kinase and protein phosphatases-1c, -2Ac (the catalytic subunits of protein phosphatase-1 and 2A, respectively) and -2B, and as inhibitors of protein phosphatase-1c. Studies of the kinetics of phosphorylation of the peptides by cAMP-dependent protein kinase indicated an important role in facilitating phosphorylation for the region COOH-terminal to the phosphorylatable threonyl residue. Studies of the dephosphorylation of the phosphopeptides demonstrated that they were effectively dephosphorylated by protein phosphatase-2A and -2B and poorly dephosphorylated by protein phosphatase-1. The active inhibitory region of phospho-DARPP-32 was analyzed by determining the effects of synthetic phosphopeptides on the activity of protein phosphatase-1c. Phospho-D32-(8-48) and phospho-D32-(8-38) inhibited protein phosphatase-1c with IC50 values of 2 x 10(-8) and 4 x 10(-8) M, respectively, compared with an IC50 of 8 x 10(-9) M for intact phospho-DARPP-32. Phospho-D32-(9-38) was equipotent with phospho-D32-(8-38); however, further NH2-terminal deletions resulted in marked reductions in IC50 values. An analog of an active DARPP-32 phosphopeptide containing a phosphoseryl residue in place of the phosphothreonyl residue also exhibited a much reduced IC50. These data identify the essential inhibitory region of phospho-DARPP-32 as residues 9-38, which contains the phosphorylation site (Thr34). This region exhibits extensive amino acid sequence identity with phosphatase inhibitor-1, a distinct inhibitor of protein phosphatase-1. Kinetic studies of the inhibition of protein phosphatase-1c by phospho-D32-(9-38), a potent inhibitor, as well as by phospho-D32-(10-38), a weak inhibitor, indicated a mixed competitive/noncompetitive mechanism of inhibition, as has been previously found for both intact phospho-DARPP-32 and intact phospho-inhibitor-1. These findings support the hypothesis that a 30-amino acid domain in the NH2-terminal region of phospho-DARPP-32 is sufficient for the inhibition of protein phosphatase-1.
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PMID:Synthetic peptide analogs of DARPP-32 (Mr 32,000 dopamine- and cAMP-regulated phosphoprotein), an inhibitor of protein phosphatase-1. Phosphorylation, dephosphorylation, and inhibitory activity. 217 4

Full length cDNA clones encoding microtubule-associated proteins (MAP) 2b and 2c from rat brain have been isolated and sequenced. The cDNA fragments spanning the coding regions for both MAP2b and MAP2c were assembled and expressed in Escherichia coli. The mobility of these bacterial expressed proteins in sodium dodecyl sulfate gels is identical to that of MAP2b and MAP2c from rat brain. The protein sequence of rat MAP2b has been compared to the full length sequence from mouse and the partial sequence from human high molecular weight MAP2. This comparison has revealed that MAP2b is composed of several highly conserved domains flanked by domains with extensive sequence divergence. Two of the conserved domains, found either at the NH2 or COOH terminus, overlap with the binding domain for the regulatory subunit of the cAMP-dependent protein kinase II and the microtubule-binding domain, respectively. A third homologous domain of unknown function lies in a central region of MAP2b. Secondary structure prediction suggests that the portion of MAP2b which extends from the microtubule surface is composed of an extensive number of alpha-helices separated by small turns which may account for the extended yet flexible structure of MAP2. Interestingly, the 4000-base pair deletion from the middle of MAP2b which generates MAP2c not only removes these helices, but also this third highly conserved MAP2b domain.
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PMID:Molecular structure of microtubule-associated protein 2b and 2c from rat brain. 217 50

In our previous report we showed cytochrome b5 to be a competitive inhibitor of cAMP-dependent protein kinase (PKA) for interaction with cytochrome P450 (P450). While P450 was phosphorylated, cytochrome b5 was not. The phosphorylation of P450 resulted in an inhibition of its catalytic activity. In this report we attempt to determine the relationship between phosphorylation of P450 from phenobarbital-induced rat and its destruction. The results indicate there is a considerable alteration of P450 IIB1 when it is put into the phosphorylation medium. This includes destruction, i.e., loss of the hemoprotein nature (Soret peak), as well as denaturation, conversion of a proportion of the P450 to P420. The extent of phosphorylation correlated best with the amount of destroyed hemoprotein, and not with the formation of P420. There did not appear to be phosphorylation-dependent formation of apo-P450. Further, prior conversion of the P450 to P420 using sodium deoxycholate showed the same extent of phosphorylation as before the conversion. Thus, intact P450 is not required for phosphorylation nor is phosphorylation a prerequisite for hemoprotein destruction. P450 CAM (CIA1), which has the PKA substrate recognition sequence internalized, likewise undergoes conversion to P420 but this denaturation does not result in phosphorylation. Destruction of CIA1 with 6 M urea, however, did permit phosphorylation by PKA. P450 IIB1 destruction was greatly diminished by cytochrome b5. This stabilization resulted in a decreased degree of phosphorylation as well as an increase in negative ellipticity in circular dichroism, indicative of an increase in the proportion of alpha-helical content in the P450. Suggestions are made that this structural modification caused by cytochrome b5 stabilizes the P450 against denaturation as well as against destruction and phosphorylation. Further, when the P450 IIB1 was kept stable as P450 in the absence of cytochrome b5 and without loss of hemoprotein during the incubation period, using phosphate-glycerol buffer containing 0.4% Emulgen 911, the phosphorylation of the P450 was greatly diminished, with only minor effects on the protein kinase reaction itself. These results suggest that the protein kinase reaction itself. These results suggest that the protein kinase substrate recognition sequence is not readily accessible to PKA in mammalian P450 IIB1 but requires a destabilization of the protein for phosphorylation to take place.
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PMID:Relationship between phosphorylation and cytochrome P450 destruction. 227 44

KT5926, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3 ,9, 10-tetrahydro-8,11-epoxy, 1H,8H, 11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde] trinden-1-one, was found to be a potent and selective inhibitor of myosin light chain kinase. The compound inhibited both Ca2+/calmodulin-dependent and -independent smooth muscle myosin light chain kinases to a similar extent. The inhibition was not affected by the concentration of calmodulin. Kinetic analyses showed that the mode of inhibition was of the competitive type with respect to ATP (Ki, 18 nM) and of the noncompetitive type with respect to myosin light chain (Ki, 12 nM). These results indicated that KT5926 directly interacted with the enzyme at the catalytic site. KT5926 also inhibited other protein kinases, but with relatively high Ki values; the values for protein kinase C, cAMP-dependent protein kinase, and cGMP-dependent protein kinase were 723, 1200, and 158 nM, respectively. Ca2(+)-ATPase, Na+/K(+)-ATPase, hexokinase, and 5'-nucleotidase were not inhibited by KT5926 at less than 10 microM. The effect of KT5926 on serotonin secretion and protein phosphorylation induced by platelet-activating factor or phorbol ester was examined in rabbit platelets. KT5926 inhibited the phosphorylation of a 20-kDa protein but had no effect on the phosphorylation of a 40-kDa protein, thereby indicating that the compound exerts its selective inhibition of myosin light chain kinase in intact cells. The compound inhibited serotonin secretion induced by platelet-activating factor, but its potency was significantly less than that of K-252a, (8R*,9S*,11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9, 10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, which inhibited the phosphorylation of both the 20-kDa protein and the 40-kDa protein. Phorbol ester-induced secretion was not suppressed by KT5926. These results provide the evidence that both the 20-kDa protein phosphorylation by myosin light chain kinase and the 40-kDa protein phosphorylation by protein kinase C substantially contribute to the secretion response in platelets.
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PMID:KT5926, a potent and selective inhibitor of myosin light chain kinase. 232 35

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.
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PMID:Hormonal regulation and identification of chicken progesterone receptor phosphorylation sites. 239 63

The ionic mechanism of the effect of intracellularly injected adenosine 3',5'-cyclic monophosphate (cAMP) on the membrane of identified neuron L5 of Aplysia kurodai was investigated with conventional voltage-clamp and ion-substitution techniques. The intracellular elevation of cAMP caused an inward current (IcAMP), which was not accompanied by a significant change in membrane conductance at potentials more hyperpolarized than -60 mV. The current increased over the voltage range (-50 to -30 mV) associated with a conductance decrease and decreased at potentials more hyperpolarized than -60 mV. Elevated intracellular cAMP was found to enhance a region of negative slope resistance in steady-state I-V relations. Duration of the IcAMP was greatly prolonged by bath-applied isobutylmethylxanthine (50 microM), but imidazole (10 mM) had an opposite effect on the IcAMP. Tolbutamide (5 mM), a protein kinase inhibitor, reduced the IcAMP. The current was not affected by the presence of bath-applied TTX (50 microM), ouabain (50 microM), or triaminopyrimidine (5 mM). Reduction of [Na+]0 reversibly decreased the IcAMP. Li+ could largely substitute for Na+. Alterations of [K+]0, and bath application of 4-AP (5 mM) and TEA (30 mM) did not affect the IcAMP. In the presence of Na+, Cl-, and divalent cations such as Ca2+ and Ba2+ inhibited the IcAMP. These results suggest that fast elevation of intracellular cAMP induces a TTX-resistant slow Na+ inward current, and the current might be due to activation of cAMP-dependent protein kinase.
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PMID:Influences of pressure-injected cyclic AMP on the membrane current and characteristics of an identified neuron of Aplysia kurodai. 242 22

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.
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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

We have studied cAMP-dependent phosphorylation of sodium channels in rat brain neurons maintained in primary culture. In back phosphorylation studies, cells were treated with drugs to increase intracellular cAMP and sodium channels were solubilized and isolated by immunoprecipitation. Surface and intracellular pools of sodium channels were isolated separately. Purified channels were then phosphorylated with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase to incorporate 32P into available cAMP-dependent phosphorylation sites. The amount of 32P incorporated in vitro is inversely proportional to the extent of endogenous phosphorylation. Incubation of cells with forskolin (0.1-100 microM), 8-Br-cAMP (0.1-10 mM), or isobutylmethylxanthine (0.01-1.0 mM) inhibited subsequent incorporation of 32P into isolated sodium channels by 70-80%, indicating that treatment of cells with these drugs had increased endogenous phosphorylation to nearly maximum levels. The phosphopeptides phosphorylated in vivo and in vitro were identical. To examine the magnitude of basal phosphorylation and the extent of stimulated phosphorylation, the amount of 32P incorporated into sodium channels from control and stimulated cells was compared to that from matched samples which had been dephosphorylated with calcineurin. Sodium channels from control cells incorporated approximately 2-fold more 32P after dephosphorylation, indicating that cAMP-dependent sites on the channel are at least 47% phosphorylated in the basal state. Sodium channels from forskolin-treated cells incorporated 7-8-fold more 32P after dephosphorylation, indicating that cAMP-dependent phosphorylation sites are 80-90% phosphorylated after stimulation. Cell surface and intracellular pools of sodium channels were phosphorylated similarly. In cells metabolically labeled with 32P, cell surface sodium channels incorporated 2.7 mol of phosphate/mol of channel. Forskolin stimulated 32P incorporation into sodium channels 1.3-fold, consistent with the results obtained by back phosphorylation. We conclude that the rat brain sodium channel is substantially phosphorylated in both the cell surface and intracellular pools in vivo in unstimulated rat brain neurons, and the extent of phosphorylation is increased to 80-90% of maximum phosphorylation by agents that elevate intracellular cAMP.
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PMID:Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons. 244 66

Cyclic AMP-dependent protein kinase catalyzes the incorporation of 3-4 mol of phosphate into the alpha subunit of rat brain sodium channels in vitro or in situ. Digestion of phosphorylated sodium channels with CNBr yielded three major phosphorylated fragments of 25, 31, and 33 kDa. These fragments were specifically immunoprecipitated with site-directed antisera establishing their location within an intracellular loop between the first and second homologous domains containing residues 448 to 630 of sodium channel RI or residues 450-639 of sodium channel RII. Five of the seven major tryptic phosphopeptides generated from intact sodium channel alpha subunits were contained in each of the 25-, 31-, and 33-kDa CNBr fragments, indicating that most cAMP-dependent phosphorylation sites are in this domain. Since CNBr digestion of sodium channels which had been metabolically labeled with 32P in intact neurons yielded the same phosphorylated fragments, the phosphorylated region we have identified is the major location of phosphorylation in situ. Only serine residues were phosphorylated by cAMP-dependent protein kinase in vitro, while approximately 16% of the phosphorylation in intact neurons was on threonine residues that must lie outside the domain we have identified. Since this domain is phosphorylated in intact neurons, our results show that it is located on the intracellular side of the plasma membrane. These results are considered with respect to models for the transmembrane orientation of the alpha subunit.
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PMID:Identification of an intracellular domain of the sodium channel having multiple cAMP-dependent phosphorylation sites. 244 73

Incubating toad bladder with 10 mU/ml vasopressin increases the amiloride-blockable Na+ flux in membrane vesicles derived from the epithelial cells by about twofold. This stimulation is further enhanced by 3-isobutyl-1-methylxanthine and can be mimicked by 8-bromoadenosine 3', 5'-cyclic monophosphate. Thus the natriferic action of cAMP involves a sustained change of the apical membrane preserved by the isolated vesicles. The possibility that transport is modulated by direct phosphorylation/dephosphorylation of the Na+ channel was tested. Trapping purified cAMP-dependent protein kinase, cAMP, and ATP in apical vesicles failed to alter Na+ transport even though the enzyme proved active and could phosphorylate intravesicular proteins. Trapping several phosphatases partially purified from toad bladder in vesicles was ineffective as well. These data suggest that the cAMP-induced increase in Na+ conductance involves processes other than phosphorylation of the channel protein or direct channel-cAMP interaction.
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PMID:Characterization of cAMP-induced activation of epithelial sodium channels. 245 30


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