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)

The primary (alpha 1) subunit of purified skeletal muscle dihydropyridine-sensitive calcium channels is present in full-length (212 kDa) and truncated (190 kDa) forms which are both phosphorylated by cAMP-dependent protein kinase (cA-PK) in vitro. In the present study, phosphorylation of the purified calcium channel by cA-PK followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and two-dimensional phosphopeptide mapping revealed differential phosphorylation of the related 190- and 212-kDa forms. The 190-kDa form of the alpha 1 subunit was phosphorylated on three major and three minor tryptic phosphopeptides; the 212-kDa form was phosphorylated on all six of these phosphopeptides plus two that were unique. Time course experiments showed that a single site on the COOH-terminal portion of the full-length form of the alpha 1 subunit is most intensely and rapidly (within 10 s) phosphorylated. Phosphorylation occurs almost exclusively on this COOH-terminal site unless harsh conditions such as treatment with denaturing detergents are employed to expose phosphorylation sites within the 190-kDa segment of the molecule. Elution of phosphopeptides from the second dimension chromatograph followed by immunoprecipitation with an anti-peptide antibody (anti-CP1) directed against the COOH-terminal amino acid sequence enabled us to identify this major phosphorylation site as serine 1854. The nearby consensus sites for cA-PK phosphorylation at serines 1757 and 1772 were phosphorylated only after denaturation or proteolytic cleavage. Phosphorylation of serine 1854 may play a pivotal role in the regulation of calcium channel function by cA-PK.
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PMID:Specific phosphorylation of a COOH-terminal site on the full-length form of the alpha 1 subunit of the skeletal muscle calcium channel by cAMP-dependent protein kinase. 132 91

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.
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PMID:Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C. 132 92

The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.
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PMID:Phosphorylation of Na,K-ATPase alpha-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C. 133 Oct 53

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.
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PMID:The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase. 133 96

Antibodies were raised against three peptides corresponding to the potential protein phosphorylation sites of rat-brain sodium channels by the cAMP-dependent protein kinase (PKA). One of the antibody against sequence (C561-575) reacted to the channel molecule. This immunoreaction occurred in a sequence-specific manner, as it was inhibited by the antigen peptide itself but not inhibited by two other peptides. Although PKA phosphorylates two synthetic peptides, C561-575 and C681-689, of the three, anti-(C561-575) antibody can only inhibit the phosphorylation of peptide (C561-575). PKA catalyzed the incorporation of 3.1-3.5 mol of phosphates into the alpha subunit of the purified sodium channel. The anti-(C561-575) antibody inhibited the channel phosphorylation by 40%. Digestion of the phosphorylated sodium channel with lysyl endoproteinase yielded four major phosphorylated fragments of 3.5, 5.0, 7.0, and 10 kDa. However, similar digestion of the channel that was phosphorylated in the presence of anti-(C561-575) antibody did not yield the phosphorylated fragment of 3.5 kDa and gave the 7.0 kDa fragment in reducing yield. Inspection of these phosphorylated fragments by the predicted sizes of the peptide fragments containing the five potential phosphorylation sites gives a conclusion that anti-(C561-575) antibody inhibits the phosphorylation on Ser-573 completely, and on either Ser-610 or Ser-623 partially, probably due to their proximity orientation in the tertiary structure.
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PMID:A site-directed antibody that inhibits phosphorylation of the rat-brain sodium channel by cyclic-AMP-dependent protein kinase. 133 1

1. The function of trout RBC Na+/H+ antiport is unrelated to cell volume or cell pH regulation. Its role is to improve oxygen transport capacity when the supply of oxygen becomes limited. 2. Antiport activation, mediated by cAMP, promotes complex changes in blood pH which have been analyzed in vivo and in vitro. 3. The regulation of antiport (activation, desensitization, control by molecular oxygen and by a newly discovered cytosolic protein, arrestin) is presented. 4. Molecular cloning of the antiport shows that two typical site motifs of phosphorylation by cAMP-dependent protein kinase are localized on the cytoplasmic region.
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PMID:Regulation of Na+/H+ exchange and pH in erythrocytes of fish. 135 21

The electrophysiological properties of a cardiac cell line (MCM1) originating from a transgenic mouse were characterized. The dominant current in these cells is a sodium current that is insensitive to concentrations of tetrodotoxin (TTX) up to 100 microM. It activates and inactivates rapidly with half-maximal activation at -40 mV and half-maximal inactivation at -79 mV. This sodium current is reduced by agents that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and activate cAMP-dependent protein kinase including isoproterenol, 8-bromo-cAMP, and isobutylmethylxanthine. The phenylalkylamine desmethoxyverapamil blocks the TTX-insensitive sodium current in MCM1 cells in both tonic and use-dependent fashion. Membrane depolarization enhances this block. It is proposed that the TTX-insensitive sodium current in these cells may be similar in origin to the embryonic type of TTX-insensitive sodium current described in other cardiac and skeletal muscle preparations.
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PMID:Tetrodotoxin-insensitive sodium channels in a cardiac cell line from a transgenic mouse. 137 81

It is universally believed that the removal of external sodium ions is without effect on calcium current. We now report that in enzymatically isolated guinea pig ventricular cells, the replacement of external sodium ions with certain other cations causes a 3- to 6-fold increase in peak L-type calcium current. The increase in current is reversibly blocked by L-type calcium-channel antagonists, not mediated by changes in internal calcium, and is inhibited by intracellular 5'-adenylyl imidodiphosphate, a nonhydrolyzable ATP analogue. The effects of sodium removal (and isoproterenol) were almost completely blocked by intracellular application of a specific (peptide) inhibitor of cAMP-dependent protein kinase. These experiments demonstrate a previously unknown effect of sodium ions to modulate calcium-channel phosphorylation via cAMP-dependent protein kinase.
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PMID:Modulation of L-type calcium channels by sodium ions. 137 2

The ubiquitous plasma membrane Na+/H+ exchanger (termed NHE1) is activated by diverse hormonal signals, with the notable exception of hormones acting through cAMP as second messenger. Therefore, the Na+/H+ exchanger found in the nucleated trout red cell is of particular interest since it is activated by catecholamines, forskolin, and cAMP analogues. We report here that a cloned cDNA encoding the red cell exchanger restores functional Na+/H+ activity when transfected into Na+/H+ antiporter-deficient fibroblasts (i.e., it regulates intracellular pH in a Na-dependent and amiloride-sensitive manner). This red cell exchanger represents an additional form of Na+/H+ exchanger (termed beta NHE), which is characterized by a specific cytoplasmic domain involved in activation by the cAMP-dependent signaling pathway. After transfection in the same cellular context, beta NHE, but not NHE1, is activated by cAMP or by hormones that increase cAMP levels. Comparison of the amino acid sequences of exchangers shows that beta NHE, but not NHE1, contains two clustered consensus motifs for phosphorylation by a cAMP-dependent protein kinase (protein kinase A; PKA). A deletion mutant devoid of the C-terminal region of the cytoplasmic loop containing the two PKA sites restores Na+/H+ activity but is no longer activated by cAMP analogues or catecholamines. In red blood cells, the Na+/H+ exchanger is also activated by another pathway involving protein kinase C (PKC). Expression of beta NHE in fibroblasts shows that these two independent signaling pathways impinge on two distinct domains of the exchanger. The cytoplasmic segment containing PKA consensus sites, which is crucial for cAMP activation, is unnecessary for stimulation by PKC activators.
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PMID:Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation. 137 18

The voltage-dependent Na+ channel of the brain is a good substrate for phosphorylation by the cAMP-dependent protein kinase (protein kinase A, or PKA), but the physiological effects of PKA on Na+ channels are poorly documented. We studied modulation by PKA of voltage-dependent Na+ channels expressed in Xenopus oocytes injected with RNA coding for the alpha-subunit of the channel protein (rat brain type IIA and its variant VA200), using the two electrode voltage-clamp technique. Intracellularly injected cAMP or catalytic subunit of PKA, or extracellularly applied forskolin, inhibited the Na+ current by 20-30%. The effect of cAMP was attenuated by prior injection of PKA inhibitors. Injection of small doses of protein phosphatase 2A increased the Na+ current by 10%, whereas larger doses of protein phosphatase 1 and alkaline phosphatase were without effect. The inhibition by PKA showed little voltage dependence, being only slightly stronger at holding potentials at which the availability of the channels was reduced. The voltage dependence of activation and inactivation processes was not altered by cAMP. Similar effects were exerted by forskolin and cAMP on the Na+ channels expressed after the injection of heterologous (total) RNA from rat brain. Thus, PKA modulates the Na+ channel by a mechanism that does not involve major changes in the voltage dependency of the current and is exerted on the channel-forming alpha-subunit.
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PMID:Protein kinase A reduces voltage-dependent Na+ current in Xenopus oocytes. 138 76


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