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 tyrosine-3-monooxygenase activity [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] of rat adrenal medulla is induced 20-24 hr after the injection of reserpine (16 mumol/kg intraperitoneally). This and other inducing stimuli increase the 3': 5'-cyclic AMP (cAMP) content in the medulla for longer than 60 min and activate the cAMP-dependent protein kinase (ATP: protein phosphotransferase; EC 2.7.1.37) for several hours. Corticotropin (ACTH), dopamine, and propranolol do not induce the monooxygenase, but elicit an increase in the cAMP content of the medulla which fails to activate protein kinase and lasts less than 1 hr. A high- and low-molecular-weight protein kinase are separated by gel filtration from the 20,000 X g pellet extract of adrenal medulla homogenate. The activity of the low-molecular-weight enzyme is expressed as its ability to phosphorylate histone. The protein kinase activity of the pellet is increased between 3 and 17 hr after reserpine injection. Our evidence indicates that this increase is due to a translocation from cytosol to subcellular structures of a kinase that utilizes lysine-rich histone as phosphate acceptor. The protein kinase activity that is extracted from a purified nuclear fraction prepared from the adrenal medulla of rats injected 7 hr previously with reserpine is greater than that extracted from medulla of saline-treated rats.
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PMID:Activation and nuclear translocation of protein kinase during transsynaptic induction of tyrosine 3-monooxygenase. 0 93

Synthetic polypeptides were employed as substrates in kinetic analyses of the reaction mechanism for the catalytic subunit of a cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from calf thymus. This enzyme preparation was shown to catalyze the transfer of phosphate from ATP to histone H1 from calf thymus, as well as to two synthetic polypeptides, Arg-Lys-Ala-Ser-Gly-Pro (H1-6) and Arg-Arg-Lys-Ala-Ser-Gly-Pro (H1-7), corresponding to the amino acid sequence about serine-38 in calf H1. A related, basic heptapeptide corresponding to a sequence from pig liver pyruvate kinase, Leu-Arg-Arg-Ala-Ser-Leu-Gly (K), was also a substrate. The stoichiometry of peptide phosphorylation was established in each case as the transfer of 1 mol of phosphate from the gamma position of MgATP to the serine hydroxyl of 1 mol of the peptide. Steady-state, initial-velocity, kinetic parameters were determined for each substrate, using various concentrations of ATP. Under the conditions used, all synthetic peptides reacted with greater maximum velocities than whole histone H1. Nevertheless, the K(m) for H1, 54 muM, was lower than the K(m) values of the synthetic substrates. The most efficient substrate was peptide K, which had a V(max) of 50.6 mumol/min per mg of kinase and a K(m) of 63 muM. In the absence of peptide substrate no ATPase activity was detectable at a sensitivity of 0.05% of the rate of peptide phosphorylation, suggesting that ATP is not cleaved to form an unstable phosphoenzyme complex. The data are consistent with a sequential reaction mechanism involving a ternary complex between enzyme, polypeptide substrate, and ATP.
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PMID:Studies on the mechanism of phosphorylation of synthetic polypeptides by a calf thymus cyclic AMP-dependent protein kinase. 20 Sep 11

The catalytic subunit of cyclic AMP-dependent protein kinase (from rabbit skeletal muscle; ATP:protein phosphotransferase, EC 2.7.1.37) was found to be irreversibly inactivated by chloromethyl ketone derivatives of lysine and phenylalanine, chemical reagents originally designed for labeling the active sites of the proteolytic enzymes trypsin and chymotrypsin. This inactivation was shown to occur at pH 7.5 and 22 degrees C, conditions under which chemically related alkylating reagents such as chloroacetamide and chloroacetic acid (which do not possess the amino acid side chain) fail to inactivate the enzyme. In the case of the chloromethyl ketone derivative of N alpha-tosyl-L-lysine, the enzyme could be protected by its nucleotide substrate (MgATP), by one of its protein substrates (histone H2b), and by its regulatory subunit which, upon binding, shields the active site of the catalytic subunit. Differential labeling experiments, together with kinetic studies of the rates of modification of the sulfhydryl groups in the enzyme before and after inactivation with the chloromethyl ketone, suggest that the loss of activity is associated with one (kinetically characterized) sulfhydryl group present either at the active site of the enzyme or at a site intimately associated with it. The general implications of these results regarding the interpretation of affinity labeling experiments carried out in complex mixtures of proteins or under in vivo conditions are discussed.
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PMID:Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone. 22 53

Bovine lung cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) is a potent and relatively specific substrate for cGMP-dependent protein kinase (cGK) as compared to cAMP-dependent protein kinase (cAK) (Thomas, M. K., Francis, S. H., and Corbin, J. D. (1990) J. Biol. Chem. 265, 14971-14978). A synthetic peptide, RKISASEFDRPLR (BPDEtide), was synthesized corresponding to the sequence surrounding the phosphorylation site in cG-BPDE. BPDEtide retained the cGK/cAK kinase specificity demonstrated by native cG-BPDE: the apparent Km of BPDEtide for cGK was 5-fold lower than that for cAK (Km = 68 and 320 microM, respectively). Vmax values were 11 mumol/min/mg for cGK and 3.2 mumol/min/mg for cAK. The peptide was not phosphorylated to a measurable extent by protein kinase C or by calcium/calmodulin-dependent protein kinase II. Thus, the primary amino acid sequence of the peptide substrate was sufficient to confer kinase specificity. Studies in crude tissue extracts indicated that BPDEtide was the most selective peptide substrate documented for measuring cGK activity. Peptide analogs of BPDEtide were synthesized to determine the contribution of specific residues to cGK or cAK substrate specificity. Substitution of a Lys for the amino-terminal Arg did not reduce cGK/cAK specificity; neither did the exchange of an Ala for the non-phosphorylated Ser nor the removal of the 3 carboxyl-terminal residues. A truncated BPDEtide (RKISASE) served equally well as substrate (Km approximately 90 microM) for both kinases. However, restoration of the Phe, to yield RKISASEF, reproduced the original cGK/cAK specificity for BPDEtide (Km = 120 and 480 microM, respectively), primarily by decreasing the affinity of cAK. Addition of a carboxyl-terminal Phe to the peptide RKRSRAE (derived from the sequence of the cGK phosphorylation site in histone H2B) or to the peptide LRRASLG (derived from the sequence of the cAK phosphorylation site in pyruvate kinase) also improved the cGK/cAK specificity by decreasing the affinity of cAK. These data suggested that the Phe in each substrate tested is a negative determinant for cAK.
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PMID:A phenylalanine in peptide substrates provides for selectivity between cGMP- and cAMP-dependent protein kinases. 131 60

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.
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PMID:Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. 161 39

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.
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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 type I form of cAMP-dependent protein kinase binds MgATP with a high affinity, and binding of MgATP decreases the affinity of the holoenzyme for cAMP [Hofmann et al. (1975) J. Biol. Chem. 250, 7795]. Holoenzyme was formed here with a mutant form of the bovine recombinant type I regulatory subunit where the essential arginine in site A, Arg-209, was replaced with Lys. Although this mutation does not significantly change the high-affinity binding of MgATP to the holoenzyme, it does abolish high-affinity binding of cAMP to site A. In the absence of MgATP, binding of cAMP to site B is sufficient to promote dissociation of the holoenzyme complex and activation of the catalytic subunit [Bubis et al. (1988) J. Biol. Chem. 263, 9668]. In the presence of MgATP however, holoenzyme formed with this mutant regulatory subunit is very resistant to cAMP. The Kd(cAMP) was greater than 1 microM, and the Ka(cAMP) increased 60-fold from 130 nM to 6.5 microM in the presence of MgATP. Thus, MgATP serves as a lock that selectively stabilizes the holoenzyme and inhibits activation. Both site A and site B are shielded from cAMP in the presence of MgATP. These results suggest that Arg-209 may play a role in stabilizing the MgATP.holoenzyme complex in addition to its role in binding the exocyclic oxygens of cAMP when cAMP is bound to the regulatory subunit. The catalytic subunit also reassociates rapidly with this mutant regulatory subunit, and in contrast to the wild-type regulatory subunit, holoenzyme formation does not require MgATP.
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PMID:Role of MgATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP binding site A. 184 4

Each regulatory (R) subunit of cAMP-dependent protein kinase contains an autoinhibitor site that lies approximately 90-100 residues from the amino terminus. In order to study the importance of this autoinhibitor site in the type I R-subunit for interacting with the catalytic (C) subunit, recombinant techniques were used to replace Ala-97 with Gln, His, Lys, and Arg and to replace Ser-99 with Gly and Lys. All of the mutant proteins having a replacement at Ala-97 showed reduced affinity for the C-subunit ranging from 14- to 55-fold. In general, the decrease in affinity of the Ala-97 mutants for the C-subunit correlated with the increase in size of the side chain. In contrast to wild type R-subunit, where MgATP facilitates holoenzyme formation, MgATP inhibits the reassociation in all of the Ala-97 mutants suggesting that the larger side chains sterically interfere with bound MgATP in the active site of the C-subunit. Whereas MgATP slowed holoenzyme formation, AMP actually accelerated the reassociation of the A97K, A97H (pH 6.0), and A97Q mutants with the C-subunit. Therefore, the side chains of Lys-97, His-97, and Gln-97 can interact either electrostatically or by hydrogen bonding with the phosphate of AMP. This interpretation is reinforced by the fact that the stimulatory effect of AMP on the A97H mutant was pH-dependent. The affinities of the S99G and S99K mutants for the C-subunit were reduced 7- and 24-fold, respectively, suggesting that Ser-99 also may contribute to interactions between the R- and C-subunits.
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PMID:Mutations in the autoinhibitor site of the regulatory subunit of cAMP-dependent protein kinase I. Replacement of Ala-97 and Ser-99 interferes with reassociation with the catalytic subunit. 184 75

Rap 1B is a low molecular weight G protein which is phosphorylated by cAMP-dependent protein kinase. In order to identify the site of phosphorylation by cAMP-dependent protein kinase, purified rap 1B from human platelets was phosphorylated and subjected to limited proteolysis with trypsin. Single digestion fragment containing the phosphorylation site was obtained and purified by reversed-phase HPLC. Sequence analysis of the phosphorylated digestion fragment demonstrated that the sequence of the phosphorylation site was -Lys-Lys-Ser-Ser-. This sequence is near the carboxy terminus and is adjacent to the site of membrane attachment of the protein.
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PMID:The localization of the cAMP-dependent protein kinase phosphorylation site in the platelet rat protein, rap 1B. 190 69

Mouse L929 cells were used to study the mechanism of cAMP induction of alkaline phosphatase (AP) activity. Following treatment with 200 microM 8-chlorophenylthio-cAMP (CPT-cAMP), alkaline phosphatase enzyme activity was observed to increase 80-fold after 24 h. The CPT-cAMP dose response of the alkaline phosphatase enzyme activity correlated well with the CPT-cAMP activation of cAMP-dependent protein kinase in L cells. A cDNA clone for the alkaline phosphatase was isolated and used to demonstrate a 10-fold increase in alkaline phosphatase mRNA levels after a 24-h treatment of L cells with CPT-cAMP. Increased mRNA levels were first detected 4-6 h, after CPT-cAMP treatment, and the level of alkaline phosphatase mRNA decreased rapidly after removal of CPT-cAMP. In vitro nuclear transcription studies showed that a 3-fold increase in alkaline phosphatase gene transcription was detectable 6 h after CPT treatment, and this increase was blocked by cycloheximide. In order to determine if the catalytic (C) subunit of cAMP-dependent protein kinase was able to mediate the induction of AP, L cells were transfected with expression vectors containing the metallothionein promoter and coding for the C alpha isoform of the catalytic subunit of cAMP-dependent protein kinase or for a catalytic subunit in which lysine 72 had been mutated to methionine (C alpha K72M). Zinc treatment of stably transfected cells expressing the wild-type C subunit showed an increase in protein kinase activity and an increase in AP activity. Zinc treatment of cells containing the mutant C subunit expression vector produced an increase in the amount of a protein which was recognized by C subunit antibodies on Western blots, but these cells showed no increase in protein kinase activity or in AP activity. We conclude that the C subunit is sufficient for transcriptional induction of the AP gene and that the phosphotransferase activity of the C subunit is required for this induction.
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PMID:Induction of alkaline phosphatase in mouse L cells by overexpression of the catalytic subunit of cAMP-dependent protein kinase. 216 96


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