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)

Two mutations of the catalytic (C) subunit of the cAMP-dependent protein kinase where His87 was changed to Ala and Asp were expressed in Escherichia coli and purified. These mutants were phosphorylated at Thr197 and were catalytically active, although some changes in their kinetic parameters were observed. The most striking differences were in their interaction with the physiological inhibitors. Both mutants were inhibited by protein kinase inhibitor with Ki values below 50 nM. Both mutants were defective in their interaction with the type I regulatory (RI) subunit as measured by (i) the rate of holoenzyme formation with cAMP bound RI-subunit and (ii) the apparent Kd with the cAMP-free RI-subunit. The rate of holoenzyme formation was impaired both in the presence and absence of ATP with the His to Asp mutant showing the greatest effect. The mutant C-subunits were also combined with RI-subunits that contained mutations in the autoinhibitor sequence at Arg94 (P-3) and Ser99 (P + 2). Complementarity between His87 and Ser99 was established, but not between His87 and Arg94. Holoenzyme formation with a Ser99-->Lys mutant RI-subunit was less dependent on ATP when combined with either of the C-subunit mutants than when it was combined with the wild-type C-subunit. The apparent Kd values in the presence of ATP for the mutant combinations were also measured. The Ser99-->Lys mutant was compensated for by both His87 mutants. The His87-->Ala C-subunit mutant was unable to form an inhibited holoenzyme complex with a mutant RI-subunit which was defective in cAMP binding to the A-site. This indicated that this R-subunit was defective in C-subunit recognition as well as in cAMP binding. The roles of His87 on the C-subunit and Ser99 and Arg209 on the RI-subunit in R-C interactions are discussed.
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PMID:Holoenzyme interaction sites in the cAMP-dependent protein kinase. Histidine 87 in the catalytic subunit complements serine 99 in the type I regulatory subunit. 807 12

The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Escherichia coli. These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites. Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, [3H]cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of [3H]cAMP from the mutant subunits at low temperature. The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation. While the presence of cAMP in site A retards dissociation of [3H]cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of [3H]cAMP from site B of the mutant subunits. The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites. A second allosteric interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242.
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PMID:Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and B of RI subunit of cyclic AMP-dependent protein kinase. 808 3

Protein phosphorylation is a key regulatory mechanism for several functions. Although the complex control of organogenesis and growth most likely includes such mechanisms, few reports have examined protein phosphorylation in the developing mammal. The identification and characterization of mammalian embryonic phosphoproteins will allow a greater understanding of the regulation and mechanisms of developmental processes. Phosphorylation of the endogenous mouse proteins during development revealed a 100-kDa protein, located in the cytosolic fraction, to be the major substrate. The Ca(2+)-calmodulin kinase inhibitors, trifluorperazine and ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid, inhibited this phosphorylation. Inhibitors of protein kinase C (H-7)- and cAMP-dependent protein kinase, as well as the tyrosine kinase inhibitor, genistein, had no effect. One- and two-dimensional phosphoamino acid analysis indicated that phosphothreonine was the major phosphorylated amino acid. To determine the identity of this protein, the 100-kDa band was isolated and submitted for amino acid analysis and N-terminal sequencing. The N-terminal sequence Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp-Lys, was identical to the N-terminal sequence of human, hamster and rat elongation factor 2 (EF-2). Western blotting analysis confirmed that the 100 kDa protein was EF-2. Our results of phosphorylated EF-2 in the developing mouse are in agreement with those reported in the avian embryo. However, our results differ in that phosphotyrosine detected in avian embryos could not be detected in murine embryos. This is the first report to demonstrate EF-2 in the developing mammalian embryo and its specific phosphorylation pattern. Our data suggest that the functional phosphoregulation of elongation factor 2 during protein synthesis in mammals is conserved from the developing embryo to the adult and thus emphasizes the importance of EF-2 in normal development and survival.
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PMID:Identification of a 100-kDa phosphoprotein in developing murine embryos as elongation factor 2. 811 99

The structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide (peptide-546) and its effects on cGMP-dependent protein kinase I alpha (G-kinase) have been studied. By primary sequence analysis and analogy to a peptide that stimulates protein kinase C, peptide-546 was predicted to form part of the protein/peptide binding site of G-kinase, and it was proposed that it would stimulate the enzyme by interaction with an autoinhibitory site. The portion of cAMP-dependent protein kinase analogous to peptide-546 forms part of the peptide substrate binding site, interacting with the peptide inhibitor residues Argp-2 and Phep-11 (where p is the pseudophosphorylation site), through residues at positions corresponding to Glu4, Pro10 and Ser13 in peptide-546. Peptide-546 is a reasonably potent G-kinase activator, increasing the turnover number with the peptide substrate Arg-Lys-Arg-Ser-Arg-Lys-Glu by about threefold with an activation constant that is about fivefold lower than the Km value of this peptide substrate. Peptide-546 does not appear to change the affinity of the enzyme for the above substrate, ATP or cGMP and does not affect the binding of [3H]cGMP to G-kinase. The activation does not seem to result from an interaction between peptide-546 and peptide substrates, and a kinetic scheme is proposed which is compatible with an action of peptide-546 on G-kinase independent of substrates. The activation is additive with that given by cGMP and causes the enzyme to enter a hitherto unrecognised superactive state. Peptide conformation has been monitored in mixed 2,2,2-trifluoroethanol/H2O solvents by circular dichroism: helical structure is observed in these mixtures when the 2,2,2-trifluoroethanol content is above 25%. The structure is lost only gradually on raising the temperature to 80 degrees C with no clear melting transition. Assignment of the resonances in the 1H-NMR spectrum has allowed the identification of elements of secondary structure from detected nuclear Overhauser effects. In particular, a helical segment from Met18 to Arg26 is observed. The four proline residues (Pro10, Pro11, Pro15 and Pro17) are all seen to be in the trans conformation, although additional, weaker peaks in the spectra may correspond to a minor conformer in which one or more of the prolines is in a cis conformation. The N-terminal residues are less structured but show some helical character.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Stimulation of cGMP-dependent protein kinase I alpha by a peptide from its own sequence. An investigation by enzymology, circular dichroism and 1H NMR of the activity and structure of cGMP-dependent protein kinase I alpha-(546-576)-peptide amide. 816 46

The response of an endogenous inhibitor of cAMP-dependent protein kinase (type I inhibitor) to tremorine was used as an index of sensitivity of control muscarinic M2-receptors. Tremorine induced a dose-dependent increase in type I inhibitor activity in the posterior hypothalamus and brain stem. The action of the compound was blocked by pretreatment with aminophylline and atropine. Prolonged, 28 days treatment with lysine vasopressin (1 U/kg/day ip) induced hypertension and modified the dose-response curve for tremorine. Five times higher doses of tremorine than in normotensive rats were necessary to induce statistically significant increase in type I inhibitor activity in the posterior hypothalamus and brain stem suggesting subsensitivity of M2-muscarinic receptors in the brain areas responsible for the regulation of blood pressure.
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PMID:The responsiveness of M2-muscarinic receptors in the posterior hypothalamus and brain stem of vasopressin hypertensive rats. 822 Jun 62

The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with Ile caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency (Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C. 842 32

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
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PMID:Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A. 855 May 70

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R- and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar Ka's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.
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PMID:Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase. 860 31

In the previous paper, we reported a sensitive method for detection of protein kinase activities in gels after SDS-polyacrylamide gel electrophoresis (Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143). This method is useful for the detection of various protein kinase activities toward protein substrates included in gels, but inapplicable to oligopeptide substrates because most of the oligopeptides eluted from the gel matrix during electrophoresis. The present study describes a new procedure for the detection of protein kinase activities toward synthetic oligopeptides in the gel. The oligopeptides which were linked to amino acid polymers such as poly-L-lysine through their amino-terminal cysteinyl residue by a heterobifunctional reagent were efficiently retained in the gel matrix and served as substrates for the protein kinases. As little as 2.5 pg of the catalytic subunit of cAMP-dependent protein kinase was detected by this in-gel assay method using a synthetic peptide as a substrate. This technique can be used for selective and sensitive detection of various protein kinases in crude tissue extracts.
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PMID:Detection of protein kinase activities toward oligopeptides in sodium dodecyl sulfate-polyacrylamide gel. 866 May 66

Three-dimensional models of the five functional modules in human protein kinase C alpha (PKC alpha) have been generated on the basis of known related structures. The catalytic region at the C-terminus of the sequence and the N-terminal auto-inhibitory pseudo-substrate have been modeled using the crystal structure complex of cAMP-dependent protein kinase (cAPK) and PKI peptide. While the N-terminal helix of the catalytic region of PKC alpha is predicted to be in a different location compared with cAPK, the C-terminal extension is modeled like that in the cAPK. The predicted permissive phosphorylation site of PKC alpha, Thr 497, is found to be entirely consistent with the mutagenesis studies. Basic Lys and Arg residues in the pseudo-substrate make several specific interactions with acidic residues in the catalytic region and may interact with the permissive phosphorylation site. Models of the two zinc-binding modules of PKC alpha are based on nuclear magnetic resonance and crystal structures of such modules in other PKC isoforms while the calcium phospholipid binding module (C2) is based on the crystal structure of a repeating unit in synaptotagmin I. Phorbol ester binding regions in zinc-binding modules and the calcium binding region in the C2 domain are similar to those in the basis structures. A hypothetical model of the relative positions of all five modules has the putative lipid binding ends of the C2 and the two zinc-binding domains pointing in the same direction and may serve as a basis for further experiments.
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PMID:Structural aspects of the functional modules in human protein kinase-C alpha deduced from comparative analyses. 891 29


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