Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

(RP)-cAMPS is known to inhibit competitively the cAMP-induced activation of cAMP-dependent protein kinase (PKA). The molecular nature of this inhibition, however, is unknown. By monitoring the intrinsic tryptophan fluorescence of recombinant type I regulatory subunit of PKA under unfolding conditions, a free energy value (delta GDH2O) of 8.23 +/- 0.22 kcal/mol was calculated. The cAMP-free form of the regulatory subunit was less stable with delta GDH2O = 6.04 +/- 0.05 kcal/mol. Native stability was recovered by treatment of the cAMP-free protein with either cAMP or (SP)-cAMPS but not with (RP)-cAMPS. Thus, (RP)-cAMPS binding to the regulatory subunit keeps the protein in a locked conformation, unable to release the catalytic subunit. This finding was further supported by demonstrating that holoenzyme formation was greatly accelerated only when bound cAMP was replaced with (RP)-cAMPS but not with cAMP or (SP)-cAMPS.
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PMID:(RP)-cAMPS inhibits the cAMP-dependent protein kinase by blocking the cAMP-induced conformational transition. 749 6

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

The regulatory R-subunit of cAMP-dependent protein kinase (cAPK) is a thermostable multidomain protein. It contains a dimerization domain at the N-terminus followed by an inhibitor site that binds the catalytic C-subunit and two tandem cAMP-binding domains (A and B). Two of the three tryptophans in the RIalpha subunit, Trp188 and Trp222, lie in cAMP-binding domain A while Trp260 lies at the junction between domains A and B. The unfolding of wild-type RIalpha (wt-RI), monitored by intrinsic fluorescence, was described previously [Leon, D. A., Dostmann, W. R. G., and Taylor, S. S. (1991) Biochemistry 30, 3035 (1)]. To determine the environment of each tryptophan and the role of the adjacent domain in folding and stabilization of domain A, three point mutations, W188Y, W222Y, and W260Y, were introduced. The secondary structure of wt-RI and the point mutants has been studied by far-UV circular dichroism spectropolarimetry (CD). The CD spectra of wt-RI and the three point mutants are practically identical, and the thermal unfolding behavior is very similar. Intrinsic fluorescence and iodide quenching in the presence of increasing urea established that: (a) Trp222 is the most buried, whereas Trp188 is the most exposed to solvent; (b) Trp260 accounts for the quenching of fluorescence when cAMP is bound; and (c) Trp222 contributes most to the intrinsic fluorescence of the wt-RI-subunit, while Trp188 contributes least. For wt-RI, rR(W188Y), and rR(W260Y), removal of cAMP causes a destabilization, while excess cAMP stabilizes these three proteins. In contrast, rR(W222Y) was not stabilized by excess cAMP.
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PMID:Probing the multidomain structure of the type I regulatory subunit of cAMP-dependent protein kinase using mutational analysis: role and environment of endogenous tryptophans. 1080 16

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.
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PMID:Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase. 1110 80

Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.
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PMID:A nonsense mutation in the NDUFS4 gene encoding the 18 kDa (AQDQ) subunit of complex I abolishes assembly and activity of the complex in a patient with Leigh-like syndrome. 1118 77

The amino terminal dimerization/docking domain and the two-tandem, carboxy-terminal cAMP-binding domains (A and B) of cAMP-dependent protein kinase regulatory (R) subunits are connected by a variable linker region. In addition to providing a docking site for the catalytic subunit, the linker region is a major source of sequence diversity between the R-subunit isoforms. The RIIbeta isoform uniquely contains two endogenous tryptophan residues, one at position 58 in the linker region and the other at position 243 in cAMP-binding domain A, which can act as intrinsic reporter groups of their dynamics and microenvironment. Two single-point mutations, W58F and W243F, allowed the local environment of each Trp to be probed using steady-state and time-resolved fluorescence techniques. We report that: (a) the tryptophan fluorescence of the wild-type protein largely reflects Trp243 emission; (2) cAMP selectively quenches Trp243 and thus acts as a cAMP sensor; (3) Trp58 resides in a highly solvated, unstructured, and mobile region of the protein; and (4) Trp243 resides in a stable, folded domain and is relatively buried and rigid within the domain. The use of endogenous Trp residues presents a non-perturbing method for studying R-subunit subdomain characteristics in addition to providing the first biophysical data on the RIIbeta linker region.
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PMID:Endogenous tryptophan residues of cAPK regulatory subunit type IIbeta reveal local variations in environments and dynamics. 1278 14

Annotation of the sequenced Drosophila genome suggested the presence of an additional enzyme with extensive homology to mammalian tryptophan hydroxylase, which we have termed DTRH. In this work, we show that enzymatic analyses of the putative DTRH enzyme expressed in Escherichia coli confirm that it acts as a tryptophan hydroxylase but can also hydroxylate phenylalanine, in vitro. Building upon the knowledge gained from the work in mice and zebrafish, it is possible to hypothesize that DTRH may be primarily neuronal in function and expression, and DTPH, which has been previously shown to have phenylalanine hydroxylation as its primary role, may be the peripheral tryptophan hydroxylase in Drosophila. The experiments presented in this report also show that DTRH is similar to DTPH in that it exhibits differential hydroxylase activity based on substrate. When DTRH uses tryptophan as a substrate, substrate inhibition, catecholamine inhibition, and decreased tryptophan hydroxylase activity in the presence of serotonin synthesis inhibitors are observed. When DTRH uses phenylalanine as a substrate, end product inhibition, increased phenylalanine hydroxylase activity after phosphorylation by cAMP-dependent protein kinase, and a decrease in phenylalanine hydroxylase activity in the presence of the serotonin synthesis inhibitor, alpha-methyl-(DL)-tryptophan are observed. These experiments suggest that the presence of distinct tryptophan hydroxylase enzymes may be evolutionarily conserved and serve as an ancient mechanism to appropriately regulate the production of serotonin in its target tissues.
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PMID:Serotonin synthesis by two distinct enzymes in Drosophila melanogaster. 1582 93

Tetrahydrobiopterin is an essential cofactor for the phenylalanine, tyrosine and tryptophan hydroxylases, and the family of nitric oxide synthases. The initial and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin is GTP cyclohydrolase I. The proximal promoter of the human GTP cyclohydrolase I gene contains the sequence motif 5'-TGACGCGA-3', resembling a cAMP response element (CRE). The objective of this study was to analyze the regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper (bZIP) transcription factors. A constitutively active mutant of the cAMP response element binding (CREB) protein strongly stimulated GTP cyclohydrolase I promoter activity, indicating that the CRE in the context of the GTP cyclohydrolase I gene is functional. Likewise, GTP cyclohydrolase I promoter/luciferase gene transcription was stimulated following nuclear expression of the catalytic subunit of cAMP-dependent protein kinase. Constitutively active mutants of activating transcription factor 2 (ATF2) and c-Jun additionally stimulated GTP cyclohydrolase I promoter activity, but to a lesser extent than the constitutively active CREB mutant. The fact that stress-activated protein kinases target the GTP cyclohydrolase I gene was corroborated by expression experiments involving p38 and MEKK1 protein kinases. We conclude that signaling pathways involving either the cAMP-dependent protein kinase or stress-activated protein kinases converge to the GTP cyclohydrolase I gene. Hence, enzymatic reactions that require tetrahydrobiopterin as cofactor are therefore indirectly controlled by signaling cascades involving the signal-responsive transcription factors CREB, c-Jun, and ATF2.
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PMID:Regulation of GTP cyclohydrolase I gene transcription by basic region leucine zipper transcription factors. 1614 46

Sir David Cuthbertson was the first to define metabolic alterations in post-aggression syndrome (PAS). From basic measurements of nitrogen loss and total protein synthesis/degradation, the current research has moved to genomics, proteomics and metabolomics. In this respect, first evidence was accumulated for the influence of acute catabolism, immobilisation by bed rest and sarcopenia of old age on the muscle-cell genome and proteome. Moreover, in post-aggression syndrome specific amino acids such as glutamine, arginine, glycine, taurine, tryptophan and cysteine are used for cell and immune modulation. Our laboratory has focused on the regulative capacity of glutamine. Glutamine deficiency as found in post-aggression syndrome reduces lymphocyte proliferation, alters monocyte/macrophage activity, decreases the formation of heat-shock proteins, stimulates cell apoptosis, shifts the cellular redox potential by altering the glutathione synthesis and increases the activity of the AMPK system. Investigating the molecular effect of glutamine on Hsp 70 induction, we tested the glutamine dependence on the formation of transfer-RNA and of heat-shock factor 1 (HSF 1), and on transcription and translation of Hsp 70. We could demonstrate that glutamine stabilises the mRNA of Hsp 70 thereby prolonging its half-life. The lecture also discusses the principal molecular targets of administered arginine, glycine, cysteine, taurine and tryptophan.
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PMID:Immune and cell modulation by amino acids. 1759 24

Human small heat shock protein with molecular mass 22 kD (HSP22, HspB8) contains two Ser residues (Ser24 and Ser57) in consensus sequence RXS and is effectively phosphorylated by cAMP-dependent protein kinase in vitro. Mutation S24D did not affect, whereas mutations S57D or S24,57D prevented phosphorylation of HSP22 by cAMP-dependent protein kinase thus indicating that Ser57 is the primary site of phosphorylation. Phosphorylation (or mutation) of Ser57 (or Ser24 and Ser57) resulted in changes of the local environment of tryptophan residues and increased HSP22 susceptibility to chymotrypsinolysis. Mutations mimicking phosphorylation decreased dissociation of HSP22 oligomer at low concentration without affecting its quaternary structure at high protein concentration. Mutations S24D, S57D, and especially S24,57D were accompanied by decrease of chaperone-like activity of HSP22 if insulin and rhodanase were used as substrates. Thus, phosphorylation by cAMP-dependent protein kinase affects the structure and decreases chaperone-like activity of HSP22 in vitro.
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PMID:Phosphorylation by cyclic AMP-dependent protein kinase inhibits chaperone-like activity of human HSP22 in vitro. 1829 77


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