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 cAMP-dependent protein kinase is a bifunctional enzyme, catalyzing the phosphorylation of the serine and threonine residues in peptides and proteins (kinase activity) as well as the phosphorylation of water (ATPase activity). We have found that several peptides, which serve as inhibitors of the kinase reaction, will either maintain or enhance the ATPase reaction catalyzed by the enzyme. Positively charged dipeptides (e.g. Arg-Arg), as well as small guanidino-containing compounds (e.g. guanethidine) block protein kinase activity yet enhance ATPase activity up to 3.5-fold over that exhibited by the enzyme in the absence of these compounds. In contrast, several nonphosphorylatable peptides, whose primary sequences are based on that of a known substrate (i.e. Leu-Arg-Arg-Ala-Ser-Leu-Gly), such as Leu-Arg-Arg-Ala-Ala-Leu-Gly, Leu-Arg-Arg-Ala-Phe-Leu-Gly, and Leu-Arg-Arg-Ala-Tyr-Leu-Gly, have little or no effect on the rate of the kinase-catalyzed hydrolysis of ATP. An exception to the latter observation is Leu-Arg-Arg-Ala-Cys-Leu-Gly, a cysteine-containing peptide that promotes the protein kinase-catalyzed ATPase reaction by 2.2-fold. We have also found that peptides that possess relatively large amino acid side chain moieties immediately following the arginine dyad (i.e. such as Phe, Tyr, Cys, or Asn at Xaa in Leu-Arg-Arg-Xaa-Ala-Leu-Gly) sharply reduce the rate of enzyme-catalyzed ATP hydrolysis. This suggests that in the presence of peptides containing an -Arg-Arg-Ala- sequence, the enzyme-bound gamma-phosphate of ATP is relatively accessible to water. In contrast, when the latter alanine moiety is replaced by a larger residue, access by water to ATP appears to be hindered. These results indicate that certain structural features associated with the substrate or substrate analog have a profound influence on the manner by which these species interact with the protein kinase. Furthermore, the work described herein demonstrates that it is possible to block the physiologically important kinase reaction and simultaneously promote the energetically wasteful ATPase reaction.
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PMID:ATPase-promoting dead end inhibitors of the cAMP-dependent protein kinase. 850 66

We have identified of set of related transcripts expressed in the germ line of male Drosophila melanogaster. Surprisingly, while one of the corresponding genes is autosomal the remainder are located on the Y chromosome. The autosomal locus, at 77F on chromosome arm 3L, corresponds to the previously described transcription unit 18c, located in the first intron of the gene for an RI subunit of cAMP-dependent protein kinase. The Y chromosome copies have been mapped to region h18-h19 on the cytogenetic map of the Y outside of any of the regions required for male fertility. In contrast to D. melanogaster, where Y-linked copies were found in nine different wild-type strains, no Y-linked copies were found in sibling species. Several apparently Y-derived cDNA clones and one Y-linked genomic clone have been sequenced. The Y-derived genomic DNA shares the same intron/exon structure as the autosomal copy as well as related flanking sequences suggesting that it transposed to the Y from the autosomal locus. However, this particular Y-linked copy cannot encode a functional polypeptide due to a stop codon at amino acid position 72. Divergence among five different cDNA clones ranges from 1.5 to 6% and includes a large number of third position substitutions. We have not yet obtained a full-length cDNA from a Y-linked gene and therefore cannot conclude that the D. melanogaster Y chromosome contains functional protein-coding genes. The autosomal gene encodes a predicted polypeptide with 45% similarity to histones of the H5 class and more limited similarity to cysteine-rich protamines. This protein may be a distant relative of the histone H1 family perhaps involved in sperm chromatin condensation.
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PMID:Drosophila melanogaster male germ line-specific transcripts with autosomal and Y-linked genes. 851 38

Affinity labels for proteins that process other proteins (e.g. proteinases and protein kinases) are an amalgam of two components, an active site-directed peptide carrier and a non-peptidic electrophilic appendage. We have synthesized several affinity labels for the cAMP-dependent protein kinase that are composed solely of L-amino acids and therefore contain only functionality present in naturally occurring proteins. We have found that 2 adjacent cysteine residues, when covalently linked via a peptide bond and an intramolecular disulfide loop (abbreviated as Cys<-->Cys), serves as a potent electrophile. The peptides Leu-Arg-Arg-Cys<-->Cys-Leu-Gly (1), Leu-Arg-Arg-Ala-Cys<-->Cys-Gly (2), and Leu-Arg-Arg-Ala-Ala-Cys<-->Cys-Gly (3) inactivate the cAMP-dependent protein kinase in a time-dependent fashion. Since dialysis does not restore activity, but dithiothreitol does, this strongly suggests that covalent modification of the target enzyme has occurred at a cysteine residue. Although there are 2 cysteine moieties contained within the protein kinase, the 14C-acetylated affinity labels modify the enzyme only once. In addition, since ATP blocks inactivation of the protein kinase, this implies that it is the active site cysteine residue (Cys-199) that has undergone covalent modification. Based on the KI(inact) values obtained from inactivation kinetics, we conclude that the optimal site on the affinity label for the electrophilic Cys<-->Cys is 1 amino acid removed from the 2 arginine residues (i.e. 2). In addition, the efficacy of these inhibitors is also dependent upon the size of the disulfide ring. The eight-membered disulfide ring-containing peptides 1-3 are relatively poor affinity labels compared to the 12-membered ring-containing inhibitor, [formula: see text]
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PMID:Covalent modification with concomitant inactivation of the cAMP-dependent protein kinase by affinity labels containing only L-amino acids. 851 45

The critical function of the neurofibromatosis type 1 (NF1) gene product (neurofibromin) is not well defined except that neurofibromin has homology with a family of the GTPase-activating proteins (GAPs). In this study, we confirmed that neurofibromin is constitutively phosphorylated and detected kinase activities which specifically phosphorylate the cysteine/serine-rich domain and the C-terminal domain of the neurofibromin in cell lysate. In vitro and in-gel kinase assays strongly indicated that cAMP-dependent protein kinase (PKA) is a candidate for the neurofibromin kinase. THe biological significance of the phosphorylation of neurofibromin is unclear at present, but we speculate that neurofibromin plays a crucial role in cellular function since it links the two major cellular pathways which are the GAP-ras and PKA-associated signals.
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PMID:Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase. 861 63

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.
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PMID:Negative regulation of Raf-1 by phosphorylation of serine 621. 881 53

We tested the hypothesis as to whether elevated arterial pressure in hypertension alters cGMP, or cAMP, mediated vasorelaxation. Relaxation to nitroglycerin and isoproterenol was determined in isolated aortic rings from one-kidney, one clip hypertensive (1K1C), coarctation hypertensive (CH) and normotensive control (C) rats. Thoracic aortas from 1K1C and CH rats, as well as abdominal aortas from 1K1C rats, but not abdominal aortas from CH rats were exposed chronically (4-6 weeks) to elevated arterial pressure. Sensitivity of rings with and without endothelium to nitroglycerin was suppressed significantly only in vessels exposed chronically to high arterial pressure. Impaired sensitivity to nitroglycerin in abdominal rings from 1K1C rats could not be abolished by exposure to 100 uM L-arginine, the substrate for production of NO by endothelial nitric oxide synthase, or 100 uM L-cysteine, the source of thiol groups required for the production of nitric oxide from nitroglycerin. Maximum relaxation to isoproterenol was impaired significantly in thoracic and abdominal rings, with and without endothelium, from 1K1C and CH rats. Relaxation to 8-bromo-cGMP and dibutyryl cAMP was similar in abdominal rings from all groups. We conclude that impaired vasorelaxation to nitroglycerin and isoproterenol in hypertension involves mechanisms prior to activation of vascular smooth muscle cGMP-dependent and cAMP-dependent protein kinase, respectively. Impaired cGMP, but not cAMP, mediated relaxation of aortas appears to result from their exposure to high arterial pressure per se. This effect does not appear to involve the vascular endothelium or vascular sources of thiols, but rather may reflect an effect of high arterial pressure to impair the ability of the artery to respond to nitric oxide derived from nitroglycerin.
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PMID:Selective effect of high arterial pressure in hypertension upon inhibition of cGMP versus cAMP mediated vascular relaxation. 884 63

We previously described the isolation of a variant subline of HL-60 cells that does not differentiate in response to nitric oxide (NO)-generating agents or to cGMP analogs. The variant cells have normal guanylate cyclase activity and normal NO-induced increases in the intracellular cGMP concentration. We now show that the variant cells have normal cGMP-dependent protein kinase (G-kinase) activity, both by an in vitro and in vivo assay, and using two-dimensional gel electrophoresis we have identified six G-kinase substrates in the parental cells. Of these six proteins, we found considerably less phosphorylation of one of the proteins in the variant cells than in parental cells, both in vitro and in intact cells, and by 35S-methionine/35S-cysteine incorporation we found much less of this protein in the variant cells than in parental cells. The protein is a shared substrate of cAMP-dependent protein kinase (A-kinase); since cAMP analogs still induce differentiation of the variant cells, it appears that the NO/cGMP/G-kinase and cAMP/A-kinase signal transduction pathways share some but not all of the same target proteins in inducing differentiation of HL-60 cells.
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PMID:Decreased phosphorylation of a low molecular weight protein by cGMP-dependent protein kinase in variant HL-60 cells resistant to nitric oxide- and cGMP-induced differentiation. 974 17

To develop an alternative approach to measure peptidyl backbone flexibility and to expand our understanding of the segmental flexibility of cAMP-dependent protein kinase (cAPK), the effect of protein kinase inhibitor peptide, PKIalpha(5-24), and MgATP on the mobility of fluorescein selectively conjugated to five sites on the catalytic subunit of cAPK was examined. Specifically, five full-length, single-site catalytic subunit mutants (K16C, K81C, I244C, C199A, and N326C) were prepared, and fluorescein maleimide was selectively attached to the side chains of each substituted cysteine or, in the case of the C199A mutant, to the unprotected native C343. The time-resolved anisotropy decay profiles of the five fluorescein maleimide-conjugated mutants were well fit to a biexponential equation. The fast rotational correlation times of the fluorescein conjugates ranged between 1.9 and 2.8 ns and were inversely correlated (r = -0.87) to the averaged crystallographic main-chain atom B factors around each site of conjugation. The slow correlation times ranged between 25 and 28 ns and were about the same magnitude as the value of 21 ns estimated from the Stokes-Einstein equation. The presence of MgATP and PKIalpha(5-24), which induces the closed conformation of cAPK, was associated with a reduction of the fast rotational correlation time of the K81C conjugate, indicating that the peptidyl backbone around K81 is measurably less flexible when the C subunit is in the closed compared with the open conformation. The results suggest (i) that time-resolved fluorescence anisotropy can assess the nanosecond flexibility of short segments of the peptidyl backbone around each site of labeling and (ii) that the substrate/pseudosubstrate binding differentially affects the backbone flexibility of cAPK.
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PMID:Backbone flexibility of five sites on the catalytic subunit of cAMP-dependent protein kinase in the open and closed conformations. 975 61

Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152-184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.
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PMID:Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation. 986 9

The conformational flexibility of peptidyl ligands may be an essential element of many peptide-macromolecular interactions. Consequently, the alpha-carbonyl backbone flexibility of the 8 kDa protein kinase inhibitor (PKI alpha) peptide of cAMP-dependent protein kinase (cAPK) free in solution and bound to cAPK was assessed by time-resolved fluorescence anisotropy. Specifically, three full-length, single-site PKI alpha mutants (V3C, S28C, and S59C) were prepared, and fluorescein iodoacetamide (FI) was selectively conjugated to the side chains of each substituted cysteine. The time-resolved anisotropy decay profiles of the labeled mutants were well fit to a model-free nonassociative biexponential equation. Free in solution, the three labeled proteins had very similar anisotropy decays arising primarily from local alpha-carbonyl backbone movements. Only a small fraction of the anisotropy decay was associated with slower, whole-body tumbling, confirming that PKI alpha is highly disordered at all three locations. Complexation of the mutants with the catalytic (C) subunit of cAPK decreased the rate of whole-body tumbling for all three mutants. The effects on the rapid decay processes, however, were dependent upon the site of conjugation. The anisotropy decay profiles of both FI-V3C- and FI-S28C-PKI alpha were associated with significantly reduced contributions from the fast decay processes, while that of FI-S59C-PKI alpha was largely unaffected by binding to the C-subunit. The results suggest that the cAPK-binding domain of PKI alpha extends from the its N-terminus to residues beyond Ser28 but does not include the segment around Ser59, which is still part of a highly flexible domain when bound to the C-subunit.
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PMID:Binding-dependent disorder-order transition in PKI alpha: a fluorescence anisotropy study. 1034 98


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