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)

Compartmentalization of the type II cAMP-dependent protein kinase is maintained by association of the regulatory subunit (RII) with A-Kinase Anchor Proteins (AKAPs). In previous studies (Scott, J. D., Stofko, R. E., McDonald, J. R., Comer, J. D., Vitalis, E. A., and Mangili J. (1990) J. Biol. Chem. 265, 21561-21566) we have shown that dimerization of RII alpha was required for interaction with the cytoskeletal component microtubule-associated protein 2. In this report we show that the localization and dimerization domains of RII alpha are contained within the first thirty residues of each RII protomer. RII des-5 (an amino-terminal deletion mutant lacking residues 1-5) was unable to bind AKAPs but retained the ability to dimerize. RII alpha I3A,I5A (a mutant where isoleucines 3 and 5 were replaced with alanine) was unable to bind a variety of AKAPs. Mutation of both isoleucines decreased AKAP binding without affecting dimerization, cAMP binding, or the overall secondary structure of the protein. Measurement of RII alpha I3A,I5A interaction with the human thyroid AKAP, Ht 31, by two independent methods suggests that mutation of isoleucines 3 and 5 decreases affinity by at least 6-fold. Therefore, we propose that two isoleucine side chains on each RII protomer are principle sites of contact with the conserved amphipathic helix binding domain on AKAPs.
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PMID:Type II regulatory subunit (RII) of the cAMP-dependent protein kinase interaction with A-kinase anchor proteins requires isoleucines 3 and 5. 792 81

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

To search for peptides which serve as substrates for protein kinases, an approach based on peptide libraries has been developed. These peptide libraries are chemically synthesized by a modified "divide-couple-recombine" strategy. After reaction with the kinase of interest, the most highly phosphorylated substrate (selected from the library) is identified using on-line liquid chromatography-electrospray mass spectrometry (LC-ESMS). Negative ion LC-ESMS with stepped collision energy is used to identify phosphorylated peptides in the enzyme reactions. As predicted, the cAMP-dependent protein kinase is shown to preferentially phosphorylate Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in a library consisting of 19 variants of Kemptide substituted at position 2. Additional experiments have been carried out on the nonreceptor tyrosine kinase v-Abl using a peptide library based on the v-Src autophosphorylation site (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly). These results indicate that Ile is the optimal residue at the position N-terminal to tyrosine. Individual peptides containing the Glu-Asp-Ala-Ile-Tyr motif have Vmax/Km values 6-fold higher than the peptide based on the autophosphorylation site itself, confirming the results of the library experiments. This motif has been identified in several tyrosine kinases at a position in the sequence not previously reported to serve as a phosphorylation or autophosphorylation site.
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PMID:Use of synthetic peptide libraries and phosphopeptide-selective mass spectrometry to probe protein kinase substrate specificity. 812 61

The type I alpha regulatory subunits of cAMP-dependent protein kinase contain an autoinhibitor site, Arg94-Arg-Gly-Ala-Ile, which serves as a pseudosubstrate. To evaluate their contribution to subunit association, Arg94 and Arg95, key determinants for peptide recognition, were replaced singly and in tandem with Ala, Glu, and His. Unlike substrate peptides in which replacement of either arginine leads to an increase in Km of approximately 3 orders of magnitude, replacement of either arginine causes only a maximal 20-fold decrease in subunit association. Replacement of both arginine residues with alanine, however, generates a regulatory subunit that can no longer recombine with the catalytic subunit under physiological conditions when the regulatory subunit is saturated with cAMP. To evaluate more fully the specific consequences of replacing these 2 arginine residues, a rapid gel filtration chromatographic method was developed so that subunit affinity could be measured independently of assaying for catalytic activity. The R94,95A mutant shows a Kd(app) = 677 nM, representing an increase of greater than 3 orders of magnitude compared with the native subunits in the presence of MgATP. In the absence of MgATP, the Kd(app) for native regulatory subunit was 125 nM, whereas the Kd(app) for the R94,R95A mutant regulatory subunit was determined to 2.87 microM. When this mutant holoenzyme is assayed at microM concentrations, no activity is observed, whereas below microM, activity is observed because of cAMP-independent subunit dissociation.
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PMID:Regulation-defective mutants of type I cAMP-dependent protein kinase. Consequences of replacing arginine 94 and arginine 95. 839 67

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

The regulatory (R) subunit of cAMP-dependent protein kinase has a well-defined domain structure including the two in-tandem cAMP-binding sites that constitute the C-terminus of the protein. The N-terminal binding site (A) has a considerably higher affinity for analogues of cAMP that are substituted with bulky and hydrophobic substituents at the 6-amino group of the adenine ring compared to the affinity observed at the second site (B). On the basis of the crystal structure of the catabolite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A could be involved in a high-affinity hydrophobic interaction, whereas a corresponding isoleucine (I368) in domain B could lead to steric hindrance in the binding of bulky N6-substituted analogues. Site-directed mutagenesis was used to construct mutations in Y244 and I368. Binding displacement experiments showed that replacing the tyrosine in site A with isoleucine (Y244I) did not affect the interaction of either N6-substituted or otherwise modified analogues with this site. However, replacing I368 with tyrosine (I368Y) led to a 3-4-fold increase in affinity for those N6-modified analogues that had a hydrophobic group attached directly or close to the 6-amino molecule. We conclude that I368 is involved in the molecular interaction between binding domain B and the 6-amino group of the adenine moiety of cAMP and that this residue is partly responsible for the reduced affinity of N6-substituted cAMP analogues for this site.
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PMID:Isoleucine 368 is involved in low-affinity binding of N6-modified cAMP analogues to site B of the regulatory subunit of cAMP-dependent protein kinase I. 864 27

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.
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PMID:Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. 864 4

The guanidinium groups of conserved arginines in the two intrachain cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase have been implicated in the allosteric interactions by which cAMP binding leads to kinase activation. We have investigated the functional role of Arg-210, the conserved arginine in site A of murine type Ialpha R subunit, by analyzing the effects of nine different substitutions at this residue on cAMP binding and allosteric properties of bacterially expressed RIalpha subunits. All substitutions reduced the cAMP binding affinity of site A, but the magnitude of reduction varied from several hundredfold to 10(6)-fold. The differential effects of the different substitutions could not easily be rationalized by interactions with cAMP and might, in part, reflect interactions with other residues in the unoccupied cAMP-binding pocket. None of the Arg-210 substitutions appeared to disrupt the allosteric interaction by which occupation of site A slows dissociation of cAMP from site B, although the effect was difficult to elicit in full with mutations that had strong effects on cAMP binding. The two weakest substitutions, Arg-210 --> Ile and Arg-210 --> Thr, could be shown to have essentially no effect on the allosteric interaction by which occupation of site A reduces the affinity of R subunit for the catalytic subunit. The weaker mutations had a smaller effect on kinase activation by the suboptimal activator Rp-adenosine cyclic 3',5'-phosphorothioate than by cAMP, suggesting that the analog largely bypasses interactions with the guanidinium group of Arg-210.
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PMID:Arginine 210 is not a critical residue for the allosteric interactions mediated by binding of cyclic AMP to site A of regulatory (RIalpha) subunit of cyclic AMP-dependent protein kinase. 891 Mar 52

The consensus substrate site for cAMP-dependent protein kinase (PKA) is Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg92-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acids form a putative pseudosubstrate site (Ser is replaced with Ala), which together with adjacent residues could competitively inhibit substrate phosphorylation by the PKA catalytic subunit (C subunit). The present studies determine the contributions of Arg92-95, Ile98, and Glu101 to inhibitory potency. Amino-terminal truncation of RI subunit through Arg92 (delta1-92) or Arg93 (delta1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg94 (delta1-94), or point mutation of Arg95 within truncated mutants (delta1-93.R95A or delta1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg95 (delta1-95) had a greater effect than did replacement or deletion of Arg94 or Arg95 alone. Using full-length RI subunit, the inhibitory potency was reduced by replacing Ile98 with Ala, Gly, or Gln, but not by replacing it with Val. The inhibitory potency of RI subunit was unchanged when Glu101 was replaced with Ala or Gln. It is concluded that Arg94, Arg95 and, to a lesser extent, Ile98 are vital constituents of PKA autoinhibition by type I alpha R subunit.
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PMID:Identification of critical determinants for autoinhibition in the pseudosubstrate region of type I alpha cAMP-dependent protein kinase. 899 72

Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue, homologous to mammalian cAMP-dependent protein kinase; the maximal level of phosphorylation achieved was around 1 mol of Pi/mol of phosphofructokinase subunit. The covalent incorporation of phosphate leads to a notable increase in the enzyme activity assayed at near-physiological concentrations of substrates and allosteric modulators and neutral pH. Tryptic digestion of labeled phosphofructokinase released a phosphopeptide whose sequence was Lys-Asp-Ser(P)-Ile-Trp-Ile-Gln-Thr-Gly-Arg. This sequence showed high homology with the phosphopeptides from other invertebrates whose phosphofructokinase is also activated by cAMP-dependent phosphorylation.
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PMID:cAMP-dependent phosphorylation activates phosphofructokinase from mantle tissue of the mollusc Mytilus galloprovincialis. Identification of the phosphorylated site. 931 95


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