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)

Temperature-sensitive mutations in the avian sarcoma virus UR2 oncogene ros, encoding a receptor protein-tyrosine kinase (PTK), were identified. The Ala385-->Gly change mapping within the highly conserved RDLAARN motif in the Ros kinase domain was responsible for the temperature-sensitive phenotype. Based on the sequence homology of all known protein kinases and the crystalline structure of the cAMP-dependent protein kinase, this conserved region probably represents the PTK catalytic loop. The same mutation when introduced into the human insulin and insulin-like growth factor I receptors made these PTKs temperature sensitive in both biological function and kinase activity. Our results support the presumed catalytic role of this highly conserved sequence in PTKs. Due to its highly conserved nature, we predict that the same mutation would probably confer temperature sensitivity on other PTKs.
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PMID:Ala-->Gly mutation in the putative catalytic loop confers temperature sensitivity on Ros, insulin receptor, and insulin-like growth factor I receptor protein-tyrosine kinases. 827 85

The substrate specificity of the cAMP-dependent protein kinase has been assessed with peptides bearing threoninol diastereomers. The threoninol residue contains both a primary alcohol and a secondary alcohol, either of which may serve as a site of phosphorylation. The enzyme-catalyzed phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2R,3R)-threoninol furnishes a Km of 498 +/- 39 microM and a Vmax of 7.8 +/- 0.2 mumol/min-mg, whereas the phosphorylation of Gly-Arg-Thr-Gly-Arg-Arg-Asn-(2S,3S)-threoninol provides a Km of 16.3 +/- 0.8 microM and a Vmax of 16.0 +/- 0.4 mumol/min-mg. Mass spectral analysis of the phosphopeptide reaction products revealed that each species is phosphorylated only once. 1H-coupled 31P NMR experiments unequivocally demonstrated that the (2R,3R)-isomer is specifically phosphorylated at the secondary alcohol, whereas the (2S,3S)-isomer is exclusively phosphorylated at the primary alcohol. This regiospecificity appears to be a consequence of the stereochemistry at C-2 in the threoninol residues. The structural attributes of the protein kinase that appear to be responsible for the observed differentiation between the C-2 stereoisomers is discussed.
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PMID:Stereochemistry specifies the regiochemistry of phosphorylation in two cAMP-dependent protein kinase substrates. 834 52

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 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

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

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 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

Vanadium oxoions have been shown to elicit a wide range of effects in biological systems, including an increase in the quantity of phosphorylated proteins. This response has been attributed to the inhibition of protein phosphatases, the indirect activation of protein kinases via stimulation of enzymes at early steps in signal transduction pathways and/or the direct activation of protein kinases. We have evaluated the latter possibility by exploring the effects of vanadate, decavanadate and vanadyl cation species on the activity of the cAMP-dependent protein kinase (PKA), a serine/threonine kinase. Vanadate, in the form of monomer, dimer, tetramer and pentamer species, neither inhibits nor activates PKA. In marked contrast, decavandate is a competitive inhibitor (Ki = 1.8 +/- 0.1 mM) of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), a peptide-based substrate. This inhibition pattern is especially surprising, since the negatively charged decavanadate would not be predicted to bind to the region of the active site of the enzyme that accommodates the positively charged kemptide substrate. Our studies suggest that decavanadate can associate with kemptide in solution, which would prevent kemptide from interacting with the enzyme. Vanadium(IV) also inhibits the PKA-catalysed phosphorylation of kemptide, but with an IC50 of 366 +/- 10 microM. However, in this case V4+ appears to bind to the Mg(2+)-binding site, since it can substitute for Mg2+. In the absence of Mg2+, the optimal concentration of vanadium(IV) for the PKA-catalysed phosphorylation of kemptide is 100 microM, with concentrations above 100 microM being markedly inhibitory. However, even at the optimal 100 microM V4+ concentration, the Vmax and K(m) values (for kemptide) are significantly less favourable than those obtained in the presence of 100 microM Mg2+. In summary, we have found that oxovanadium ions can directly alter the activity of the serine/threonine-specific PKA.
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PMID:Vanadium oxoanions and cAMP-dependent protein kinase: an anti-substrate inhibitor. 902 Aug 63

A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family.
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PMID:Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase. 920 6

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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