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 hexapeptide (Arg)2-Pro-Thr-Pro-Ala (T1) and octapeptide (Arg)2-Pro-Thr-Pro-Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor-1, a physiological target of cAMP-dependent protein kinase, and five related peptides were synthesized by the method in solution. The phosphorylation rates of such peptides by cAMP-dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2-Ala-Ser-Val-Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C-terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N-terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T1 on its C-terminal side to give the derivative (Arg)2-Pro-Thr-Pro-Ala-Thr-Val-Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced. Therefore the local structural features of the phosphorylatable site of inhibitor-1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.
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PMID:Synthetic peptides reproducing the site phosphorylated by cAMP-dependent protein kinase in protein phosphatase inhibitor-1. Effect of structural modifications on the phosphorylation efficiency. 661 51

Rat liver ribosomes and 40 S ribosomal subunits were phosphorylated with the purified catalytic subunit of cAMP-dependent protein kinase. Phosphorylation of ribosomal protein S6 plateaued at around 2 mol of phosphate/mol of protein with both substrates. Peptide map analyses showed that the most prominent phosphorylation sites associated with 40 S substrates were the adjacent serines in the Arg-Leu-Ser-Ser-Leu-Arg segment of S6. The first serine residue appeared to be the preferred site as has been established previously for 80 S ribosomes (Wettenhall, R.E.H., and Cohen, P. (1982) FEBS Lett. 140, 263-269). Additional phosphorylation sites were apparent from the peptide maps. One of these was associated with the triphosphopeptide (termed T1a) having the sequence Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys. A larger fragment of S6 (termed Tlc) isolated from mild tryptic digests of whole ribosomes, consisted of the T1a sequence extended by the sequence Ser-Glu-Glu-Ser-Gln-(Lys) at the COOH terminus. A comparison of the size and chromatographic and isoelectric focusing properties of the T1a/T1c peptides and prominent tryptic peptides of S6 from insulin-stimulated hepatocytes indicated a relationship between these peptides. Thus, it appeared that some of the potential phosphorylation sites in the T1a/T1c region of S6 are phosphorylated by an insulin-regulated kinase in hepatocytes.
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PMID:Phosphorylation of hepatic ribosomal protein S6 on 80 and 40 S ribosomes. Primary structure of S6 in the region of the major phosphorylation sites for cAMP-dependent protein kinases. 669 58

The kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase has been investigated employing the heptapeptide Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as substrate. Initial velocity measurements performed over a wide range of ATP and Kemptide concentrations indicated that the reaction follows a sequential mechanistic pathway. In line with this, the results of product and substrate inhibition studies, the patterns of dead end inhibition obtained employing the nonhydrolyzable ATP analogue, AMP X PNP (5'-adenylylimidodiphosphate), and equilibrium binding determinations, taken in conjunction with the patterns of inhibition observed with the inhibitor protein of the cAMP-dependent protein kinase that are reported in the accompanying paper (Whitehouse, S., and Walsh, D.A. (1983) J. Biol. Chem. 258, 3682-3692), are best fit by a steady state Ordered Bi-Bi kinetic mechanism. Although the inhibition patterns obtained employing the synthetic peptide analogue in which the phosphorylatable serine was replaced by alanine were apparently incompatible with this mechanism, these inconsistencies appear to be due to some element of the structure of this latter peptide such that it is not an ideal dead end inhibitor substrate analogue. The data presented both here and in the accompanying paper suggest that both this substrate, analogue and the ATP analogue, AMP X PNP, do not fully mimic the binding of Kemptide and ATP, respectively, in their mechanism of interaction with the protein kinase. It is proposed that, as with some other kinase reactions, the configuration of the terminal anhydride bond of ATP assumes a conformation once the nucleotide is bound to the protein kinase that assists in the binding of either Kemptide or the inhibitor protein but not the alanine-substituted peptide and that AMP X PNP, because of its terminal phosphorylimido bond, cannot assume this conformation which favors protein (or peptide) binding.
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PMID:Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein kinase. 683 26

Co3+ and Cr3+ complexes of beta, gamma-methylene-ATP (AMPPCP), which are substitution-inert substrate analogues inactive in phosphoryl transfer reactions, have been used in binding and structural studies of cAMP-dependent protein kinase. Dissociation constants of enzyme complexes with Co(NH3)4AMPPCP and CrAMPPCP and with Mn2+, which binds at an inhibitory site, were determined by electron paramagnetic resonance and by proton relaxation rate enhancement techniques. Nuclear relaxation rate measurements at 100 and 360 MHz were used to determine the distance between Mn2+ and the beta, gamma-methylene protons of Co-(NH3)4AMPPCP, yielding 7.4 +/- 0.6 A in the absence of enzyme and 5.0 +/- 0.9 A when both Mn2+ and Co-(NH3)4AMPPCP were bound to the enzyme. The effect of the paramagnetic CrAMPPCP on the electron spin relaxation time of the enzyme-bound Mn2+ was used used to calculate the distance between the two metal ions of 4.8 +/- 0.4 A. This distance and the Mn2+-methylene distance are consistent with the previous finding that the inhibitory metal bridges the enzyme to the triphosphate chain of the enzyme-bound nucleotide [Granot, J., Kondo, H., Armstrong, R. M., Mildvan, A. S., & Kaiser, E. T. (1979) Biochemistry 18, 2339]. From the paramagnetic effects on the relaxation rates of the protons of the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly, distances from Mn2+ and Cr3+ to the serine methylene protons of 9.1 +/- 0.9 and 8.1 +/- 0.8 A, respectively, were calculated. These and previous measurements were used to estimate a distance of 5.3 +/- 0.7 A along the reaction coordinate between the gamma-phosphorus of ATP and the serine hydroxyl oxygen. This distance is 2 A greater than that required for molecular contact. The mechanistic implications of these findings are discussed.
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PMID:Magnetic resonance measurements of intersubstrate distances at the active site of protein kinase using substitution-inert cobalt(III) and chromium(III) complexes of adenosine 5'-(beta, gamma-methylenetriphosphate). 689 73

A synthetic heptadecapeptide corresponding to part of the NH2-terminal 17 residues of chicken gizzard myosin light chain (Mr = 20,000), Ser-Ser-Lys-Thr-Thr-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser-(P)-Asn-Val-Phe-Ser-NH2, was readily phosphorylated by the myosin light chain kinase isolated from the same tissue. The synthetic peptide was phosphorylated stoichiometrically at serine 13, the same residue phosphorylated in the parent protein. The apparent Km and Vmax for peptide phosphorylation was 90 microM and 1.3 mumol min-1 mg-1 compared to 10 microM and 22 mumol min-1 mg-1, respectively, for the myosin light chain. The synthetic heptadecapeptide acted as a competitive inhibitor for myosin light chain phosphorylation with Ki approximately 600 microM. Acetylation of the heptadecapeptide alpha-amino group of serine 1 had little effect on Vmax (0.8 mumol min-1 mg-1) and increased the apparent Km 2-fold. The smooth muscle myosin light chain kinase did not phosphorylate the synthetic heptadecapeptide analog of the corresponding skeletal muscle myosin light chain (Mr = 18,500), nor did it phosphorylate synthetic peptide substrates specific for the cAMP-dependent protein kinase or phosphorylase b kinase. These findings support the idea that the myosin light chain kinase has particular protein substrate specificity requirements and that some of these are derived from the region of primary structure around the phosphorylation site in its native substrate.
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PMID:Phosphorylation of a synthetic heptadecapeptide by smooth muscle myosin light chain kinase. 689 43

The regulatory subunit of cAMP-dependent protein kinase I has been cleaved proteolytically into two structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent, the two protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the two major domains, a 15-residue peptide that links the two domains has been isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1, susceptible to cleavage by both trypsin and thermolysin, has the following sequence: LysArg-Arg-Gly-Ala-Ile-Ser-Ala-. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site. The sequence at Site 2 was Val-Arg-Arg-Val-Ile-Ala. Cleavage here generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; however, in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. In contrast to the dissociated regulatory subunit, the holoenzyme is partially protected from proteolytic degradation.
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PMID:The structural domains of cAMP-dependent protein kinase I. Characterization of two sites of proteolytic cleavage and homologies to cAMP-dependent protein kinase II. 743 94

The RII beta regulatory subunit of cAMP-dependent protein kinase (PKA) contains an autophosphorylation site and a nuclear location signal, KKRK. We approached the structure-function analysis of RII beta by using site-directed mutagenesis. Ser114 (the autophosphorylation site) of human RII beta was replaced with Ala (RII beta-P) or Arg264 of KKRK was replaced with Met (RII beta-K). ras-transformed NIH 3T3 (DT) cells were transfected with expression vectors for RII beta, RII beta-P, and RII beta-K, and the effects on PKA isozyme distribution and transformation properties were analyzed. DT cells contained PKA-I and PKA-II isozymes in a 1:2 ratio. Over-expression of wild-type or mutant RII beta resulted in an increase in PKA-II and the elimination of PKA-I. Only wild-type RII beta cells demonstrated inhibition of both anchorage-dependent and -independent growth and phenotypic change. The growth inhibitory effect of RII beta overexpression was not due to suppression of ras expression but was correlated with nuclear accumulation of RII beta. DT cells demonstrated growth inhibition and phenotypic change upon treatment with 8-Cl-cAMP. RII beta-P or RII beta-K cells failed to respond to 8-Cl-cAMP. These data suggest that autophosphorylation and nuclear location signal sequences are integral parts of the growth regulatory mechanism of RII beta.
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PMID:Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts. 747 55

Phosphorylation of purified Na+,K(+)-ATPase by cAMP-dependent protein kinase (protein kinase A) decreases the activity of this enzyme. We have now shown, using several experimental approaches, that a highly conserved seryl residue on the catalytic (alpha) subunit of Na+,K(+)-ATPase, corresponding to Ser943 of the rat alpha 1 isoform, is the phosphorylation site for protein kinase A. cDNAs corresponding to wild-type Na+,K(+)-ATPase and Na+,K(+)-ATPase in which Ser943 was mutated to Ala were transfected into COS cells. Treatment of the transfected cells with forskolin plus 3-isobutyl-1-methylxanthine resulted in a decrease in the activity of the wild-type enzyme but not in that of the mutated enzyme. The results suggest that, in intact cells, the activity of the Na+,K(+)-ATPase is regulated in part by signal transduction pathways that use protein kinase A-dependent phosphorylation of the Na+,K(+)-ATPase alpha subunit.
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PMID:Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K(+)-ATPase and effects of site-directed mutagenesis. 751 Jul 9

Hormonal regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is largely mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10 dibasic consensus sites for potential PKA phosphorylation ((R/K) (R/K)X(S*/T*)). Previous studies (Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., Hanrahan, J. W., and Riordan, J.R (1993) J. Biol. Chem. 268, 11304-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphorylation by in vitro mutagenesis of all 10 dibasic consensus sites (10SA CFTR). To identify potential additional sites responsible for the residual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-32P]ATP. Cyanogen bromide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8-kDa fragment of the R domain between residues 722-773. Removal of serines at potential PKA sites in this fragment showed that Ser-753 accounted for all of the gamma-32P labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced the level of residual CFTR activity by a further 40%, indicating that phosphorylation at this previously unidentified site contributes to the activation of 10SA CFTR.
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PMID:cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue Ser-753 and its role in channel activation. 753 Jul 19

Among water channel proteins (aquaporins), aquaporin-collecting duct (AQP-CD) is the vasopressin-regulated water channel. Vasopressin causes cAMP production in the renal collecting duct cells, and this is believed to lead to exocytic insertion of water channel into the apical membrane (shuttle hypothesis). AQP-CD contains a consensus sequence for cAMP-dependent protein kinase, residues at positions 253-256 (Arg-Arg-Gln-Ser). To determine the role of this site, Ser-256 was substituted for Ala, Leu, Thr, Asp, or Glu by site-directed mutagenesis. In Xenopus oocytes injected with wild-type or mutated AQP-CD cRNAs, osmotic water permeability (Pf) was 4.8-7.7 times higher than Pf of water-injected oocytes. Incubation with cAMP plus forskolin or direct cAMP injection into the oocytes increased Pf of wild-type, but not mutated, AQP-CD-expressing oocytes, whereas the amounts of AQP-CD expression were similar in wild and mutated types as identified by Western blot analysis. In vitro phosphorylation studies of AQP-CD proteins expressed in oocyte showed that cAMP-dependent protein kinase phosphorylated wild-type, but not mutated, AQP-CD proteins. Phosphoamino acid analysis revealed that this phosphorylation occurred at the serine residue. Moreover, phosphorylation of AQP-CD protein in intact rat kidney medulla tissues was stimulated by incubation with cAMP. Our data suggest that cAMP stimulates water permeability of AQP-CD by phosphorylation. This process may contribute to the vasopressin-regulated water permeability of collecting duct in addition to the apical insertion of AQP-CD by exocytosis.
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PMID:cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes. 753 30


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