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)

Protein kinase C, purified to near homogeneity from the brain, has been tested toward a variety of synthetic peptide substrates including different phosphorylatable residues. While it proved totally inactive toward the tyrosyl peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly, as well as toward several more or less acidic seryl peptides, it phosphorylates with a Ca2+/phospholipid-dependent mechanism, at seryl and/or threonyl residues, many basic peptides, some of which are also good substrates for cAMP-dependent protein kinase (A-kinase). Among the peptides tested, however, the best substrate for protein kinase C, with kinetic constants comparable to those of histones, is the nonapeptide Gly-Ser-Arg6-Tyr, which is not a substrate for A-kinase. Moreover, although the peptide Pro-Arg5-Ser-Ser-Arg-Pro-Val-Arg is a good substrate for both kinases, its derivative with ornitines replacing arginines is phosphorylated only by protein kinase C. Some typical substrates of A-kinase on the other hand, like the peptides Phe-Arg2-Leu-Ser-Ile-Ser-Thr-Glu-Ser and Arg2-Ala-Ser-Val-Ala, are phosphorylated by protein kinase C rather slowly and with unfavourable kinetic constants. It is concluded that, while both protein kinase C and A-kinase need basic groups close to the phosphorylatable residues, their primary structure determinants are quite distinct.
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PMID:Distinct structural requirements of Ca2+/phospholipid-dependent protein kinase (protein kinase C) and cAMP-dependent protein kinase as evidenced by synthetic peptide substrates. 315 99

Three major 32P-labeled polypeptides were found in the soluble fraction of bovine lenses cultured in a medium containing [32P]orthophosphate. Two of the polypeptides corresponded to the phosphorylated A and B chains of alpha-crystallin. In this communication, the third polypeptide is now identified. This polypeptide is characterized by a molecular weight of 27,000 and a pI of 6.6, eluted exclusively in the beta Low fraction of a CL-6B gel filtration separation of lens soluble material, and could be further purified by DE52 anion exchange chromatography. The only 32P-labeled amino acid detected was phosphoserine. A single 32P-labeled peptide was observed after tryptic digestion and two-dimensional mapping. The amino acid sequence of the purified peptide is Gly-Ala-Phe-His-Pro-Ser-Ser. This sequence exactly matches the expected C-terminal tryptic fragment, residues 198-204, of the bovine beta-crystallin B2. The results of carboxypeptidase A digestion of the 32P-labeled peptide suggest that only Ser203 is phosphorylated. By using the catalytic subunit of the cAMP-dependent protein kinase, purified beta B2 was phosphorylated in vitro, generating a single 32P-labeled polypeptide with the identical pI as the phosphorylated polypeptide obtained from lens culture. On the basis of these data, the Mr 27,000 32P-labeled polypeptide is identified as the phosphorylated form of the beta-crystallin B2.
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PMID:Phosphorylation of beta-crystallin B2 (beta Bp) in the bovine lens. 317 May 71

In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.
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PMID:Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1. 328 29

Oligonucleotide-directed mutagenesis was used to produce mutants in the hinge region of the regulatory subunit (R) of the Saccharomyces cerevisiae cAMP-dependent protein kinase. The mutant proteins were expressed in Escherichia coli, purified, urea treated to produce cAMP-free regulatory (R), and analyzed in vitro for catalytic (C) subunit inhibitory activity in the presence and absence of cAMP. When assayed in the absence of cAMP, wild type R dimer inhibited C with an IC50 of 40 nM. Replacement of amino acid residue Ser-145 (the autophosphorylation site of yeast R) with Ala or Gly produced mutants which were 2-10-fold better inhibitors of C, while replacement with Glu, Asp, Lys, or Thr produced mutants which were 2-5-fold worse inhibitors of C relative to wild type R. When assayed in the presence of cAMP, all R subunits had a decreased affinity for C subunit, with Ser-145 and Thr-145 undergoing autophosphorylation. These results suggest that the amino acid at position 145 of R contributes to R-C interaction and therefore influences the equilibrium of yeast protein kinase subunits in vitro.
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PMID:Mutagenesis of the regulatory subunit of yeast cAMP-dependent protein kinase. Isolation of site-directed mutants with altered binding affinity for catalytic subunit. 328 30

The phosphorylation of sperm specific histone H1 in the sea urchin Strongylocentrotus purpuratus occurs both in vivo and in vitro on a single serine site in the sequence Arg-Lys-Gly-Ser(P)-Ser-Asn-Ala-Arg. This is a preferred sequence for cAMP-dependent protein kinase. The in vitro phosphorylation is completely dependent on cAMP and is inhibited by the peptide protein kinase inhibitor. The protein kinase inhibitor H-8 blocks the in vivo phosphorylation of H1 without damaging motility, the acrosome reaction or the ability of sperm to fuse with and activate eggs.
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PMID:CAMP-dependent protein kinase of sea urchin sperm phosphorylates sperm histone H1 on a single site. 334 29

A peptide-based photoaffinity label for the catalytic subunit of the cAMP-dependent protein kinase was prepared from the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)]. By using solid-phase peptide synthesis methodology, DL-Phe(pBz) was incorporated into the cAMP-dependent protein kinase substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly in place of the phosphorylatable serine. The diastereomeric peptides were separated by reverse-phase HPLC. The peptide substrate analog containing L-Phe(pBz) had a Ki of approximately 110 microM at pH 7.5. When photolyzed at 350 nm in the presence of the enzyme, this peptide caused time- and concentration-dependent inactivation. Radioactive acetylated L-Phe(pBz) peptide was used to establish the binding stoichiometry of peptide to enzyme; these results, together with protection experiments, showed the photoaffinity labeling to be specific (approximately 1:1). To identify the residues that were modified on the catalytic subunit, the photoinactivated enzyme was cleaved with CNBr and V8 protease (Staphylococcus aureus). The resulting peptide fragments were purified by HPLC and were sequenced; these experiments identified the modified residues as Gly-125 and Met-127. This region of the cAMP-dependent protein kinase catalytic subunit contains many residues that are conserved in serine- and tyrosine-protein kinases.
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PMID:Probing the peptide binding site of the cAMP-dependent protein kinase by using a peptide-based photoaffinity label. 339 99

The structure of the inhibitory domain of the inhibitor protein of the cAMP-dependent protein kinase has been assessed by circular dichroism studies of synthetic inhibitory peptides. Using the inhibitory peptide PKI(5-22)amide (Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn- Ala-Ile22) [Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., & Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992] and shorter peptides of this sequence, it has been estimated that this parent peptide is composed of approximately 30% alpha-helix with the remainder being random coil with one beta-turn. The pseudosubstrate arginine cluster (Arg15-Arg19) is within the suggested region of random coil and beta-turn, representing one critical region of binding recognition by the protein kinase. The alpha-helix region proposed between Thr6 and Ile11 likewise contributes to the full biological potency and specificity of the inhibitor peptide and inhibitor protein. The removal of the two N-terminal threonines, for example, causes both a marked conformational change in the peptide and a diminishment by an order of magnitude of inhibitory activity. It is proposed that this alpha-helix region could serve one of several possibilities, including that it may provide a suitable constraint on the Tyr7 such that the hydroxyl is oriented in a position proximal to the pseudosubstrate domain, and/or may allow the optimal location of other protein kinase recognition signals. These data provide an initial description of some of the structural features of the inhibitor protein that could contribute to its high biological potency.
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PMID:Circular dichroic investigations of secondary structure in synthetic peptide inhibitors of cAMP-dependent protein kinase: a model for inhibitory potential. 342 97

The amino acid sequence of the heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) was determined recently [Scott, J. D., Fischer, E. H., Takio, K., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5732-5736]. An earlier report [Scott, J. D., Fischer, E.H., Demaille, J. G. & Krebs, E. G. (1985) Proc. Natl. Acad. Sci. USA 82, 4379-4383] showed that at least part of the inhibitory domain of PKI is located in a 20-residue segment extending from residue 11 to residue 30: Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile-His-Asp-Ile-Leu-Val-Ser- Ser-Ala . In the present study, we further mapped the inhibitory region of PKI by addition or deletion of residues at both ends of this peptide and by substitutions for specific amino acids. The results show that (i) deletion of residues 25-30 did not change inhibitory activity but addition of residues toward the amino terminus increased the inhibitory potency up to 150-fold (Ki 4.8 nM), to a level approaching that of PKI; (ii) replacement of alanine-21 by serine converted the inhibitor into a substrate having a relatively low affinity (Km 280 microM) for the enzyme; (iii) replacement of alanine-21 by phosphoserine or alpha-aminobutyric acid decreased inhibitory activity by a factor of 120 and 20, respectively; (iv) replacement of serine-13 had essentially no effect, whereas substitution of threonine-16 decreased inhibitory activity. The greatest decreases of inhibitory potency occurred with replacements of the arginines in positions 18 and 19.
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PMID:Primary-structure requirements for inhibition by the heat-stable inhibitor of the cAMP-dependent protein kinase. 345 5

As an important new reagent for studying the cAMP-dependent protein kinase, a 20-residue peptide has been synthesized that corresponds to the active site of the skeletal muscle inhibitor protein. This synthetic peptide inhibits the protein kinase competitively with a Ki = 2.3 nM; its sequence, Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr- Gly-Arg-Arg-Asn-Ala-Ile-His-Asp, is that of a peptide previously reported by us which was derived from the native inhibitor protein by V8 protease digestion (Cheng, H. C., Van Patten, S. M., Smith, A. J., and Walsh, D. A. (1985) Biochem. J. 231, 655-661). Studies with analogues of this peptide show that its high affinity binding to the protein kinase (as also of the inhibitor protein) appears to be due to it mimicking the protein substrate by binding to the catalytic site via the arginine-cluster basic subsite (Formula: see text), and also to a critical contribution from one or more of the 6 N-terminal residues (Formula: see text). The availability of this high affinity synthetic peptide should open up a variety of avenues to probe the cellular actions of cAMP.
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PMID:A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. 351 Oct 44

Incubation of Saccharomyces cerevisiae strain JR153 with either [3H]myristate or [3H]palmitate demonstrates the synthesis of proteins that contain covalently bound fatty acids. A unique set of proteins is labeled by each fatty acid. Detailed analysis of a 20-kDa protein labeled with myristic acid demonstrates that myristate is linked to the amino-terminal glycine. We describe an enzymatic activity in yeast that will transfer myristic acid to the amino terminus of the octapeptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg, whose sequence was derived from a known N-myristoylated acyl protein, the catalytic subunit of cAMP-dependent protein kinase of bovine cardiac muscle. The acylation reaction is dependent on ATP and CoA, is enriched in a crude membrane fraction, and will use myristate but not palmitate as the acyl donor. Specificity of the glycyl peptide substrate is demonstrated by the observation that other glycyl peptides do not competitively inhibit myristoylation of Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg.
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PMID:Protein fatty acid acylation: enzymatic synthesis of an N-myristoylglycyl peptide. 351 77


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