Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.
 ...
PMID:A solid-phase screen for protein kinase substrate selectivity. 141 23

Phosphorylation of immunopurified chicken oviduct progesterone receptor (PR) was studied in intact cells and under cell-free conditions. Cytosol PR was isolated by incubation with anti-PR monoclonal antibody alpha PR22 adsorbed to protein A-Sepharose and suspended in a reaction mixture containing 10 mM Mg2+, 0.1 mM [gamma-32P]ATP, and the catalytic subunit of cAMP-dependent protein kinase (cAMP-PK) from bovine heart. All three major proteins of avian PR (PR-A, 79 kDa; PR-B, 110 kDa; 90 kDa) incorporated 32P-radioactivity on serine residues. The phosphorylation reaction was inhibited by synthetic inhibitors of protein kinases, H-8 and 20-residue peptide IP20. A 40 degrees C preexposure of PR oligomer increased phosphorylation of the 90-kDa protein, known to be a heat-shock protein (hsp-90). The extent of the phosphorylation reaction was temperature-dependent as the 32P-incorporation into PR-A and PR-B increased gradually, showing a maximum at 37 degrees C. Multiple phosphopeptides (4-7) were resolved by two-dimensional electrophoresis chromatography following cleavage of 32P-labeled peptides with trypsin. Both A and B forms of receptor showed similar phosphorylation patterns with B receptor digestion exhibiting two to three additional peptides. Under physiological conditions, preincubation of oviduct mince with forskolin, a regulator of intracellular cAMP levels, caused a greater extent of phosphorylation of PR-A and PR-B proteins. The results of this study demonstrate that chicken oviduct PR is an excellent substrate for the action of cAMP-PK in vitro and that this enzyme may be a physiological regulator of progesterone action in the oviduct.
 ...
PMID:Phosphorylation of chicken oviduct progesterone receptor by cAMP-dependent protein kinase. 141 66

The cAMP response element-binding protein (CREB) mediates transcriptional activation of genes in response to the cAMP signal transduction pathway. There are two different isoforms of CREB, which are generated by alternative RNA splicing. There is evidence that the two isoforms may have different biological activities. As the longer isoform (CREB341) contains a potential phosphorylation site that is not present in the shorter isoform (CREB327), we examined the possible differential phosphorylation of the two CREB isoforms. Recombinant CREB was prepared and used as substrate for phosphorylation by the cAMP-dependent protein kinase in vitro. Phosphopeptide mapping and mutagenesis studies demonstrated that CREB341 contains two sites, serine 133 and serine 98, that can be phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase. In contrast, CREB327 contains only a single phosphorylation site at serine 119 (equivalent position to serine 133 in CREB341). A kinase titration experiment demonstrated that serine 98 of CREB341 was phosphorylated only at relatively high concentrations of the cAMP-dependent protein kinase. Transient transfection studies were used to test for any possible function of the phosphorylation of serine 98 of CREB341. These studies used GAL4-CREB fusion proteins. We found that mutation of serine 98 to alanine (which would block phosphorylation) has little or no effect on the ability of the CREB fusion protein to activate transcription. These findings suggest that differences in the biological activity of the two CREB isoforms are probably not mediated by differential phosphorylation by the cAMP-dependent protein kinase.
 ...
PMID:Phosphorylation of cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein isoforms by the cAMP-dependent protein kinase. 148 Jan 75

We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene.
 ...
PMID:Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii. 151 58

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.
 ...
PMID:The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase. 151 35

Rat submandibular and parotid gland exocytosis is primarily controlled by beta-adrenergic receptor stimulation. Although its precise role in the regulation of salivary gland exocytosis is not fully understood, protein phosphorylation, mediated by the activation of cAMP-dependent protein kinase, may be directly involved. Previous studies suggest that analogous 26-kDa integral membrane phosphoproteins may play a direct role in regulating exocytosis. Studies were here undertaken to purify and partially characterize both phosphoproteins. After endogenous phosphorylation with 32P, subcellular fraction and solubilization of the microsomal fraction in n-octyl beta-glucopyranoside, the 26-kDa integral membrane phosphoproteins were purified by high performance liquid chromatography (HPLC), followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroelution of the proteins. Amino acid analysis indicated a significant number of serine amino acids: N-terminal sequence data demonstrated a high level of homology; and trypsin digestion followed by reversed-phase HPLC indicated the possibility of multiple phosphorylation sites.
 ...
PMID:Purification and partial characterization of analogous 26-kDa rat submandibular and parotid gland integral membrane phosphoproteins that may have a role in exocytosis. 152 94

The intracellular events which are involved in controlling the G1 to S phase transition during the eucaryotic cell cycle are important to define in order to understand the mechanisms by which mitogenic and growth arrest-inducing agents control cell growth. Because a change in protein kinase activity is associated with the initial response of cells to mitogenic stimulants and growth factors, we used a kinase renaturation assay to identify specific protein kinases which are modulated as human T cells make the G1 to S phase transition after mitogenic stimulation with lectin. We identified four protein serine/threonine kinases of 180, 97, 85, and 38 kilodaltons which are increased in activity as these cells enter S phase. A-55 kDa serine/threonine kinase (PK55) was shown to have maximal activity during G0 and its activity was reduced by 95% upon movement into S phase. PK55 is inducible in human T cells by removal of interleukin 2 and low serum incubation which arrests cells in G1 phase, indicating that it is closely associated with G1 phase growth arrest. Furthermore, a similar PK55 activity was induced upon growth arrest in HL-60 cells treated with dimethyl sulfoxide and in Daudi cells treated with interferon alpha. Because the cAMP-dependent protein kinase (PK-A) family has been shown to be antiproliferative to lectin stimulated T cells, we were interested in determining whether PK55 was in fact an isozyme of PK-A. Comparative analysis using a specific peptide inhibitor of PK-A activity revealed that PK55 is catalytically distinct from PK-A. This data suggest that increases in PK55 may be associated with the growth-arrested state and further that PK55 is distinct from PK-A.
 ...
PMID:Specific protein kinases modulated during T cell mitogenesis. Activity of a 55-kDa serine kinase is associated with growth arrest in human T cells. 153 85

We describe here a protein kinase from the promastigote form of the parasitic protozoan, Leishmania donovani, purified to near homogeneity to a single-subunit, 34-kDa protein. This enzyme does not require a cofactor, and has several characteristics in common with the catalytic subunit of mammalian cAMP-dependent protein kinase, for example, preference for kemptide as a substrate, phosphorylation of serine residues of protamine and inhibition by the mammalian heat-stable inhibitor. The leishmanial enzyme can associate with the regulatory subunit of mammalian cAMP-dependent protein kinase to form an inactive holoenzyme that is activated by cAMP and is protected from inhibition by thiol reagents. From these results it is concluded that L. donovani promastigotes possess a protein kinase which has similar characteristics with the mammalian catalytic subunit of cAMP-dependent protein kinase.
 ...
PMID:Isolation and characterization of a cyclic nucleotide-independent protein kinase from Leishmania donovani. 162 Jan 59

Essential to signal transduction are mechanisms of "cross-talk" to coordinate different pathways. This study shows that stimulation of serine/threonine protein kinases activates protein-tyrosine phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). More than 95% of intracellular PTPase was in the particulate fraction of various cell lines and was extracted with detergent as a 150-kDa complex that contained a 55-kDa catalytic subunit. The complex was activated by protease digestion, which changed its substrate specificity coincident with reduction in size. The complex was dissociated by treatment of the membrane fraction with 3 M LiBr. Treatment of intact cells with isoproterenol, forskolin, or cAMP analogues to stimulate cAMP-dependent protein kinase (PKA) or with phorbol ester or dioctanoylglycerol to stimulate Ca2+/phospholipid-dependent protein kinase (PKC) produced activation of membrane PTPase complex without a change in its size. Inhibition of protein-serine/threonine phosphatases with okadaic acid or fluoride also resulted in activation of the membrane PTPase. These results support a model for regulation of PTPase by phosphorylation and dephosphorylation of serine/threonine residues in a regulatory component complexed with the 55-kDa PTPase catalytic subunit. This mechanism may be important in regulating sensitivity to extracellular signals transduced via tyrosine phosphorylation and in the synchronization of events during the cell cycle.
 ...
PMID:Activation of membrane protein-tyrosine phosphatase involving cAMP- and Ca2+/phospholipid-dependent protein kinases. 165 Apr 78

The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 mumol/mg of protein/min, with a Km of 4.7 microM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 microM), sodium vanadate (IC50 of 150 microM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 microM ZnCl or 10 mM NaF. Poly-(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.
 ...
PMID:Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor. 165 33


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>