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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the construction of vectors for expressing in Escherichia coli DNA fragments obtained by progressive deletions of DNA inserts in single-stranded sequencing vectors as M13 or pTZ according to the methode of Dale et al. (Plasmid 1985, 13, 31-40). These vectors, pIMS1, pIMS5, and pIMS6, harbor all of the elements required for the regulated expression of any open reading frame flanked by EcoRI restriction sites. The encoded peptides contain only a few vector-derived amino acids. A method is described for direct selection of recombinant clones by in situ RNA hybridization. The properties of the expression vector have been analyzed with a DNA deletion series obtained from the cDNA coding for the regulatory subunit of Dictyostelium discoideum cAMP-dependent protein kinase.
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PMID:Vectors for expression of truncated coding sequences in Escherichia coli. 304 59

The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.
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PMID:cAMP-dependent protein kinase from Dictyostelium discoideum. 307 32

The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase 'b' form requires 5' AMP for activity and is present in early development. The active phosphorylase 'a' form is 5' AMP independent and occurs during later development. We here show that the 92 kd 'b' enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn2+ and ATP, the phosphorylase 'b' shows apparent conversion into a 5' AMP independent form as measured by enzyme activity. In addition, Mn2+ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase 'b' subunit. We also demonstrate phosphorylation of the 'b' enzyme subunit in vivo by 32-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase 'b' subunit is characterized.
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PMID:Glycogen phosphorylase 'b' in Dictyostelium: stability and endogenous phosphorylation. 314 89

cDNA clones encoding the regulatory subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from Dictyostelium discoideum were isolated by immunoscreening of a cDNA library constructed in the expression vector lambda gt11. High-affinity cAMP-binding activity was detected in extracts from bacteria lysogenized with these clones. Nucleotide sequence analysis of three overlapping clones allowed the determination of a 1195-base-pair cDNA sequence coding for the entire regulatory subunit and containing nontranslated 5' and 3' sequences. The open reading frame codes for a protein of 327 amino acids, with molecular weight 36,794. The regulatory subunit from Dictyostelium shares a high degree of homology with its mammalian counterparts, but is lacking the NH2-terminal domain required for the association of regulatory subunits into dimers in other eukaryotes. On the basis of the comparison of the regulatory subunits from Dictyostelium, yeast, and bovine tissues, a model for the evolution of these proteins is proposed.
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PMID:Cloning and cDNA sequence of the regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum. 346 59

It has been known for 20 years that during cellular differentiation of Dictyostelium discoideum, glycogen is degraded to provide the glucose precursors that are required for the synthesis of the end-products of development. Because this pathway provided a distinct developmentally regulated event, a number of laboratories have investigated the regulation of the first step in glycogen degradation, glycogen phosphorylase. Of particular interest was the possible regulation of this enzyme by cAMP. Cyclic AMP is know to act as a signal in this organism for both chemotaxis and cell differentiation. The phosphorylase activity was found to increase during development and, therefore, it has been used in many studies as a marker for late stage development. However, only one form of the phosphorylase was found, and therefore it was concluded that cAMP was not involved in regulation of this key step in the developmental pathway. Here we report the discovery of a second form of the enzyme. This form is completely dependent on AMP for activity and is found only in the undifferentiated stage. This second form contains several of the properties of the nonphosphorylated enzyme that occurs in systems that are regulated by cAMP. This result and the recent discovery of a cAMP-dependent protein kinase has rekindled the possibility that at least one of the effects of cAMP in this organism occurs via a cAMP-dependent cascade of phosphorylation; that is, the activation of glycogen phosphorylase and subsequent production of the precursors for the end-products of development.
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PMID:Identification of two forms of glycogen phosphorylase in Dictyostelium. 349 Aug 29

A search for nuclear substrates of the cAMP-dependent protein kinase (cAMP-d PK) of Dictyostelium discoideum led to the identification of several such proteins. Identification was based initially on increased phosphorylation of the proteins in nuclear extracts catalyzed by added cAMP-d PK. One protein of Mr 38,000 was phosphorylated also in intact nuclei and in vivo; the amount of phosphoprotein or the level of phosphorylation increased during development. Both the Mr 38,000 protein and another substrate of Mr 195,000 were found in the nuclei of prespore and prestalk cells. Phosphorylation of other potential substrates of the cAMP-d PK was either prespore or prestalk cell-specific.
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PMID:Identification of nuclear substrates of the cyclic AMP-dependent protein kinase in Dictyostelium discoideum. 358 Nov 72

cAMP is an important effector of the development of Dictyostelium discoideum amoebae and could exert its effects on gene expression through the cytosolic cAMP-dependent protein kinase (cAK). Antibodies, specific for the regulatory subunit (R) of the cAK, were used to investigate the developmental regulation of the corresponding mRNA (R-mRNA) by in vitro translation and immunoprecipitation. Under such conditions, a single polypeptide of the same mol. wt. as R (42 kd) is detected, showing that the protein is not synthesized as a large precursor. The level of the R-mRNA, which is low in vegetative cells, increases 10- to 20-fold during the first hours of development. Its expression is stimulated by the treatment of AX3 cells with cAMP either added to a concentration of 1 mM or given as 0.1 microM pulses every 5 min, whereas such treatments have little or no effect in cells of strain AX2. The R-mRNA remains highly expressed (0.01-0.03% of translatable mRNA) throughout post-aggregative development; it is not affected by mechanical disaggregation of the multicellular organism. The parallel developmental time courses of the translatable R-mRNA and the R protein produced in vivo suggest that the expression of this polypeptide is regulated at the level of mRNA synthesis.
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PMID:Detection and developmental regulation of the mRNA for the regulatory subunit of the cAMP-dependent protein kinase of D. discoideum by cell-free translation. 370 16

The level of the regulatory subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum was analyzed in subcellular fractions of cells at various stages of development by Western blotting. The protein was found only in the cytosolic fraction. A small amount of regulatory subunit was present in vegetative cells, and its level increased sharply during the first hours of aggregation; a further increase also occurred during culmination. Analysis of mature spores and of the stalky mutant HL 65 revealed that the protein is present only in prespore cells.
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PMID:The regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum: cellular localization and developmental regulation analyzed by immunoblotting. 390 61

A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type 1. In addition, the activation of the kinase by these analogs was monitored. The binding data indicate that cAMP is bound to both sites in a comparable manner: the adenine appears to have no hydrogen-bond interactions with the binding sites, whereas the ribose may be bound by three hydrogen bonds involving the 2', 3' and 5' positions of cAMP. The binding data are not conclusive about the nature of the interaction with the exocyclic oxygen atoms on phosphorus, though a charge interaction seems to be absent. The cAMP molecule seems to be bound in the syn conformation. The results of activation experiments show that modifications in the adenine and ribose moiety do not affect the maximal activation level, while alteration of the two exocyclic oxygen atoms may result in a reduced maximal activation level and in one case, (Rp)-adenosine 3', 5'-monophosphorothioate [Rp-cAMPS], in total absence of activation even at concentrations at which the analog saturates both binding sites. Since occupancy of the cAMP-binding sites by this derivative apparently did not lead to activation of the enzyme, we examined whether this compound could antagonize the activation by cAMP. Indeed (Rp)-cAMPS was found to inhibit cAMP stimulated kinase activity at concentrations compatible to its binding affinity. Also with mammalian protein kinase type II (Rp)-cAMPS showed antagonistic activity, while with a cAMP-dependent protein kinase from Dictyostelium discoideum partial agonistic activity was observed. Previously a mechanism for activation of protein kinase type I was proposed involving a charge interaction between the equatorial exocyclic oxygen atom and the binding site [De Wit et. al. (1982) Eur. J. Biochem 122, 95-99]. This was based on measurements with impure preparations of (Rp)-cAMPS and the Rp and Sp isomers adenosine 3', 5'-monophosphodimethylamidate. cAMPN(CH3)2. The present work using highly purified compounds suggests the absence of a charge interaction, since the uncharged analog (Sp)-cAMPN(CH3)2 activates the kinase effectively. The data seem compatible with an activation model involving the formation of a covalent bond with phosphorus in both cAMP binding sites.
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PMID:Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases. 608 45

The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and RP-cAMPN(CH3)2. We have investigated the agonistic and antagonistic activities of these compounds in four cAMP-dependent reactions: activation of the cellular slime mold Dictyostelium discoideum via its cell surface cAMP receptor, and phosphorylation by cAMP-dependent protein kinases type I, type II (both mammalian enzymes), and type D (derived from D. discoideum). The results show that 1) the compounds (SP)-cAMPS and (SP)-cAMPN(CH3)2 are (mostly full) agonists for the four proteins. Half-maximal activation is at micromolar concentrations (0.8-7 microM). 2) (RP)-cAMPS is a full antagonist for the cell surface receptor and protein kinases type I and II, with apparent inhibition constants between 0.8 and 8 microM. This compound is a partial agonist for protein kinase type D, where it induces maximally 50% activation of the enzyme if compared with cAMP. 3) (RP)-cAMPN(CH3)2 is a full antagonist for the cell surface receptor, and for protein kinase type II. This compound is a partial agonist for protein kinase type I (at least 50% activation if compared with cAMP), and inactive for protein kinase type D. This derivative is at least 25-fold less active as an antagonist than (RP)-cAMPS. 4) The activity of mixtures of different concentrations of the antagonist (RP)-cAMPS with different concentrations of cAMP reveals that the compound is a competitive antagonist of cAMP at micromolar concentrations.
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PMID:Competitive cAMP antagonists for cAMP-receptor proteins. 608 78


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