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)

lambda gt11 phages harboring five different cDNA fragments for the regulatory (R) subunit of Dictyostelium discoideum cAMP-dependent protein kinase (CAK) directed the synthesis of this protein in Escherichia coli cells. Crude bacterial extracts were probed with an antiserum against the Dictyostelium R subunit. The presence of specific epitopes for the R subunit in a given extract was compared with high-affinity cAMP-binding activity and with the ability to inhibit the catalytic (C) subunit through protein-protein interaction. The expression and the biochemical properties of these proteins were correlated with their cDNA nucleotide sequence. The results show that the Dictyostelium R subunit can be functionally expressed in E. coli cells either as a fusion protein with beta-galactosidase or as a nonfusion protein. In both cases, the products of cDNA clones containing the entire coding sequence retained high-affinity cAMP-binding activity and the capacity to interact with the catalytic subunit. One of the fusions, lacking the 94 N-terminal residues, failed to inhibit catalytic activity, although it bound cAMP with an affinity similar to that of the native R protein from D. discoideum.
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PMID:Expression and properties of the regulatory subunit of Dictyostelium cAMP-dependent protein kinase encoded by lambda gt11 cDNA clones. 245 May 71

During the aggregation of Dictyostelium discoideum extracellular cAMP is known to act as a chemotractant and as an inducer of cellular differentiation. However, its intracellular role as a second messenger remains obscure. We have constructed a fusion gene consisting of the cDNA encoding the regulatory subunit (R) of the cAMP-dependent protein kinase fused to the promoter and N-terminal-proximal sequences of a Dictyostelium actin gene. Stable transformants, containing multiple copies of this gene, overproduce the R subunit which accumulates prematurely relative to the endogenous protein. These transformants fail to aggregate. Detailed analysis has shown that they are blocked at interphase, the period prior to aggregation, and that they are severely defective in most responses to cAMP including the induction of gene expression. Our observations suggest that intracellular cAMP acts, presumably by activation of the catalytic subunit of the cAMP-dependent protein kinase, to facilitate early development.
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PMID:Overproduction of the regulatory subunit of the cAMP-dependent protein kinase blocks the differentiation of Dictyostelium discoideum. 255 73

We have previously shown that several genes expressed during Dictyostelium development could be induced in shaking culture by exogenous cAMP, even though the accumulation of intracellular cAMP was inhibited. The use of selected cAMP analogs indicated that the exogenous cAMP functioned by activating the cell surface cAMP receptor and not by interacting with the regulatory subunit of the intracellular cAMP-dependent protein kinase. Although some genes in Dictyostelium appear to be regulated by intracellular cAMP, these data suggest that this is not the case for all genes regulated by cAMP. Intracellular second messengers other than cAMP may, therefore, promote the expression of these other genes. Here, we have examined inositol trisphosphate and diacylglycerol as candidates for such mediators of signal transduction. We have studied three genes that exhibit disparate modes of temporal and spatial expression during development of Dictyostelium. In shaking cultures, maximal levels of expression of each are dependent on the accumulation of or exposure to extracellular cAMP. We show that the addition of inositol trisphosphate and/or diacylglycerol to cells in shaking culture has distinct effects on the expression of each gene and, under specific conditions, can bypass the requirement for extracellular cAMP. These data suggest that extracellular cAMP interacting with its cell surface receptor may promote synthesis of inositol trisphosphate and diacylglycerol to regulate gene expression and aspects of differentiation in Dictyostelium.
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PMID:Inositol trisphosphate and diacylglycerol can differentially modulate gene expression in Dictyostelium. 255 9

cAMP is a mediator of inter- and intracellular events in Dictyostelium discoideum and is thought to act through specific receptors. Eight forms of cAMP-binding proteins have been described in this organism: four forms of a cell surface receptor, a cell surface and extracellular phosphodiesterase, an intracellular cAMP-dependent protein kinase (CAK), and a recently identified cAMP-binding protein (CABP1) that is present on the cell surface, in the cytoplasm, and in the nucleus. In this study we have analyzed the cyclic nucleotide specificity of these cAMP-binding proteins using 13 derivatives of cAMP with modifications in the adenine, ribose, and phosphate moiety. The results suggest that the cAMP-binding proteins belong to three groups: (i) four forms of the cell surface receptor, (ii) two forms of an intracellular receptor (CABP1 and CAK), and (iii) cell surface and extracellular phosphodiesterase. cAMP is probably bound to the surface receptors in the anti conformation in a hydrophobic cleft of the receptor with essential interactions at N6H2' and O3'. In contrast, cAMP is probably bound to CAK and CABP1 in the syn conformation with essential interactions at O2', O3', O5', and exocyclic oxygen. Finally, binding of cAMP to phosphodiesterase involves only O3' and exocyclic oxygen. The cyclic nucleotide specificity of cAMP-induced processes in D. discoideum indicates that the cell surface receptors participate in the transduction of the cAMP signal during chemotaxis and cell differentiation. Functions for CABP1 and CAK in these processes are presently elusive.
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PMID:The cyclic nucleotide specificity of eight cAMP-binding proteins in Dictyostelium discoideum is correlated into three groups. 272 97

A protein kinase with unusual characteristics has been found in Dictyostelium discoideum. This kinase can use histone H1 as exogenous substrate, and the activity is stimulated by phospholipids, but not by Ca2+. This enzyme has been partially purified by using chromatography on DEAE-cellulose DE-52, spermine-agarose and phosphatidylserine-polyacrylamide. The protein kinase activity is very labile, even in the presence of protease inhibitors, making further purification difficult. In the activity-containing fractions, an endogenous protein of 140 kDa is labelled in vitro with [gamma-32P]ATP under conditions in which intramolecular rather than intermolecular reactions are favoured. This protein is labelled only in the presence of phospholipids, but not of Ca2+. We propose that the 140 kDa phosphoprotein might be the autophosphorylated enzyme.
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PMID:A phospholipid-stimulated protein kinase from Dictyostelium discoideum. 276 88

The regulatory subunit of the cAMP-dependent protein kinase expressed in clones isolated by immunoscreening of a lambda gt11 cDNA library from Dictyostelium discoideum exhibits high affinity for cAMP [Mutzel et al., Proc. Natl. Acad. Sci. USA 84 (1987) 6-10]. Based on this property, we have developed a screening procedure to detect in situ cAMP-binding activity directly on phage plaques transferred to nitrocellulose filters. Highly radioactive cAMP was synthesized using [alpha-32P]ATP at 3000 Ci/mmol as the substrate of purified adenylate cyclase from Bordetella pertussis. Filter replicas of the library plated at 3 X 10(4) pfu/dish, were incubated in the presence of 2 nM [32P]cAMP and then washed thoroughly. Three clones out of 1.2 X 10(5) were detected, all of which coded for the regulatory subunit, as judged by hybridization with a specific DNA probe. The cAMP binding to the purified clones was characterized in situ by displacement with specific analogues. The ability to displace labelled cAMP was in accord with the affinities of the analogues previously reported for the regulatory subunit of the Dictyostelium cAMP-dependent protein kinase. We are able to detect fmol levels of regulatory subunit contained in phage plaques and therefore the method could be used to screen libraries from other organisms for proteins exhibiting high affinities for cyclic nucleotides.
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PMID:Gene isolation by direct in situ cAMP binding. 282 98

We report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[32P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO4/PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca2+/calmodulin, Ca2+/phospholipid, or EGTA. Similarities with the beta-adrenergic receptor protein kinase are discussed.
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PMID:An unusual protein kinase phosphorylates the chemotactic receptor of Dictyostelium discoideum. 283 47

The cellular slime mold, Dictyostelium disoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum, glycogen degradation provides precursors for the synthesis of developmentally regulated structural products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The active, or 'a' form, is independent of 5'adenosine monophosphate (5'AMP) while the inactive, or 'b' form, is 5'AMP-dependent. The activity of the 'b' form predominates early in development, while the activity of the 'a' form peaks in mid-late development; their combined specific activities remain constant at any point. Polyclonal antibodies raised to the purified forms of this enzyme showed low cross-reactivity. The anti-'a' serum reacted with a 104-kDa protein that was associated with phosphorylase 'a' activity; the anti-'b' serum reacted with a 92-kDa protein that was associated with phosphorylase 'b' activity and weakly cross-reacted with the 104-kDa protein. Immunoblots of peptide maps of the purified enzyme forms showed that each antibody was specific for the proteolytic fragments of its respective antigen. We also demonstrated in vitro phosphorylation of the 'b' form by an endogenous protein kinase. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase-'a' activity and the 104-kDa protein. Immunotitration data suggested that the 'a' form accumulates due to de novo protein synthesis, although this result must be interpreted with caution.
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PMID:The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum. 284 92

A key step in the cellular differentiation of Dictyostelium is the degradation of glycogen to provide the precursors for synthesis of the structural end products of development. We have found that the enzyme that initiates this degradative pathway, glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase; EC 2.4.1.1), is developmentally regulated and exists as two forms. During the time course of development, a previously undescribed activity, the "b" form, decreases, while that of the "a" form increases. The "b" form is inactive unless 5'AMP is included in the reaction mixture. The two forms differ in their elution from DE52 cellulose, affinity constants, thermal stability, affinity for 5'AMP Sepharose, subunit molecular weight, and peptide maps. In crude extracts, anti-a antiserum stains a 104-kD protein that is associated with phosphorylase "a" activity and appears late in development, while anti-b antiserum stains a 92-kD protein that is associated with phosphorylase "b" activity and is present throughout development. We have also demonstrated in vitro phosphorylation of the "b" form by an endogenous protein kinase and a corresponding loss of 5'AMP dependence. If intact cells were exposed to exogenous cAMP, "b" activity decreased and was replaced by "a" activity, as well as the 104-kD protein band on SDS-PAGE. In order to determine if the two forms of the enzyme are different gene products, we screened lambda gt11 expression libraries with antibodies against the purified "a" and "b" forms. Three clones were found to be overlapping by Southern analysis. A yeast glycogen phosphorylase cDNA clone (gpy) and a human muscle glycogen phosphorylase clone (HM-11) cross-hybridized with the Dictyostelium inserts, and gpy shared a few common restriction fragments with the Dictyostelium clones on genomic blots. Northern analysis of Dictyostelium total RNA showed that the Dictyostelium inserts and gpy recognize an mRNA of 3.2 kb, while on poly A-enriched RNA, the yeast clone detects preferentially a 3.6-kb message.
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PMID:Glycogen phosphorylase in Dictyostelium discoideum: demonstration of two developmentally regulated forms, purification to homogeneity, immunochemical analysis, cAMP induction, in vitro translation, and molecular cloning. 285 25

Extracts of aggregation-competent cells of Dictyostelium discoideum have an S6 protein kinase activity which is inhibited in the presence of the inhibitor of the cAMP-dependent protein kinase. The phosphorylation of S6 is rapid, and decays rapidly. The S6 kinase activity is detectable in the 150,000g supernatant only in the presence of phosphatase inhibitors known for preserving the S6 kinase in other systems, indicating that the activated form of the enzyme is phosphorylated by the cAMP-dependent protein kinase. S6 kinase elutes as a peak from DEAE-Sephacel at 100 mM NaC1, with an activity that is cAMP-dependent.
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PMID:Phosphorylation of ribosomal protein S6 is dependent on cyclic AMP in Dictyostelium discoideum. 285 12


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