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)

The effects of specific anti-calmodulin monoclonal antibodies on the conformation and interaction of calmodulin with two enzymes, the insulin receptor tyrosine kinase and casein kinase II, are examined. Addition of the anti-calmodulin antibody 2D1 in vitro augments phosphorylation of calmodulin by rat hepatocyte insulin receptors 4.9 +/- 0.5-fold (n = 7). Nonimmune immunoglobulin has no effect. Maximal phosphorylation is observed at a molar ratio of calmodulin:antibody of approx. 2:1, with higher concentrations of antibody producing lesser enhancement. Increasing Ca2+ concentrations in the physiological range progressively inhibit phosphorylation both in the absence and presence of antibody 2D1. Phosphate is incorporated predominantly on Tyr-99, which is distant from the antibody binding site. Enhancement of casein kinase II-catalyzed calmodulin phosphorylation is also produced by the antibody 2D1, implying that antibody binding induces a change in calmodulin conformation. In contrast, two other anti-calmodulin monoclonal antibodies, 4F4 and 4G2, decrease phosphorylation of calmodulin by both the insulin receptor kinase and casein kinase II. These data indicate that secondary and tertiary structures are important in enzyme-substrate interactions and suggest that the antibodies may be useful in investigating the mechanism of calmodulin function.
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PMID:Alteration of calmodulin-protein interactions by a monoclonal antibody to calmodulin. 818 41

In the preceding report (Ladner, R.D., McNulty, D.E., Carr, S.A., Roberts, G.D., and Caradonna, S.J. (1996) J. Biol. Chem. 271, 7745-7751), we identified two distinct isoforms of dUTPase in human cells. These isoforms are individually targeted to the nucleus (DUT-N) and mitochondria (DUT-M). The proteins are nearly identical, differing only in a short region of their amino termini. Despite the structural differences between these proteins, they retain identical affinities for dUTP (preceding article). In previous work, this laboratory demonstrated that dUTPase is posttranslationally phosphorylated on serine residue(s) (Lirette, R., and Caradonna, S. (1990) J. Cell. Biochem. 43, 339-353). To extend this work and determine if both isoforms of dUTPase are phosphorylated, a more in depth analysis of dUTPase phosphorylation was undertaken. [32P]Orthophosphate-labeled dUTPase was purified from HeLa cells, revealing that only the nuclear form of dUTPase is phosphorylated. Electrospray tandem mass spectrometry was used to identify the phosphorylation site as Ser-11 in the amino-terminal tryptic peptide PCSEETPAIpSPSKR (the NH2-terminal Met is removed in the mature protein). Mutation of Ser-11 by replacement with Ala blocks phosphorylation of dUTPase in vivo. Analysis of the wild type and Ser-11 --> Ala mutant indicates that phosphorylation does not regulate the enzymatic activity of the DUT-N protein in vitro. Additionally, experiments with the Ser-11 --> Ala mutant indicate that phosphorylation does not appear to play a role in subunit association of the nuclear form of dUTPase. The amino acid context of this phosphorylation site corresponds to the consensus target sequence for the cyclin-dependent protein kinase p34(cdc2). Recombinant DUT-N was specifically phosphorylated on Ser-11 in vitro with immunoprecipitated p34(cdc2). Together, these data suggest that the nuclear form of dUTPase may be a target for cyclin-dependent kinase phosphorylation in vivo.
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PMID:Identification of a consensus cyclin-dependent kinase phosphorylation site unique to the nuclear form of human deoxyuridine triphosphate nucleotidohydrolase. 863 17

Phosphate starvation induces the transcription of several genes involved in phosphate metabolism in the budding yeast Saccharomyces cerevisiae. The signal transduction pathway that mediates this response consists of components that resemble those used to regulate the eukaryotic cell cycle; these include a cyclin-dependent kinase or CDK (Pho85), a cyclin (Pho80) and a CDK inhibitor (Pho81). The possibility that this pathway mediates cell-cycle responses to phosphate starvation is discussed.
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PMID:Signaling phosphate starvation. 891 92

Nuclear respiratory factor 1 (NRF-1) is a transcriptional activator that acts on a diverse set of nuclear genes required for mitochondrial respiratory function in mammalian cells. These genes encode respiratory proteins as well as components of the mitochondrial transcription, replication, and heme biosynthetic machinery. Here, we establish that NRF-1 is a phosphoprotein in vivo. Phosphorylation occurs on serine residues within a concise NH2-terminal domain with the major sites of phosphate incorporation at serines 39, 44, 46, 47, and 52. The in vivo phosphorylation pattern can be approximated in vitro by phosphorylating recombinant NRF-1 with purified casein kinase II. Phosphate incorporation at the sites utilized in vivo results in a marked stimulation of DNA binding activity which is not observed in mutated proteins lacking these sites. Pairwise expression of the wild-type protein with each of a series of truncated derivatives in transfected cells results in the formation of a dimer between wild-type and mutant forms demonstrating that a homodimer is the active binding species. Although NRF-1 can dimerize in the absence of DNA, phosphorylation does not enhance the formation of these dimers. These findings suggest that phosphorylation results in an intrinsic change in the NRF-1 dimer enhancing its ability to bind DNA.
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PMID:Serine phosphorylation within a concise amino-terminal domain in nuclear respiratory factor 1 enhances DNA binding. 922 45

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.
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PMID:Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry. 938 46

Oxysterol binding protein (OSBP), a high affinity receptor for 25-hydroxycholesterol that localizes to a Golgi/vesicular compartment, migrated on SDS-PAGE as a doublet of 96 and 101 kDa. The reduced mobility of the upper band of this doublet is the result of phosphorylation on multiple serine residues. Phosphorylation of rabbit OSBP stably overexpressed in CHO-K1 cells was altered by staurosporine and okadaic acid, while other protein kinase activators and inhibitors such as TPA, sphingosine and bis-indolylmaleimide were without affect. Treatment of overexpressing and control cells with brefeldin A (BFA) caused dephosphorylation of OSBP that coincided with disruption of the Golgi apparatus. [32P]Phosphate pulse-chase and immunoprecipitation experiments showed that BFA inhibited phosphorylation of OSBP, but not its rate of dephosphorylation. Phosphopeptide maps of OSBP from overexpressing and control CHO-K1 cells were similar, and BFA promoted dephosphorylation of all five peptides. Compared to overexpressing cells, one tryptic phosphopeptide was more abundant in control CHO-K1 cells and was preferentially dephosphorylated by BFA treatment. OSBP was phosphorylated in vitro by the Golgi enriched fraction of CHO-K1 cells or rat liver by a staurosporine- and BFA-insensitive kinase. The phosphorylation status of OSBP was not affected by 25-hydroxycholesterol and did not alter in vitro 25-[3H]hydroxycholesterol binding. Furthermore, dephosphorylation of OSBP by staurosporine did not affect 25-hydroxycholesterol-mediated localization to the Golgi apparatus. Rapid phosphorylation/dephosphorylation of OSBP requires interaction with the Golgi apparatus and an associated kinase. (c) 1998 Elsevier Science B.V.
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PMID:Inhibition of phosphorylation of the oxysterol binding protein by brefeldin A. 948 39

Staphylococcal leukocidin (Luk) consists of two protein components, LukF and LukS, which cooperatively lyse human and rabbit polymorphonuclear leukocytes. Here, we demonstrate that the phosphorylation of LukS by protein kinase A is crucial for the LukS-specific leukocytolytic function of Luk on HPMNLs by using N-[2(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), which is a potent and selective inhibitor of protein kinase A. At 0.5 microM H-89 completely prevented the Luk-induced cell lysis accompanied by blocking of the incorporation of exogenous 32P-H3PO4 into LukS on HPMNLs. However, with LukS and LukF together, 0.5 microM H-89 did not inhibit the cell swelling which takes place before the cell lysis. HPMNLs also became swollen upon treating with both LukF and LukS mutants which could not be phosphorylated.
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PMID:Phosphorylation of LukS by protein kinase A is crucial for the LukS-specific function of the staphylococcal leukocidin on human polymorphonuclear leukocytes. 980 89

Milk caseins have been phosphorylated by a recombinant protein kinase CK2 catalytic subunit from Schizosaccharomyces pombe (rspCK2alpha). Phosphate incorporation stoechiometries into purified caseins and into native phosphocaseinate, a substrate exhibiting a micellar-like structure, were determined. We incorporated 2.01 mol of P/mol of alpha-casein, 6.46 mol of P/mol of beta-casein, up to 0. 29 mol of P/mol of kappa-casein in 4 h, and more than 1.36 mol of P/mol of casein into phosphocaseinate under optimized conditions. Phosphocaseinate was sequentially phosphorylated; beta-caseins being labeled at first; alpha-caseins being labeled later; and to a lower extend, kappa-caseins were the last to be phosphorylated. The solubility of phosphocaseinate micelles increased by 1.34 from 65 to 87%, and its rennetting time was increased 2.88 times. These results are discussed in terms of plausible structural organization of caseins micelles and the effect of phosphorylation on their structure.
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PMID:Overphosphorylation of milk caseins by a recombinant protein kinase CK2 catalytic subunit. 1055 56

The influence of nucleic acids (DNA, tRNA), synthetic oligonucleotides, and polysaccharides (lipopolysaccharides from Escherichia coli, heparin) on protein kinase and lipid kinase activities of preparations of human secretory immunoglobulin A (sIgA) has been studied. The preparations of sIgA were isolated from human milk by chromatography on the column with Protein A-Sepharose and DEAE-sorbent (sIgA1), by affinity chromatography of sIgA1 on DNA-cellulose (sIgA2), and by gel-filtration of sIgA1 in buffer containing 5% dioxane (sIgA3). Two 32P-labeled products with high and low electrophoretic mobility in polyacrylamide gel containing SDS were found after incubation of sIgA1 and sIgA2 with [gamma-32P]ATP. The product with low electrophoretic mobility was degraded in 10% trichloroacetic acid giving a radioactive background in lanes of the polyacrylamide gel. 32P-Labeled phospholipids were found among the phosphorylation products. Soluble and immobilized DNA increase lipid kinase activity of preparations of sIgA. In this case the secretory component and H-chains of sIgA were degraded. Fractions possessing lipid kinase activity were precipitated in the presence of heparin (1 mg/ml), and lipid kinase activity was separated from sIgA by gel-filtration in buffer containing 5% dioxane. 32P-Labeled products were formed in the presence of [gamma-32P]ATP as well as [32P]ortho-phosphoric acid. The influence of heparin and synthetic deoxy- and ribooligonucleotides on casein kinase activity of sIgA3 was studied. It was observed that deoxyribooligonucleotides in micromolar concentrations increased the rate of casein phosphorylation in the presence of sIgA3 and [gamma-32P]ATP. It has been proposed that catalytically active sIgA have an affinity to DNA (anti-DNA sIgA) and can be present in human milk as a part of lipoprotein complexes.
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PMID:Influence of nucleic acids and polysaccharides on phosphotransferase activity of preparations of secretory immunoglobulin A from human milk. 1071 54

The 5-hydroxytrptamine3 (5-HT3) receptor is a pentameric complex belonging to the family of ligand-gated ion channels. A variety of studies have suggested that phosphorylation regulates the rate of desensitisation and the size of amplitude of the receptor current. In this study we have examined the phosphorylation of the myc-tagged wild-type 5-HT3A receptor subunits from guinea-pig expressed in HEK293 cells (human embryonic kidney). Stably transfected cells were metabolically labelled with 32P-phosphoric acid. The results of immunoprecipitation and autoradiography demonstrate that both splicc variants of the 5-HT3A receptor subunit are phosphorylated in HEK293 cells. Site-specific mutagenesis revealed that phosphorylation occurs at serine 409, a potential target of protein kinase A. Thus the 5-HT3 receptor might be modulated by intracellular pathways, that allow variable 5-hydroxytryptamine action as responses to different extracellular stimuli.
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PMID:Phosphorylation of the 5-hydroxytryptamine3 (5-HT3) receptor expressed in HEK293 cells. 1080 Jul 72


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