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 protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.
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PMID:Catalysis of serine and tyrosine autophosphorylation by the human insulin receptor. 138 4

The Ag-NOR proteins are defined as markers of "active" ribosomal genes. They correspond to a set of proteins specifically located in the nucleolar organizer regions (NORs), but have not yet been clearly identified. We adapted the specific detection method of the Ag-NOR proteins to Western blots in order to identify these proteins. Using a purified protein, Western blots, and immunological characterization, the present study brings the first direct evidence leading to the identity of one Ag-NOR protein. We found that nucleolin is specifically revealed by Ag-NOR staining. Using different nucleolin fragments generated by CNBr cleavage and by overexpression in Escherichia coli, we demonstrate that the amino-terminal domain of nucleolin and not the carboxy-part of the protein is involved in silver staining. Moreover, as the pattern of staining does not vary using casein kinase II- and cdc2-phosphorylated nucleolin or dephosphorylated nucleolin, we conclude that the reduction of the silver ions is not linked to the phosphorylation state of the molecule. We propose that the concentration of acidic amino acids in the amino-terminal domain of nucleolin is responsible for Ag-NOR staining. This hypothesis is also supported by the finding that poly L-glutamic acid peptides are silver stained. These results provide data that can be used to explain the specificity of Ag-NOR staining. Furthermore, we clearly establish that proteolysis of the amino-terminal Ag-NOR-sensitive part of nucleolin occurs in vitro, leading to the accumulation of the carboxy-terminal Ag-NOR-negative part of the protein. We argue that this cleavage occurs in vivo as already proposed, bearing in mind that nucleolin is present in the fibrillar and in the granular component of the nucleolus, whereas no Ag-NOR staining is observed in the latter nucleolar component.
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PMID:Nucleolin is an Ag-NOR protein; this property is determined by its amino-terminal domain independently of its phosphorylation state. 138 90

Protein kinase N (PKN) is a protein kinase rapidly activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma and additional cell types. PKN is selectively inhibited by purine analogs, and this property has served both as a diagnostic for PKN activity and to establish its apparent involvement in certain pathways of the NGF mechanism of action. The present work has focused on further characterization, identification, and purification of NGF-activated PKN. We show here that PKN can be substantially enriched by elution from ion exchange resins with ATP. We exploited this novel technique (nucleotide affinity exchange chromatography) to devise two alternative isolation schemes for PKN. One utilizes sequential chromatographic steps and provides a preparation that is apparently 60% homogeneous for PKN and represents a total enrichment of approximately 10,000-fold. The other is a single column procedure and includes prewashes with NAD. This method yields material that is about 5-10% homogeneous for PKN, requires about 1 h, and can be applied to multiple samples in parallel. The ATP elution technique furthermore distinguishes NGF-regulated from basal PKN activity and thereby suggests the presence of distinct PKN isoforms. The applications of sucrose gradient centrifugation, gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/silver staining, affinity labeling with 8-azido-ATP/SDS-PAGE, and autophosphorylation (after SDS-PAGE, blotting and renaturation) all indicate that PKN has an apparent molecular mass of 45-47 kDa and is mainly monomeric in solution. These and additional properties appear to distinguish PKN from many previously described protein kinases.
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PMID:Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography. 140 Apr 78

A second messenger-independent serine/threonine protein kinase from lactating goat mammary gland is purified and characterized. The purification steps include: homogenization, ultracentrifugation, ammonium sulphate precipitation, DEAE-Sepharose, phosphocellulose, hydrophobic and Mono Q columns. On the final step of purification the enzyme is revealed as a single band of mol wt 45,000 on silver-stained SDS-PAGE. Mg2+ and K+ are necessary for its optimum activity. Phosvitin and casein are substrates for the enzyme but kemptide, RRREEETEEE, protamine and histone mixture are all poorly phosphorylated. The kinase is inhibited by quercetin, heparin, random tyrosine- and glutamic acid-containing polymers, Ca2+, NaF, 2,3-bis-phosphoglycerate. 1 mM Mn2+ affects positively the basal level of the kinase activity but 5 mM Mn2+ completely suppress the effect of 10 mM Mg2+. Km of this enzyme for ATP is 1.57 microM and pH optimum is from 6 to 7. Isolation of this kinase is facilitated by its unusually high affinity for phosphocellulose.
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PMID:Identification and purification of a novel serine/threonine messenger-independent growth-related protein kinase from lactating goat mammary gland. 162 98

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.
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PMID:Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669. 165 22

The functional significance of the oxidation/reduction state of sulfhydryl groups of cGMP-dependent protein kinase (cGMP kinase) was studied at 30 degrees C using different metal ions as oxidizing agents. Mn2+, Zn2+, Fe2+, Ni2+, and Co2+ failed to activate cGMP kinase, whereas Cu2+, Cu+, Fe3+, Hg2+, and Ag+ activated cGMP kinase by oxidation with an activity ratio (-cGMP/+cGMP) of about 0.7. The activation was not caused by degradation of the enzyme to a cGMP-independent constitutively active form. Reduction of the Cu(2+)-activated and gel-filtered enzyme with dithiothreitol lowered the activity ratio in the absence of cGMP to 0.17. Oxidation did not change the kinetic and binding parameters of cGMP kinase significantly but reduced the number of titratable sulfhydryl groups from 9.5 +/- 0.7 to 6.0 +/- 0.4 cysteines/75-kDa subunit. The free cysteinyl residues of the native and Cu(2+)-oxidized cGMP kinase were labeled with 4-dimethylaminoazobenzene-4'-iodoacetamide or N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Tryptic peptides of the labeled proteins were isolated and sequenced. The cysteinyl residues oxidized by Cu2+ were identified as disulfide bonds between Cys-117 and Cys-195 and Cys-312 and Cys-518, respectively. Cu2+ activation of cGMP kinase was prevented by mild carboxymethylation of the reduced enzyme with iodoacetamide, which apparently modified these four cysteinyl groups. The results show that cGMP kinase is activated by the formation of at least one intrachain disulfide bridge.
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PMID:Oxidation of cysteines activates cGMP-dependent protein kinase. 165 29

The rat testicular galactolipid sulfotransferase has been purified by affinity chromatography using 3'5'-adenosine diphosphate-agarose affinity chromatography. Both galactosyl glycerolipid and galactosyl ceramide were effective substrates with Km values of 4.8 and 1.1 microM respectively. A single protein of molecular mass 56 kDa was present in the purified sulfotransferase preparation as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Specific photoaffinity substrate labeling, using an azido derivative of galactosyl ceramide, was used to identify this protein, both in crude extracts and when purified. The protein was also selectively phosphorylated in the presence of the rat testicular galactolipid sulfotransferase stimulatory protein kinase.
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PMID:Purification of the testicular galactolipid: 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase. 173 Jul 10

A rat liver cytosolic cholesteryl ester hydrolase (CEH) was purified 12,600-fold by ammonium sulfate precipitation, cation exchange chromatography and gel permeation high-performance liquid chromatography, with an overall yield of 20%. Its properties are compared to those of pancreatic CEH, with which it has sometimes been identified. Liver CEH exhibited a single silver stained band following SDS-polyacrylamide gel electrophoresis (Mr = 66 kDa), was activated by 0.5-10 mM taurocholate but was strongly inhibited by higher levels of taurocholate, which activate pancreatic CEH. Whereas bile salts are known to induce formation of a hexamer of pancreatic CEH, in the current study, 0.5 mM taurocholate dissociated a multimeric form of liver CEH to monomer. Liver CEH did not coelute with pancreatic CEH from cation exchange and chromatofocusing columns, exhibited no immunoreactivity with anti-rat pancreatic CEH IgG in Western blots, was not inhibited by anti-rat pancreatic CEH IgG and had a different amino acid composition from pancreatic CEH. In contrast to liver CEH, which is known to be activated by protein kinases A and C, pancreatic CEH was unaffected by cofactors for protein kinase A and was inhibited by cofactors for protein kinase C.
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PMID:Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme. 179

The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.
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PMID:Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity. 190 Aug 38

Infection of mouse L cells with mengovirus resulted in the activation of a protein kinase (PK) that selectively phosphorylated the small, 38,000-molecular-weight alpha subunit of eucaryotic initiation factor 2 (eIF-2) in vitro. The mengovirus-activated kinase was detected in vitro approximately 3 h after virus adsorption. The ratio of phosphorylated to unphosphorylated eIF-2 also increased in vivo between 3 and 7 h after adsorption. The virus-activated kinase fractionated with the ribosomal pellet and had a high affinity for DEAE-cellulose and Mono Q ion-exchange columns. Gel electrophoresis of the kinase activity eluting from the Mono Q column and silver staining of the gel revealed only one protein band with a molecular mass of 70 kilodaltons. The optimal assay conditions for the mengovirus-activated kinase paralleled those of the double-stranded RNA-activated PK (dsRNA-PK). Lysates from infected cells contained elements capable of activating partially purified dsRNA-PK. These elements were identified as double-stranded RNA by their sensitivity to double-stranded RNase. The phosphorylation of the alpha subunit of eIF-2 coincided with the synthesis of dsRNA in infected cells, suggesting that the mengovirus-activated kinase is the dsRNA-PK. The phosphorylation of the alpha subunit of eIF-2 correlated with the global inhibition of protein synthesis that occurs at late times after infection.
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PMID:The alpha subunit of eucaryotic initiation factor 2 is phosphorylated in mengovirus-infected mouse L cells. 216 23


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