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)

Particulate fractions prepared from concanavalin A-activated murine T lymphocytes contain an endogenous protein kinase that phosphorylates an endogenous protein substrate of Mr 112 000. The phosphorylation of 112 kDa protein is greatly reduced or absent in unstimulated T cells. Phosphoamino acid analysis indicates that 112 kDa protein is labeled on a serine. Add-back experiments using purified protein kinases indicate that 112 kDa protein serves as a substrate for casein kinase II. Phosphorylation of 112 kDa protein by the endogenous kinase is inhibited by heparin, a known casein kinase II inhibitor. The site or sites modified by the endogenous kinase and exogenous casein kinase II appear identical by peptide-mapping experiments. A time-course of the appearance of phosphorylated 112 kDa protein following stimulation with concanavalin A, measured in the presence or absence of added casein kinase II, suggests that 112 kDa protein is induced in activated T cells. Subcellular localization studies suggest that 112 kDa protein is a nuclear protein. Silver-binding and purification studies suggest that 112 kDa protein is of the nucleolar organizing region.
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PMID:Induction of a substrate for casein kinase II during lymphocyte mitogenesis. 660 22

The biological activities of cyclin-dependent, proline-directed protein kinases (PDPKs) are highly regulated by a complex series of protein phosphorylation/dephosphorylation reactions involving both catalytic and regulatory subunits. In this paper we report on the enzymatic activation of p34cdc2/p58Cyclin A PDPK by a protein kinase present in human cells that targets threonine-161 of Cdc2. An assay for this Cdc2 kinase-kinase (PK161) was developed and specific enzyme activity was detected in a variety of mammalian cells and tissues. PK161 activity was rapidly stimulated by epidermal growth factor in human A431 epidermoid carcinoma cells. The development of an assay selective for PK161 phosphotransferase activity afforded the partial purification of the enzyme from human Wilms' tumors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of highly purified enzyme preparations revealed the presence of phosphoproteins migrating at approximately 42-44 and approximately 95 kDa, respectively, which correlated with enzyme activity upon fast-protein liquid chromatography gel permeation chromatography. Further purification was accomplished by immobilized peptide substrate affinity chromatography. The ability of PK161 to phosphorylate and activate p34cdc2/p58Cyclin A PDPK was confirmed by the use of purified recombinant subunits. Polyclonal antibodies directed against the Xenopus MO15 gene product (a putative Cdc2-activating kinase) cross-reacted with the purified 42- to 44-kDa phosphoprotein, thus identifying the catalytic subunit of human PK161 as a human homologue of Xenopus p40MO15. Subsequent immunoprecipitation experiments with metabolically labeled A431 cells identified a approximately 95-kDa phosphoprotein that coprecipitated with the approximately 42-kDa catalytic subunit. Taken together, these findings identify a human Cdc2-activating kinase as a growth factor-responsive enzyme system that may participate in the acute activation of cyclin-dependent protein kinases observed in mammalian somatic cells.
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PMID:Identification, assay, and purification of a Cdc2-activating threonine-161 protein kinase from human cells. 794 11

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by a dysfunctional cellular immune response. We have previously identified a metabolic disorder of the adenylate cyclase/cAMP/protein kinase A (AC/cAMP/PKA) pathway characterized by impaired cAMP-inducible, PKA-catalyzed protein phosphorylation in intact T lymphocytes from subjects with severe SLE disease activity. Because this metabolic disorder may contribute to abnormal T cell immune effector functions, we tested the hypothesis that impaired PKA-dependent protein phosphorylation is the result of a PKA isozyme deficiency in SLE T lymphocytes. Compared with healthy and rheumatoid arthritis (RA) controls, subjects with severe SLE activity exhibited reduced PKA-catalyzed phosphorylation of proteins in the T lymphocyte plasma membrane where the type I isozyme of PKA (PKA-I) is predominantly localized. Both silver staining and biosynthetic labeling of membrane-associated proteins with [35S]methionine demonstrated that reduced protein phosphorylation was not due to either an altered distribution of or absence of proteins. Moreover, phosphorylation of SLE membrane-associated proteins with the PKA catalytic (C) subunit showed a similar distribution and extent of phosphorylation compared with membrane proteins from healthy T cells, suggesting that SLE T cell membrane proteins could be phosphorylated. Sequential column chromatography of the type I and type II isozymes of PKA (PKA-I, PKA-II) demonstrated a deficiency of PKA-I isozyme activity. Compared with a ratio of PKA-I to PKA-II activity of 4.2:1 in healthy T cells, the activity ratio in T cells from subjects with severe SLE disease activity was 0.99:1 (P = 0.01, SLE versus healthy controls for PKA-I). The deficient PKA-I activity was associated with a significant increase of free C-subunit activity (P = 0.04, SLE versus healthy controls for C-subunit). T cells from subjects with mild/moderate SLE disease activity also exhibited diminished PKA-I activity, yielding a ratio of PKA-I to PKA-II activity of 2.4:1. By contrast, T cells from RA controls possessed increased PKA-I, PKA-II, and free C-subunit activities compared with healthy controls, resulting in a ratio of PKA-I to PKA-II activity of 3.6:1. We conclude that the reduced PKA-catalyzed protein phosphorylation in the plasma membrane of SLE T cells is the result of deficient PKA-I isozyme activity. This is the first identification of a deficiency of PKA activity in SLE T lymphocytes; the deficiency, resulting in diminished protein phosphorylation, may alter cellular homeostasis, contributing to the cellular immune dysfunctions observed in SLE.
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PMID:Deficient type I protein kinase A isozyme activity in systemic lupus erythematosus T lymphocytes. 804 Feb 83

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.
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PMID:Purification and characterization of casein kinase I from broccoli. 832 68

Gene UL13 of herpes simplex virus type 1 (HSV-1) has previously been proposed to encode a protein kinase. An HSV-1 mutant with UL13 inactivated by insertion of the Escherichia coli lacZ gene was constructed. This UL13-lacZ mutant was found to grow to near wild-type (wt) titres in tissue culture. Comparison of silver-stained SDS-PAGE profiles of wt and UL13-lacZ virions demonstrated that the UL13 protein is a readily detectable component of wt virions, located in the tegument and probably equivalent to the previously described species VP18.8. Studies of in vitro phosphorylation with nuclear extracts of virus-infected cells and with detergent-treated virions showed that the UL13 protein is involved in phosphorylation of the tegument protein VP22. Extracts of cells engineered to express UL13, and infected with UL13-lacZ virus, were also capable of VP22 phosphorylation.
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PMID:A mutant of herpes simplex virus type 1 in which the UL13 protein kinase gene is disrupted. 838 74

Acetoxymethyl esters of alkyl or aryl phosphates can be prepared by reacting their trialkylammonium or silver salts with acetoxymethyl bromide. Because acetoxymethyl esters are rapidly cleaved intracellularly, they facilitate the delivery of organophosphates into the cytoplasm without puncturing or disruption of the plasma membrane. In addition, acylation of free hydroxyls, for example with butyryl groups, is useful both for synthetic convenience and increased hydrophobicity of the permeant derivatives. The highly polar intracellular messengers cAMP and cGMP were thus converted into uncharged membrane-permeant derivatives. Extracellularly applied N6,2'-O-dibutyryl cAMP acetoxymethyl ester (Bt2cAMP/AM) is shown to simulate intracellular cAMP in three model systems, namely dissociation of cAMP-dependent protein kinase in fibroblasts, activation of Cl- secretion of monolayers of the human colon epithelial cell line T84, and dispersion of pigment granules in angel fish melanophores. Bt2cAMP/AM is effective at concentrations two or three orders of magnitude less than those required for commonly used membrane-permeant cAMP derivatives such as Bt2cAMP, 8-Br-cAMP, and 8-pCPT-cAMP lacking the acetoxymethyl ester. This methodology should be of general utility for the intracellular delivery of phosphate-containing second messengers.
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PMID:Acetoxymethyl esters of phosphates, enhancement of the permeability and potency of cAMP. 838 7

In cells, insulin stimulates autophosphorylation of the insulin receptor on tyrosine and its phosphorylation on serine and threonine by poorly characterized kinases. Here we describe methods for the purification of an insulin-stimulated insulin receptor serine kinase from human placenta and rat liver by sequential chromatography of solubilized membranes on wheat germ agglutinin-agarose, Mono Q, phenyl-Superose, and Superose 12. On silver-stained SDS-polyacrylamide gels, the resulting kinase was homogeneous (human) or near-homogeneous (rat) and had an apparent M(r) of 40000. The apparent M(r) determined by gel filtration was also 40000, suggesting that the kinase exists as a monomer. The kinase could be reconstituted back to the insulin receptor stripped of the kinase to yield a high stoichiometry of serine phosphorylation of the insulin receptor in the presence of insulin (0.75 +/- 0.15 mol/mol of beta-subunit, mean +/- SEM, n = 3). The activity of the reconstituted kinase toward the insulin receptor was insulin-regulated, being stimulated > 5-fold by insulin. Insulin increased the catalytic activity of the reconstituted kinase. The purified kinase specifically phosphorylated serine 1078 of the insulin receptor, a major site of insulin-stimulated serine phosphorylation in vivo, showing that the purified kinase phosphorylated a physiologically relevant site on the insulin receptor. Phosphorylation of serine 1078 of the insulin receptor to high stoichiometry by the kinase did not affect insulin-stimulated exogenous protein tyrosine kinase activity of the insulin receptor. Similarly, insulin receptor phosphorylated with or without the purified kinase exhibited the same levels of tyrosine autophosphorylation and of the tyrosine kinase-activating tris-phosphorylated kinase domain species. Properties of the kinase distinguished it from kinases known to act on the insulin receptor and other kinases that are insulin-stimulated, indicating that the kinase is a novel entity. The serine kinase underwent autophosphorylation on serine and immunoprecipitated with the insulin receptor. The availability of the purified kinase should facilitate cloning of the kinase, determination of the mechanism of activation of the kinase, and study of the wider potential role of the kinase in insulin signalling, and the ability to be able to phosphorylate serine 1078 to high stoichiometry should facilitate further studies into the function of this serine phosphorylation site.
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PMID:Purification and characterization of an insulin-stimulated insulin receptor serine kinase. 891 21

An ultrastructural examination of mRNA within adult rat CA1 hippocampal dendrites was conducted using two different methods. The messages for the alpha and beta forms of the calcium-calmodulin-dependent protein kinase II were localized in ultracryosections using silver-intensified gold detection of isoform-specific oligonucleotide probes. Labeling for both isoforms was observed within the cell bodies and proximal dendrites of pyramidal neurons, but only the alpha form was observed in more distal dendrites. Unfortunately, the morphological preservation of the tissue was not sufficient to determine the localization of labeling relative to subcellular features such as dendritic spines. To address this issue, a preembedding peroxidase-based method was developed, resulting in better preservation of the neuropil. The total population of polyadenylated [poly(A)] mRNA was localized in hippocampus using a biotinylated poly(dT) probe. Poly(A) mRNA was present in the nucleus and throughout the cell body of all hippocampal cells and within isolated dendrites and glial processes within the neuropil. Within pyramidal neurons, labeling was distributed in a longitudinal pattern in proximal apical dendrites. More distally, the amount of labeling diminished, and smaller foci of labeling were observed, particularly near the plasma membrane. Concentrated labeling was present at the base of dendritic spines and, less frequently, near synapses onto the dendritic shaft. These results suggest that dendritic mRNA is found in the vicinity of postsynaptic sites and provide additional evidence that local protein synthesis may play an important role in establishing and maintaining synaptic specializations.
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PMID:Ultrastructural localization of dendritic messenger RNA in adult rat hippocampus. 892 99

Protein kinase from Streptomyces lincolnensis was purified nearly to homogeneity using a high performance liquid chromatography (HPLC) and a Pharmacia FPLC system. The procedure used employed column chromatography on DE-53, followed by FPLC affinity chromatography with serine- or threonine-Sepharose (prepared as described in this paper) and gel filtration using a Superose 12 or TSK G3000SW column. Starting with 3.5 g of mycelial proteins, approximately 1 mg of pure enzyme was obtained. The procedure is simple and highly reproducible. The protein kinase thus obtained was nearly pure by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein kinase phosphorylated substrate proteins at the seryl residues.
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PMID:Improved method for rapid purification of protein kinase from streptomycetes. 892 41

Bovine Trp-tRNA synthetase is a dimer with subunit molecular mass of 60 kDa (p60) which catalyzes ATP-dependent formation of tryptophanyl-tRNA. Evidence is presented that Trp-tRNA synthetase whose homogeneity had been proven by SDS/PAGE and silver staining of the gel is autophosphorylated in vitro. Anti-(Trp-tRNA synthetase) antibodies, whose specificity was verified by using a combination of different approaches, were able to effectively inhibit and immunoprecipitate the Trp-tRNA-synthetase-associated kinase activity. The two-dimensional tryptic phosphopeptide map of autophosphorylated p60 Trp-tRNA synthetase was found to be similar to that of its major 40-kDa degradation fragment bearing resemblance to previously demonstrated unlabeled peptide patterns of the Trp-tRNA synthetase forms. Trp-tRNA synthetase which had undergone denaturation during SDS/PAGE, regained serine/threonine specific protein kinase activity (PK 60) after guanidine treatment. Trp-tRNA synthetase induced phosphorylation of specific substrate such as 100-kDa protein in non-immune but not in anti-(Trp-tRNA synthetase) sera which distinguishes Trp-tRNA-synthetase-associated kinase from other protein kinases. Sequence analysis permitted the identification of regions of bovine Trp-tRNA synthetase sharing similarity with the catalytic domains of known protein kinases. These findings suggest that PK 60 and Trp-tRNA synthetase (p60) are either closely related or identical.
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PMID:A mammalian tryptophanyl-tRNA synthetase is associated with protein kinase activity. 910 48


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