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 glucocorticoid hormone receptor (92 kDa), purified 9000-fold from rat liver cytosol by steroid affinity chromatography and DEAE-Sephacel chromatography, was assayed for the presence of protein kinase activity by incubations with [gamma-32P]ATP and the photoaffinity label 8-azido-[gamma-32P]ATP. Control preparations isolated by affinity chromatography in the presence of excess steroid to prevent the receptor from binding to the affinity matrix were assayed for kinase activity in parallel. The receptor was not labeled by the photoaffinity label under photoactivation conditions in the presence of Ca2+ or Mg2+. A Mg2+-dependent protein kinase (48 kDa) that could be photoaffinity labeled with 8-azido-ATP copurified with the receptor. This kinase was also present in control preparations. The kinase could phosphorylate several minor contaminants present in the receptor preparation, including a protein (or proteins) of similar molecular weight to the receptor. The phosphorylation of 90-92-kDa proteins was independent of the state of transformation or steroid-binding activity of the receptor. These experiments provide direct evidence that neither the glucocorticoid receptor nor the 90-92-kDa non-steroid-binding protein associated with the molybdate-stabilized glucocorticoid receptor possesses intrinsic Ca2+- or Mg2+-dependent protein kinase activity.
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PMID:Rat liver glucocorticoid receptor isolated by affinity chromatography is not a Mg2+- or Ca2+-dependent protein kinase. 380 33

Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase. 396 81

The cytoplasmic glucocorticoid receptor of X. laevis liver has a high affinity for [3H]dexamethasone (Kd, 0.3 x 10(-8) M), and its binding specificity for a variety of steroids is similar to that found for mammalian glucocorticoid receptors. The ability of this receptor to bind [3H]-dexamethasone is stable at 0 degrees C but is rapidly lost at 10 and 20 degrees C. Alkaline phosphatase increases, whereas molybdate and tungstate decrease, the rate at which the binding activity is lost. These results are consistent with the loss of binding activity being due to dephosphorylation of the receptor. Binding of [3H]dexamethasone to the receptor does not alter the rate at which the binding activity is lost but does increase the stabilizing effect of molybdate. 100 mM molybdate lowers the apparent affinity of the receptor for [3H]dexamethasone, suggesting that molybdate can interact with the X. laevis glucocorticoid receptor. Addition of UTP, but not ATP, GTP or CTP, reactivates the receptor-binding activity, which indicates that the receptor may be phosphorylated by a UTP-dependent protein kinase.
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PMID:Glucocorticoid receptor of X. laevis: possible effect of phosphorylation on hormone binding. 628 68

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5 m and DEAE-Sephacel. The purified GR sedimented in the 9-10S region in 5-20% sucrose gradients containing 0.10M KCl and 20mM Na2MoO4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 +/- 2,000. Affinity labeling of GR with [3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50 microM [gamma-32P]-ATP and divalent cations. Radioactive phosphate from [gamma-32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg2+ and Ca2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.
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PMID:Protein kinase activity of purified rat liver glucocorticoid receptor. 651 35

Glucocorticoid receptor was purified from rat liver cytosol using a dexamethasone affinity column. The receptor thus purified displayed a single protein band when subjected to SDS-polyacrylamide gel electrophoresis. It had a molecular weight of 90,000 which was consistent with the reported value for other glucocorticoid receptor preparations. Incubation of the purified preparation with [gamma 32P] ATP and Mg2+ resulted in transfer of [32P] to the receptor protein indicating the presence of an endogeneous protein kinase activity capable of phosphorylating the receptor molecule. Phosphorylation of the glucocorticoid receptor by the endogenous protein kinase might serve as a direct mechanism for the activation of the receptor.
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PMID:Phosphorylation of purified glucocorticoid receptor from rat liver by an endogenous protein kinase. 671 52

Prorenin (Pro) is synthesized in a number of human utero-placental tissues, including chorion, decidua, villous placenta and probably mesenchymal cells. The release of Pro from these extra-renal tissues follows new protein synthesis and appears to utilize the constitutive secretory pathway. Unlike processing in the kidney, very little of the Pro is subsequently cleaved to the smaller product (active renin). Primary signals which regulate Pro include protein hormones and peptides (relaxin, endothelin, hCG), amines (epinephrine, norepinephrine, and related beta adrenergic agents), and eicosanoids. These agents increase the mRNA for prorenin at a time before peak secretory effects are noted. Other extracellular signals have negative regulatory effects. These include angiotensin, endotoxin and cytokines (TNF-alpha and interleukin-1 B). There is also evidence that glucocorticoid receptor activation has an inhibitory effects on Pro release in placenta. Second messengers involved in the regulation of Pro include cyclic AMP and protein kinase A (PKA), protein kinase C (PKC), and calcium. The possible biological effect(s) of the extracellular Pro are unknown but may be due to direct generation of angiotensin I. Since angiotensin-peptides have a number of trophic effect on both vascular and non-vascular tissues, regulation of utero-placental Pro by autocrine, paracrine or endocrine signalling may be critical in normal fetal and/or placental development.
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PMID:Regulation of utero-placental prorenin. 748 44

The Golli-mbp gene complex contains two overlapping transcription units with two distinct promoters, of which the downstream (myelin basic protein [mbp]) promoter is more frequently used. A previous comparison of the downstream promoter sequences from shark and mouse allowed the identification of two DNA sequences called the boxes I and II and the wobble zone. The boxes I and II sequence is a composite cis-acting motif that is thought to be involved in the regulation of the downstream promoter. It contains sequences similar to T-antigen, MyoD/E2A, and glucocorticoid receptor-binding sites. The wobble zone codes for an exon (5a in the nomenclature of Campagnoni et al., 1993) that is included in messenger RNAs transcribed from the upstream promoter. The polypeptides encoded by this exon from shark and mouse are 86 and 84 amino acids long, respectively. These polypeptides are overall 59% identical and include a region (residues 41-75 in shark and 39-73 in mouse) that is 89% identical between the two species. A primary sequence analysis showed that each of these polypeptides contains an N-glycosylation site, phosphorylation sites for Ca2+/calmodulin-dependent protein kinase, protein kinase C and casein kinase II, and partial ATP- and GTP-binding sites. The shark polypeptide also contains a phosphorylation site for proline-directed protein kinase. These observations are consistent with the notion that the intricate structure and regulation of the Golli-mbp gene complex arose during vertebrate evolution within a common ancestor to sharks and mammals.
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PMID:The structural complexities of the myelin basic protein gene from mouse are also present in shark. 752 2

The rat tyrosine aminotransferase gene is a model system to study transcriptional regulation by glucocorticoid hormones. We analyzed transcription factor binding to the tyrosine aminotransferase gene glucocorticoid-responsive unit (GRU) at kb -2.5, using in vivo footprinting studies with both dimethyl sulfate and DNase I. At this GRU, glucocorticoid activation triggers a disruption of the nucleosomal structure. We show here that various regulatory pathways affect transcription factor binding to this GRU. The binding differs in two closely related glucocorticoid-responsive hepatoma cell lines. In line H4II, glucocorticoid induction promotes the recruitment of hepatocyte nuclear factor 3 (HNF3), presumably through the nucleosomal disruption. However, the footprint of the glucocorticoid receptor (GR) is not visible, even though a regular but transient interaction of the GR is necessary to maintain HNF3 binding. In contrast, in line FTO2B, HNF3 binds to the GRU in the absence of glucocorticoids and nucleosomal disruption, showing that a "closed" chromatin conformation does not repress the binding of certain transcription factors in a uniform manner. In FTO2B cells, the footprint of the GR is detectable, but this requires the activation of protein kinase A. In addition, protein kinase A stimulation also improves the recruitment of HNF3 independently of glucocorticoids and enhances the glucocorticoid response mediated by this GRU in an HNF3-dependent manner. In conclusion, the differences in the behavior of this regulatory sequence in the two cell lines show that various regulatory pathways are integrated at this GRU through modulation of interrelated events: transcription factor binding to DNA and nucleosomal disruption.
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PMID:Glucocorticoids and protein kinase A coordinately modulate transcription factor recruitment at a glucocorticoid-responsive unit. 756 84

We have stably introduced expression vectors for the glucocorticoid receptor and a sensitive, hormone-responsive reporter (mouse mammary tumor virus-luciferase) into a human breast carcinoma-derived cell line. Employing this cell line, we have conducted a detailed examination of the induction of glucocorticoid-regulated genes and the phosphorylation of glucocorticoid receptor following pharmacologic manipulation of cell signaling pathways. The hormone response can be enhanced from 2 to 10-fold by activators of protein kinase A, protein kinase C, and inhibitors of protein phosphatase. Forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP), but not BrcGMP, enhance the hormone effect, yet surprisingly, phosphodiesterase inhibitors, isobutylmethylxanthine and Ro20-1724, strongly inhibit hormone-mediated induction of the reporter gene. These treatments do not alter cellular receptor content, dexamethasone binding, nor hormone-mediated receptor down-regulation. Tryptic peptide analysis of 32P-labeled receptor reveals that neither BrcAMP, isobutylmethylxanthine, nor the tumor promoter and protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate, detectably alter the state of glucocorticoid receptor phosphorylation. The only agent which alters receptor phosphorylation is the protein phosphatase inhibitor okadaic acid, but only at concentrations higher than required for maximum effects on glucocorticoid receptor transactivation. We propose that these effectors do not modify receptor directly but alter its interaction with transcription complexes.
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PMID:Modulation of cell signaling pathways can enhance or impair glucocorticoid-induced gene expression without altering the state of receptor phosphorylation. 769 81

Activation of protein kinase A potentiates the transcriptional response mediated by the glucocorticoid receptor in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs in responsive fibroblasts and in mammary carcinoma cells. This potentiation is ligand-dependent and occurs without detectable change in the phosphorylation of receptor. The transcriptional response to glucocorticoid or progestin agonists can be blocked by potent antagonists like RU 486. However, upon activation of protein kinase A, the antagonist action of RU 486 on both receptors is blunted. Indeed, RU 486 can itself activate transcription of a hormone-responsive promoter. The conditional agonist activity is observed with type II antagonists, those which recapitulate many of the early steps of ligand-dependent receptor activation, but not type I antagonists, which do not. These studies have now been extended to antimineralocorticoids. In COS-1 cells transfected with a mineralocorticoid receptor expression vector, treatment with 8-BromocAMP potentiates the response to the agonist aldosterone and elicits additional agonist activity in mineralocorticoid antagonists. A model is proposed wherein type II antagonist-receptor complexes occupy receptor binding sites on the genome. The antagonist, however, fails to promote a receptor conformation that can interact productively with a coactivator mediating the communication between receptor and the basal transcription apparatus. Activation of protein kinase A results in the recruitment or activation of a coactivator that permits recovery of receptor-mediated activation function.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The two faces of a steroid antagonist: when an antagonist isn't. 779 25


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