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)

Heme-hemopexin or cobalt protoporphyrin (CoPP)-hemopexin (a model ligand for hemopexin receptor occupancy) is shown to increase transcription of the metallothionein-1 (MT-1) gene by activation of a signaling pathway. Promoter deletion analysis followed by transient transfection assays show that 110 base pairs (-153 to -43) of 5'-flanking region of the murine MT-1 promoter are sufficient for increasing transcription in response to heme-hemopexin or to CoPP-hemopexin in mouse hepatoma cells. The protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7), prevented the increase in MT-1 transcription by heme-hemopexin, CoPP-hemopexin, or phorbol 12-myristate 13-acetate, but the protein kinase A inhibitor, HA1004, was without effect. N-Acetylcysteine (NAC) and glutathione, as well as superoxide dismutase and catalase, inhibited both the increase in endogenous MT-1 mRNA and the activation of reporter gene activity by heme-hemopexin, CoPP-hemopexin, and phorbol 12-myristate 13-acetate. In sum, these data suggest that reactive oxygen intermediates are generated by heme-hemopexin via events associated with receptor binding, including protein kinase C activation. Induction of heme oxygenase-1 expression, in contrast to MT-1, is significantly less sensitive to NAC. Deletion and mutation analyses of the MT-1 proximal promoter revealed that the sequence 5'-GTGACTATGC-3' (from -98 to -89 base pairs) is, in part, responsible for the hemopexin-mediated regulation of MT-1 which is inhibited by H7. Regulation via this element is also induced by H2O2 showing that it is an antioxidant response element. Heme itself acts via more distal elements on the MT-1 promoter. In contrast to NAC and glutathione, diethyl dithiocarbamate and pyrrolidine dithiocarbamate, which inactivate reactive oxygen intermediates and chelate Zn(II), synergistically augment the induction of MT-1 mRNA levels and reporter gene activity in response to heme-hemopexin via the antioxidant response element by both metal-responsive element-dependent and -independent mechanisms.
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PMID:Mechanism of metallothionein gene regulation by heme-hemopexin. Roles of protein kinase C, reactive oxygen species, and cis-acting elements. 759 95

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.
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PMID:Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein. 785 55

We have recently shown, using antisense strategy, that the RII beta regulatory subunit of cAMP-dependent protein kinase is essential for cAMP-induced growth inhibition and differentiation of HL-60 human leukemia cells. We constructed a retroviral vector for RII beta (MT-RII beta) by inserting human RII beta complementary DNA into the OT1521 retroviral vector plasmid that contains an internal mouse metallothionein-1 promoter and a neomycin resistance gene. The PA317 packaging cell line was then transfected with MT-RII beta plasmid to produce the amphotrophic stock of MT-RII beta retroviral vector. The infection with MT-RII beta and treatment with CdCl2 brought about growth arrest in HL-60 human leukemia and Ki-ras-transformed NIH 3T3 clone DT cells in monolayer culture with no sign of toxicity. The growth inhibition correlated with the expression of RII beta and accompanied changes in cell morphology; cells became flat, exhibiting enlarged cytoplasm. The growth of these cells in semisolid medium (anchorage-independent growth) was almost completely suppressed. In contrast, overexpression of the RI alpha subunit of protein kinase enhanced the cell proliferation in DT cells. The MT-RII beta-infected cells exhibited an increased sensitivity toward treatment with cAMP analogues, such as 8-Cl-cAMP and N6-benzyl-cAMP, as compared with the parental noninfected cells. In MT-RII beta HL-60 cells, N6-benzyl-cAMP treatment greatly enhanced the expression of monocytic surface markers. These results suggest that the RII beta cAMP receptor, by binding to its ligand, cAMP, acts as a tumor suppressor protein exerting growth inhibition, differentiation, and reverse transformation.
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PMID:Retroviral vector-mediated overexpression of the RII beta subunit of the cAMP-dependent protein kinase induces differentiation in human leukemia cells and reverts the transformed phenotype of mouse fibroblasts. 794 90

Superior cervical ganglion neurons from neonatal rats are dependent on nerve growth factor for their survival both in vivo and in vitro. In culture this requirement can be largely replaced by cAMP or its analogues. Since activation of protein kinase A by cAMP is likely to be the pathway by which it exerts its survival-promoting effect, we have tested the feasibility of using herpes simplex virus (HSV) as a vector for expressing survival-promoting genes in neurons by cloning the catalytic subunit of the cAMP-dependent protein kinase (PKAcat) with a metallothionein gene promoter into the HSV thymidine kinase gene by homologous recombination. About 95% of the neurons became infected using 2.5 p.f.u. per cell. When this construct was used to express PKAcat in superior cervical ganglion neurons, in the presence of nerve growth factor (NGF) increases of 1.9- to 2.4-fold in PKA activity were found 8-10 h after infection; levels remained elevated (1.4- to 2.1-fold) up to 18 h, returning to basal by 24 h. After infection in the absence of NGF, cumulative activity over 24 h was approximately 3.5-fold lower in the first 24 h. Although the level of the inhibitory regulatory subunit type I was raised by 18 h, this is unlikely to completely explain the transient activity of PKAcat. When neurons were induced to express maximum PKAcat levels in the presence of NGF and then deprived of NGF, survival was extended by up to 2 days, demonstrating a direct role for PKA in promoting survival. By this time, some neurite degeneration was beginning which appeared to be partly due to toxic effects of the virus. However, replenishment with NGF supported further survival, showing that at this time the neurons were still viable. Similar rates of survival were obtained using a tsK-based PKAcat vector, but no significant survival was obtained with parental HSV or tsK virus strains. These data demonstrate the feasibility, and highlight some of the problems, of using HSV-based vectors as tools for expressing functional survival proteins in sympathetic neurons.
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PMID:Expression of the cyclic AMP-dependent protein kinase (PKA) catalytic subunit from a herpes simplex virus vector extends the survival of rat sympathetic neurons in the absence of NGF. 798 74

The activity of several proteins involved in the development of antitumor drug resistance is regulated by protein phosphorylation. These proteins include the mdr-1-encoded P-glycoprotein (Pgp) and topoisomerase II (topo II). The corresponding evidence is reviewed and attempts to modulate multidrug resistance (MDR) by protein kinase C inhibitors are described. The expression of several proteins which are essential in drug resistance is regulated at the transcriptional level, involving protein phosphorylation by members of the protein kinase C (PKC) family, casein kinase II (CKII), and others. These proteins include mdr-1-encoded P-glycoprotein, metallothionein, glutathione S-transferase (GST), dTMP synthase, and the proteins Fos and Jun. The corresponding genes are under positive regulation of ras, which in turn requires the activation of a protein kinase cascade for its function. Protein kinases are therefore potentially useful targets in reducing the expression of proteins involved in the development of multifactorial drug resistance caused by the expression of transforming ras-genes. Attempts to inhibit the ras-induced fos expression by an inhibitor of protein kinase C (ilmofosine) are described. Protein kinase inhibitors are also able to synergistically enhance the cytotoxicity of cis-platinum, which is discussed as resulting from a reduction of PKC-dependent fos expression.
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PMID:Role of protein kinases in antitumor drug resistance. 806 Nov 7

Engagement of the T cell receptor/CD3 complex activates the serine/threonine kinase, Raf-1, but the physiologic consequences of its activation have not been determined. The effects of Raf-1 on interleukin 2 (IL2) production in T cells were examined using activated and inhibitory forms of Raf-1. A truncated active form of Raf-1 was expressed constitutively from the metallothionein promoter in a malignant T cell line, Jurkat. Treatment of the cells with zinc and cadmium greatly increased active Raf-1 expression. This increase in Raf-1 expression allowed antibodies to CD3 and to CD28 to stimulate IL2 production in the absence of phorbol myristate acetate (PMA) and enhanced IL2 production stimulated by these antibodies in the presence of PMA. The action of active Raf-1 was to increase IL2 gene transcription as it enhanced transcription of a reporter gene linked to IL2 promoter. Finally, the dominant negative form of Raf-1 inhibited transcription directed by the IL2 promoter that was induced by the mitogen phytohemagglutinin (PHA) and PMA. We conclude that Raf-1 activity is necessary for IL2 gene transcription and secretion. These data indicate a role for Raf-1 in the immune response.
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PMID:Raf-1 is required for T cell IL2 production. 822 46

The true rate-limiting step in steroidogenesis is the delivery of cholesterol to the inner mitochondrial membrane where it is converted to pregnenolone by the cholesterol side-chain cleavage complex. This process is known to require de novo protein synthesis. We have previously described the synthesis of a family of 37, 32, and 30 kilodalton mitochondrial proteins in response to hormone stimulation in MA-10 mouse Leydig tumor cells and have proposed that these proteins are involved in the acute regulation of steroidogenesis. In this study we have used two subclones of MA-10 cells to further demonstrate the correlation between the quantity of these proteins and the production of steroids in response to hormone treatment. One of these, designated MA-10(K3), has been transfected with a mutant gene of the type 1 regulatory subunit of the cAMP-dependent protein kinase under the control of a metallothionein promoter, whereas the other, designated MA-10(P+29), is a constitutive overproducer of a cAMP-phosphodiesterase (PDE). MA-10 parent cells designated (P), produce large amounts of progesterone in response to LH, human CG, and (Bu)2cAMP. The MA-10(K3) cells, on the other hand, whereas significantly higher than controls, produce much less steroid than the parent cells in response to hormone stimulation. Activation of the mutant gene with Zn+2 results in yet a further decrease in the amount of steroid produced. The MA-10(P+29) cells display greatly reduced progesterone production when stimulated with LH, because of the presence of high amounts of PDE, but return to maximally stimulated levels when a PDE inhibitor is present. Quantitation of the synthesis of the mitochondrial proteins described above in MA-10(K3) cells in the presence and absence of Zn+2 and in MA-10(P+29) cells in the presence and absence of PDE inhibitor clearly demonstrate that the amount of the 30 kilodalton mitochondrial proteins present in these cells closely parallels that of progesterone production. The high degree of correlation between the appearance and quantity of these mitochondrial proteins and the production of steroids make them strong candidates for the putative proteins involved in acute regulation of steroidogenesis.
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PMID:The use of genetic manipulation of MA-10 Leydig tumor cells to demonstrate the role of mitochondrial proteins in the acute regulation of steroidogenesis. 838 3

The roles of calcium and/or of the other cellular transduction pathways, and of nitric oxide (NO) on the induction of metallothionein (MT) mRNA by lipopolysaccharide (LPS) has been studied in rat primary cell culture, using inhibitors of protein kinase pathways (H-7, W-7 and TMB-8) and NO production inhibitors (L-NAME, PTIO). LPS exposure led to a rapid increase of MT-mRNA and a peak level revealed 2.5-fold induction as compared to control for 6h incubation at a dose of 3.0 mg/L. A dose of 5.0 and 10.0 mg/L of LPS also provided the same level of MT-mRNA induction. The inhibition of MT induction by LPS was observed with L-NAME, PTIO, but not H-7, W-7. These findings indicate that the alteration of cellular calcium concentration and distribution does not relate to the induction of MT-mRNA by LPS in hepatocytes and that protein kinase C and calmodulin dependent protein kinase pathways have not contributed to MT-mRNA induction by LPS. Finally, the present results show that NO plays an important role in MT induction by LPS.
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PMID:Nitric oxide mediated metallothionein induction by lipopolysaccharide. 858 48

Growth hormone (GH) secretion is under the control of the hypothalamic hormones GH-releasing hormone (GHRH) and somatostatin (SRIF), and is regulated by feedback effects of GH and insulin-like growth factor (IGF-1). GHRH and SRIF act on somatotropes by binding to G-protein-coupled receptors. GHRH activates the stimulatory G protein (Gs), leading primarily to activation of adenylyl cyclase and protein kinase A. SRIF activates the inhibitory G protein (Gi). Several animal models enable the study of various disorders of GH secretion in vivo. Genetic models of impaired GH secretion include the little (lit) mouse, the dwarf (dw) rat, the fatty (fa) rat, and the high-growth (hg) mouse. Transgenic models of impaired and excessive GH secretion, respectively, include the tyrosine hydroxylase-human GH (TH-hGH) transgenic mouse and the metallothionein-human GHRH transgenic mouse. These models encompass a wide spectrum of disorders of GH secretion, involving defects of hypothalamic regulation, feedback control at the pituitary level, or the mechanism of GHRH action in the somatotrope. They may provide insights into our understanding of human GH secretory disorders.
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PMID:New insights into the regulation of somatotrope function using genetic and transgenic models. 876 67

The sequential transcriptional activation of cyclins, the regulatory subunits of cell cycle specific kinases, regulates progress through the cell cycle. In mitogen-stimulated cells cyclin D1 induction in early G1 is followed by induction of cyclin E, activation of the cyclin-dependent kinase Cdk2, and hyperphosphorylation of the retinoblastoma gene product (pRB) in mid-to-late G1 phase. T-47D breast cancer cells expressing cyclin D1 under the control of a metal-responsive metallothionein promoter were used to determine whether Cdk2 activation and pRB hyperphosphorylation are consequences of cyclin D1 induction. A 4-5-fold increase in cyclin D1 protein abundance was followed by approximately 2-fold increases in cyclin E protein abundance and Cdk2 activity and by hyperphosphorylation of pRB. These responses were apparent approximately 3 h after the increase in cyclin D1 protein, and approximately 3 h prior to the entry of cyclin D1-stimulated cells into S phase 12 h after zinc treatment. Cyclin D1 immunoprecipitates contained Cdk4 but no detectable Cdk2 and displayed pRb but not histone H1 kinase activity. Cdk2 activation was therefore likely to be due to increased abundance of cyclin E/Cdk2 complexes rather than formation of active cyclin D1/Cdk2 complexes. The sequence of events following zinc induction of cyclin D1 thus mimicked that following mitogen induction of cyclin D1. These data show that cyclin D1 induction is sufficient for Cdk2 activation and pRB hyperphosphorylation in T-47D human breast cancer cells, providing evidence that cyclin D1 induction is a critical event in G1 phase progression.
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PMID:Inducible expression of cyclin D1 in T-47D human breast cancer cells is sufficient for Cdk2 activation and pRB hyperphosphorylation. 886 12


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