Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: DrugBank:BIOD00001 (DNase I)
 8,324 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Physarum EGTA-resistant actin-fragmin complex, previously named cap 42(a+b), is phosphorylated in the actin subunit by an endogenous kinase [Maruta and Isenberg (1983) J. Biol. Chem., 258, 10151-10158]. This kinase has been purified and characterized. It is an 80 kDa monomeric enzyme, unaffected by known kinase regulators. Staurosporine acts as a potent inhibitor. The actin-fragmin complex is the preferred substrate. The phosphorylation is inhibited by micromolar Ca2+ concentrations, but only in the presence of additional actin. Polymerized actin (vertebrate muscle and non-muscle isoforms) and actin complexes with various actin-binding proteins are poorly phosphorylated. The heterotrimer consisting of two actins and one fragmin, which is formed from cap 42(a+b) and actin in the presence of micromolar concentrations of Ca2+, is also a poor substrate. From the other substrates tested, only histones were significantly phosphorylated, in particular histone H1. In the same manner, casein kinase I could also phosphorylate the actin-fragmin complex. The major phosphorylation site in actin is Thr203. A second minor site is Thr202. These residues constitute one of the contact sites for DNase I [Kabsch et al. (1990) Nature, 347, 37-44] and are also part of one of the predicted actin-actin contact sites in the F-actin model [Holmes et al. (1990) Nature, 347, 44-49].
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PMID:Physarum actin is phosphorylated as the actin-fragmin complex at residues Thr203 and Thr202 by a specific 80 kDa kinase. 132 66

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.
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PMID:Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. 133 64

Transcription of the ornithine decarboxylase (ODC) gene is rapidly elevated by activation of protein kinase A (PKA). The additive influence of three cis-acting elements is responsible for this regulation in an adrenal carcinoma cell line. Two sites, CRE2 at -48 base pairs (bp) relative to the start of transcription and CRE3 at +95 bp, are identical to the core motif of the cAMP-responsive element (CRE) of the somatostatin gene and are conserved in the mouse, rat, and human ODC genes. Mutation of CRE2 resulted in a substantial decrease in basal promoter activity, as well as a 5-fold decrease in inducibility of the ODC promoter by PKA. CRE3 did not contribute to the basal activity of the ODC promoter, but mutation of this site resulted in a 2-fold decrease in inducibility by PKA. Deletion of a 45-bp sequence (GC-box) located 5' of CRE2, also resulted in a 2-fold decrease in inducibility of the ODC promoter. DNase I protection revealed the presence of protein binding at CRE2, the TATA box, and the GC-box of the ODC promoter. Mutation of CRE2 resulted in loss of protection of this sequence, as well as the 3' extension of the footprint over the TATA box, without affecting interactions at the GC box. Antibodies to the well characterized CRE-binding protein CREB recognized proteins binding to CRE2, suggesting that binding of CREB, or an antigenically related protein, is important for the activity of CRE2. Additionally, recombinant CREB bound to a DNA probe containing the CRE2 sequence.
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PMID:Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A. 135 8

PMA treatment of human leukemic cells resulted in a significant increase in the phosphorylation of a 72-kDa protein, which was abrogated by treating the nuclear extracts with DNase I, but additionally stimulated by adding DNA. To be active, DNA must be double-stranded with an average size of 300 base pairs, but shows no apparent species- or sequence-specificity. NP-72 isolated from control or PMA-treated nuclei with 1 mM ATP lacked phosphorylating activity, suggesting it to be a substrate for a dsDNA-stimulated protein kinase(s). Simultaneous exposure of HL-60 cells to PMA and the protein kinase C inhibitor staurosporine diminished the phosphorylation of NP-72. These data suggest that leukemia cell differentiation is accompanied by the induction and/or activation of a dsDNA-stimulated protein kinase whose protein substrates include NP-72 and whose activity is directly or indirectly influenced by protein kinase C.
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PMID:dsDNA-stimulated phosphorylation of a 72-kDa nucleoprotein accompanies PMA-induced HL-60 leukemic cell differentiation. 177 55

We have studied the regulation of urokinase-type plasminogen activator gene expression by cAMP in LLC-PK1 cells. We found a cAMP responsive region 3.4 kb upstream of the transcription initiation site, which comprised three protein-binding domains designated A, B, and C. Domains A and B both contain a sequence, TGACG, homologous to a consensus cAMP response element (CRE; TGACGTCA). Effective cAMP-mediated induction was achieved when these two domains were linked with domain C, which by itself did not confer cAMP responsiveness to a heterologous promoter nor contained CRE-like sequence, suggesting a functional cooperation among these domains. Results of competition studies using gel retardation and DNase I footprinting assays suggest that there is a protein-protein interaction between a CRE binding protein and a domain C binding protein. In gel retardation assays, binding of a nuclear protein to domains A and B was strongly augmented by addition of the catalytic subunit of cAMP-dependent protein kinase, whereas the protein binding to domain C was slightly inhibited, suggesting that protein phosphorylation is involved in the regulation of protein-DNA interaction.
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PMID:Macromolecular interaction on a cAMP responsive region in the urokinase-type plasminogen activator gene: a role of protein phosphorylation. 215 33

We have studied the mechanisms that regulate the expression of the mouse gene encoding steroid 11 beta-hydroxylase (11 beta-OHase), a steroidogenic cytochrome P450 enzyme that is expressed only in the adrenal cortex. DNase I footprinting and gel-mobility shift analyses revealed potential regulatory elements at -370 and -310 in the 11 beta-OHase promoter region. To determine the contributions of these elements to expression, we altered their sequences by site-selected mutagenesis and studied promoter activity after transfection into Y1 mouse adrenocortical tumor cells. Mutation of either element markedly decreased basal promoter activity but did not affect the response to treatment with 8-bromo cAMP. These experiments thus document the functional roles of these elements, within the context of the intact promoter, in constitutive expression of 11 beta-OHase. Moreover, addition of either of these elements to p-40GH, a 5'-deletion plasmid containing 11 beta-OHase sequences from -40 to +8 upstream of a growth hormone reporter gene, significantly increased promoter activity but did not confer cAMP responsiveness. Finally, increased expression was seen after transfection of Y1 derivatives deficient in cAMP-dependent protein kinase, indicating that neither element required cAMP-dependent protein kinase activity. These studies thus define two regulatory elements that play important roles in 11 beta-OHase expression.
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PMID:Identification and characterization of two upstream elements that regulate adrenocortical expression of steroid 11 beta-hydroxylase. 223 42

A 43-kDa DNA binding protein which recognizes the TGACGTCA element of the rat somatostatin promoter has been purified from rat brain. Purification of the protein involved initial separation of three sequence-specific binding activities, b1-b3, from each other using DEAE-Sepharose chromatography. The protein corresponding to the b2 complex was further purified to apparent homogeneity by two cycles of sequence-specific DNA affinity chromatography, yielding a single species with an apparent mass of 43,000 daltons on a silver-stained polyacrylamide gel. Sequence-specific DNA binding of this purified protein was demonstrated by Southwestern blotting, renaturation, and DNase I footprinting studies. The 43-kDa protein was phosphorylated on serine residue(s) by the catalytic subunit of cAMP-dependent protein kinase, as shown by phosphoamino acid analysis. Furthermore, the purified protein specifically stimulated transcription from the rat somatostatin promoter in an in vitro transcription system. These results indicate that this 43-kDa protein is a transcription factor required for somatostatin gene expression.
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PMID:Purification and characterization of a 43-kDa transcription factor required for rat somatostatin gene expression. 256 50

The Escherichia coli OmpR protein is an activator protein specific for the ompF and ompC genes, which respectively encode the outer membrane proteins, OmpF and OmpC. The EnvZ protein is a protein kinase specific for the OmpR protein. In this study, we compared the in vitro DNA-binding ability of the phosphorylated form of the OmpR protein with that of the non-phosphorylated form by means of non-denaturing gel retardation analysis and DNase I footprinting analysis. The results indicate that the phosphorylation of the OmpR protein results in stimulation of its in vitro DNA-binding ability as to both the ompF and ompC promoter DNAs.
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PMID:Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability. 267 13

Many hormones act on neuroendocrine cells by activating second messenger pathways. Two of these, the phosphoinositol and cAMP-dependent pathways, cause changes in cellular activity through specific protein kinases. By phosphorylating cytoplasmic and nuclear proteins, these kinases apparently coordinate cellular processes, including the biosynthesis and release of neuropeptides. Somatostatin biosynthesis and release, for example, are both positively regulated by the second messenger cAMP in hypothalamic cells, and cAMP also induces somatostatin gene transcription 8-10-fold in transfected PC12 pheochromocytoma cells. Transcriptional induction requires a 30-nucleotide cAMP response element (CRE) which is conserved in other cAMP-responsive genes. This element also confers cAMP responsiveness when placed upstream of the heterologous simian virus 40 (SV40) promoter. The somatostatin gene does not, however, respond to cAMP in mutant PC12 cells which lack cAMP-dependent protein kinase type II activity. Activation of somatostatin gene transcription may consequently require the phosphorylation of a nuclear protein which binds to the CRE. Using a DNase I protection assay, we have characterized a nuclear protein in PC12 cells which binds selectively to the CRE in the somatostatin gene. We have purified this protein which is of relative molecular mass 43,000 (Mr 43K) by sequence-specific DNA affinity chromatography. This 43K CRE binding protein (CREB) is phosphorylated in vitro when it is incubated with the catalytic subunit of cAMP-dependent protein kinase. Stimulating PC12 cells with forskolin, an activator of adenyl cyclase, causes a 3-4-fold increase in the phosphorylation of this protein. We conclude that the cAMP-dependent pathway may regulate gene transcription in response to hormonal stimulation by phosphorylating this CREB protein.
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PMID:Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. 288 56

During chondrogenesis in vivo and in vitro, a family of nonhistone proteins (Mr 35,500), designated PCP 35.5, is lost from the nuclei of precartilage mesenchyme cells. A basic subcomponent of this family, designated PCP 35.5b, is phosphorylated during the first few hours of chondrogenesis in vitro by a phosphorylating system whose activity is enhanced 12- to 15-fold by exposure of differentiating precartilage cells to dibutyryl cyclic AMP. This phosphorylating system is present in isolated precartilage cell nuclei, where it retains its dependence on cyclic AMP and its specificity for PCP 35.5b. Assays for nuclear cyclic AMP inhibitable protein phosphatase activity capable of dephosphorylating PCP 35.5b were negative, indicating that the system responsible for phosphorylating this protein is a cyclic AMP-dependent protein kinase. Chromatin fractionation studies indicate that PCP 35.5b is localized at sites previously shown to be closely associated with DNase I-sensitive domains of precartilage cell chromatin. These studies define PCP 35.5b as a strategically located component of precartilage cell chromatin which is the major or sole chromatin target of cyclic AMP-dependent phosphorylation during chondrogenesis. This chromatin modification occurs prior to overt cartilage differentiation and may therefore play a regulatory role in the acquisition of the cartilage cell phenotype.
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PMID:Nuclear events during early chondrogenesis: phosphorylation of the precartilage 35.5-kDa domain-specific chromatin protein and its regulation by cyclic AMP. 302 88


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