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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Estramustine (EM) is an anti-microtubule drug used in the treatment of hormone-refractory advanced prostate cancer. Since microtubules are the targets for EM cytotoxicity, we investigated the effects of EM on the microtubule-associated protein tau to determine what role it may play in drug resistance. We have compared tau expression in human prostate cancer cells (DU145) and an EM-resistant derived cell line (E4). Reverse transcriptase polymerase chain reaction has established that tau is expressed in both cell lines but increased 1.9-fold in E4 compared with DU145 cells. This result was confirmed at the protein level by Western blotting. Tau is a phosphoprotein, most of its reported phosphorylation sites being serine or threonine residues. We have shown, however, that tau is also phosphorylated at tyrosine residues in DU145 cells and that the phosphotyrosine level of tau is significantly increased in E4 cells. Moreover, DU145 cells exposed to short term micromolar drug concentrations enter a phase of microtubule depolymerization, display an increased level of tau phosphorylation and follow a pattern similar to that observed in EM-resistant E4 cells. EM is therefore able to induce a very rapid change in the posttranslational state of tau. Our results show that the acquisition of EM resistance in E4 cells, which is accompanied by changes at the tubulin level, is also associated with important changes in tau expression and phosphorylation.
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PMID:Estramustine resistance correlates with tau over-expression in human prostatic carcinoma cells. 967 68

The dengue virus NS5 RNA-dependent RNA polymerase has been detected in the nucleus of virus-infected mammalian cells. We demonstrate here for the first time using in vitro and in vivo assay systems that the 37-amino-acid linker interdomain of NS5 (residues 369 to 405) contains a nuclear localization sequence (NLS) which is capable of targeting b-galactosidase to the nucleus. Further, we show that the linker is recognized by subunits of the NLS-binding importin complex with an affinity similar to that of the bipartite NLS of the retinoblastoma protein and, in analogous fashion to proteins such as the SV40 large tumor antigen, contains a functional protein kinase CK2 phosphorylation site (threonine 395). Interestingly, this site appears to inhibit NS5 nuclear targeting, probably through a cytoplasmic retention mechanism. The linker may have an important role in targeting NS5 to the nucleus in a regulated manner during the dengue virus infectious cycle.
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PMID:The 37-amino-acid interdomain of dengue virus NS5 protein contains a functional NLS and inhibitory CK2 site. 1020 52

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a key subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphorylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic domain I of P protein must be phosphorylated for in vivo transcription activity, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of the P protein in transcription and replication, we have used a panel of mutant P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combinations. Analyses of the mutant proteins for their ability to support replication of a VSV minigenomic RNA suggest that phosphorylation of either Ser-226 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-type (wt) protein. Substitution of alanine for Ser-233 did not have any adverse effect on replication activity of the protein. In contrast, all the mutant proteins showed activities similar to that of the wt protein in transcription. These results indicate that phosphorylation of the carboxy-terminal domain II residues of P protein are required for optimal replication activity but not for transcription activity. Furthermore, substitution of glutamic acid residues for Ser-226 and Ser-227 resulted in a protein that was only 14% active in replication but almost fully active in transcription. Taken together, these results, along with our earlier studies, suggest that phosphorylation of residues at two different domains in the P protein regulates its activity in transcription and replication of the VSV genome.
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PMID:Optimal replication activity of vesicular stomatitis virus RNA polymerase requires phosphorylation of a residue(s) at carboxy-terminal domain II of its accessory subunit, phosphoprotein P. 1036 10

Protein kinase C (PKC), a key component of the signaling pathways leading to proliferation and differentiation, consists of a family closely related serine/threonine protein kinases. The mRNA expression of these PKC isoforms has been characterized during hematopoietic differentiation. Using the reverse-transcriptase polymerase chain reaction technique, we have analyzed the levels of isoform transcripts in bone marrow CD34(+) hematopoietic progenitors and their progeny differentiated along erythroid, megakaryocyte, or granulocyte/monocyte lineages, upon exposure to growth factors. In contrast with isoforms alpha, beta(I), beta(II), delta, and epsilon, ubiquitously expressed, isoforms theta, eta/L, zeta, and iota/lambda exhibited a lineage-restricted expression. These qualitative changes, which allow to distinguish the erythroid and megakaryocyte phenotypes from the granulocyte/monocyte phenotype, include zeta exclusively upregulated in granulocytes/monocytes and theta, eta/L, and iota/lambda exclusively expressed in megakaryocytes and erythroblasts. In contrast, erythroblasts and megakaryocytes, which supposedly share a common bipotential progenitor, displayed only quantitative changes. These results evidence the selective expression of PKC isoforms at transcriptional and/or posttranscriptional levels in hematopoietic progenitors induced to differentiate, which may suggest a differential contribution of individual isoforms to cellular signaling.
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PMID:Differential expression of protein kinase C isoform transcripts in human hematopoietic progenitors undergoing differentiation. 1051 25

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.
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PMID:Cloning and expression of glycolipid transfer protein from bovine and porcine brain. 1067 54

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.
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PMID:Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system. 1088 Mar 40

nsP3 is one of the four RNA replicase subunits encoded by alphaviruses. The specific essential functions of nsP3 remain unknown, but it is known to be phosphorylated on serine and threonine residues. Here we have completed mapping of the individual phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by point mutational analysis of threonine residues. This showed that threonines 344 and 345 represented the major threonine phosphorylation sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be phosphorylated by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells infected with mutant viruses produced close to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defective in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesis also exhibited greatly reduced pathogenicity in mice.
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PMID:Elimination of phosphorylation sites of Semliki Forest virus replicase protein nsP3. 1110 56

Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76 kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80 kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress.
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PMID:Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK. 1128 15

Adrenergic regulation of the pineal enzyme serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87] accounts for the circadian rhythm in melatonin formation. In the present study, the role of protein phosphatases in the adrenergic regulation of rat pineal AA-NAT was investigated using specific inhibitors. In cultured pineals, the serine/threonine phosphatase type 1 and type 2A inhibitors okadaic acid and calyculin A significantly decreased adrenergically or cAMP-induced AA-NAT activity, whereas the serine/threonine phosphatase type 2B inhibitor cypermethrin and tyrosine phosphatase inhibitor dephostatin were ineffective. Reverse transcriptase-polymerase chain reaction (RT-PCR) data indicate that okadaic acid exerts its effect on cAMP-dependent AA-NAT induction by downregulating the amount of AA-NAT transcript. The 'third' messengers, inducible cAMP early repressor (ICER) and Fos-related antigene-2 (Fra-2), are believed to play a negative role in pineal AA-NAT transcription. Okadaic acid increased the cAMP responsiveness of neither ICER mRNA nor Fra-2 mRNA. Therefore, the regulatory role of pineal serine/threonine phosphatases in adrenergically stimulated AA-NAT expression probably does not depend on ICER or Fra-2.
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PMID:Serine/threonine phosphatase inhibitors decrease adrenergic arylalkylamine n-acetyltransferase induction in the rat pineal gland. 1144 72

Human endogenous retroviruses (HERVs) have been implicated as causative agents in diseases characterized by inflammation and macrophage activation, such as multiple sclerosis. Because monocyte activation and differentiation influence retroviral transcription and replication, we investigated the contribution of these processes to the expression of four HERV families (HERV-W, HERV-K, HERV-E, and HERV-H) in human monocytes, and autopsied brain tissue from patients with brain diseases associated with increased macrophage activity. Reverse transcriptase-polymerase chain reaction analysis of primary macrophages and U937 monocytoid cells stimulated with phorbol-12-myristate-13-acetate or lipopolysaccharide revealed three- to ninefold increases in HERV-W, HERV-K, and HERV-H RNA levels. In addition, elevated reverse transcriptase activity and HERV RNA were detectable in supernatants from PMA-stimulated U937 cultures, properties that could be attenuated with the inhibitor of monocyte differentiation threonine-lysine-proline. In contrast, stimulation of monocytes decreased or had no effect on HERV-E expression. Compared with controls, HERV-W and HERV-K expression was increased in brain tissue from patients with multiple sclerosis or human immunodeficiency virus infection or AIDS, with concomitant elevated tumor necrosis factor-alpha levels. Similarly, elevated HERV-W levels were detected in patients with Alzheimer's dementia only when tumor necrosis factor-alpha expression was also evident (2 of 6 cases). The detection of several HERVs in inflammatory brain diseases and the capacity to augment HERV expression in monocytes with compounds that influence cellular activity suggest that increased expression of these viruses is a consequence of increased immune activity rather than causative of distinct diseases.
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PMID:Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases. 1160 92


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