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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cell cycle progression requires activation of different cyclin-dependent kinases (CDKs) which are positively regulated by cyclins and negatively regulated by CDK inhibitors. Growth inhibition of the Calu-1 lung carcinoma cells induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, is associated with G2/M arrest and induction of expression of a novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein, an inhibitor of cyclin-dependent kinases. This faster-migrating p21 protein was also expressed in TPA-treated A549 lung carcinoma cells which also exhibited G2/M arrest but not in TPA-treated U937 leukemia cells, which only expressed a slower-migrating form of p21 protein. However, reverse transcriptase-polymerase chain reaction and Southern analysis demonstrated no evidence of novel splice in TPA-treated Calu-1 cells. On the other hand, immunoblotting analysis demonstrated that the faster-migrating p21 protein could be detected only by peptide antibody directed against the N terminus but not the C terminus, suggestive of truncation of the latter or protein modification that results in the loss of the C-terminal epitope. Correlation of G2/M arrest with expression of the faster-migrating p21 protein suggests that this novel form of p21 protein may be a mediator of G2/M arrest and growth inhibition.
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PMID:Novel form of p21(WAF1/CIP1/SDI1) protein in phorbol ester-induced G2/M arrest. 893 83

The ethylene signal is transduced in plant cells via phosphorylation events. To identify protein kinases whose levels of expression are modulated by the plant hormone ethylene, we utilized a differential reverse transcriptase-polymerase chain reaction approach using mRNA extracted from ethylene-treated and untreated tobacco leaves. An ethylene-induced cDNA clone, PK12, encoding a protein kinase, was isolated. PK12 is a new member of the recently defined LAMMER family of protein kinases, which has been identified in mammals, flies, yeasts, and plants. The LAMMER kinases are related to the cell cycle-dependent CDC2-type kinases and are characterized by their similarity at kinase subdomain X. The recombinant PK12 protein autophosphorylates in vitro on serine, threonine, and tyrosine residues, thereby making it a member of the dual-specificity protein kinases. Immunoprecipitation of PK12 from plant extracts and kinase assay revealed that the apparent PK12 activity is rapidly and transiently increased when plants are treated with ethylene. By using in situ hybridization, we detected accumulation of the PK12 transcript in leaves after ethylene treatment and in the untreated flower abscission zone. The tissue in this zone is known to constitutively express ethylene-regulated genes.
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PMID:PK12, a plant dual-specificity protein kinase of the LAMMER family, is regulated by the hormone ethylene. 898 79

CaMK-II (the (type II) multifunctional Ca2+/CaM-dependent protein kinase) has been implicated in diverse neuronal and non-neuronal functions, including cell growth control. CaMKII expression was evaluated in a variety of human tumor cell lines using RT-PCR (reverse transcriptase coupled polymerase chain reaction). PCR primers which flanked the CaMK-II variable domain were used so that all possible variants of the four mammalian CaMK-II genes (alpha, beta, gamma and delta) could be identified. 8 distinct CaMK-II isozymes were identified from human mammary tumor and neuroblastoma cell cDNA, each of which represented a variant of beta, gamma or delta CaMK-II. They included 2 beta isozymes (beta e, beta 'e), 4 gamma isozymes (gamma B, gamma C, gamma G, gamma H) and 2 delta isozymes (delta C, delta E) This is the first report of human beta and delta CaMK-II sequences. A panel of human cell types was then screened for these CaMK-II isozymes. As expected, cerebral cortex predominately expressed alpha, beta and delta A CaMK-II. In contrast, tumor cells, including those of neuronal origin, expressed an entirely different spectrum of CaMK-II isozymes than adult neuronal tissue. Tumor cells of diverse tissue origin uniformly lacked alpha CaMK-II and expressed 1-2 beta isozymes, at least 3 gamma isozymes and 1-2 delta isozymes. When compared to undifferentiated fibroblasts, beta e, beta'e, gamma G and gamma H were preferentially expressed in tumor cells. CaMK-II immunoblots also indicated that neuroblastoma and mammary tumor cells express isozymes of CaMK-II not present in their non-transformed cell or tissue counterpart. The identification of these new, potential tumor-specific CaMK-II variants supports previous indications that CaMK-II plays a role in growth control. In addition, these results provide insight into both splice variant switching and variable domain structural similarities among all CaMK-II isozymes.
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PMID:Identification of novel human tumor cell-specific CaMK-II variants. 906 Sep 99

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
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PMID:Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells. 907 71

We used a catecholaminergic neuron-like cell line (CATH.a cells) as a model system to investigate the likelihood that pituitary adenylate cyclase-activating polypeptide (PACAP) may participate in the regulation of specific gene expression in catecholaminergic neurons. Analysis by reverse transcriptase-PCR amplification revealed the presence in these cells of type I PACAP receptors, with a short isoform, together with a heavier so-called Hop splice variant. PACAP38 and PACAP27 enhanced, in a dose-dependent manner, both cyclic AMP formation and phosphoinositide breakdown, with EC50 values of, respectively, 0.6 x 10(-10) and 2 x 10(-9) M. These peptides, in addition, also elevated [Ca2+]i by mobilizing intracellular calcium pools. Vasoactive intestinal peptide (VIP) was approximately 1,000-fold less potent in stimulating cyclic AMP (with EC50 = 2 x 10(-7) M) and failed to change the turnover of phosphoinositides and to alter [Ca2+]i. Both forms of PACAP, as well as forskolin, stimulated transcriptional induction of tyrosine hydroxylase (TH) and c-fos promoters fused to a chloramphenicol acetyltransferase (CAT) reporter gene in transiently transfected cells (p < 0.01 vs. controls). Induction of CAT activity linked to both TH and c-fos promoters was obliterated upon coexpression of a dominant inhibitory mutant (Mt-RAB) of cyclic AMP-dependent protein kinase. We conclude that CATH.a cells do express functional PACAP type I receptors, the activation of which impinges on TH and c-fos transcription according to a process that is primarily dependent on the cyclic AMP-PKA pathway.
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PMID:Pituitary adenylate cyclase-activating polypeptide triggers dual transduction signaling in CATH.a cells and transcriptionally activates tyrosine hydroxylase and c-fos expression. 908 43

A homologue of human protein kinase C (PKC)-related kinase-2, PRK2, which had previously escaped identification in normal mammalian tissues, was isolated from rat liver as the protease-activated kinase (PAK) originally named PAK-2. The 130-kDa cytosolic enzyme was purified to homogeneity and shown by tryptic peptide and reverse transcriptase- polymerase chain reaction (RT-PCR)-amplified rat cDNA sequence analyses to be structurally related to the 116-kDa rat hepatic PAK-1/protein kinase N (PKN) and, even more closely (95% sequence identity) to the 130-kDa human PKC-related kinase, PRK2. Rat myeloma RNA was used as the RT-PCR template because of its relative abundance in PAK-2/PRK2 mRNA compared with liver and other rat tissues. The catalytic properties of PAK-2/PRK2 in many respects resembled those of hepatic PAK-1/PKN, but were distinguished by more favorable kinetics with several peptide substrates, and greater sensitivity to PKC pseudosubstrate and polybasic amino acid inhibitors. PAK-2/PRK2 was also activated by lipids, particularly cardiolipin and to a lesser extent by other acidic phospholipids and unsaturated fatty acids. Cardiolipin activation was most evident with autophosphorylation and histone H2B phosphorylation, but only marginally evident with the favored ribosomal S6-(229-239) peptide substrate for the protease-activated kinase activity. It was concluded that PAK-2 is the rat homologue of human PRK2, with biochemical properties distinct from although overlapping those of the PAK-1/PKN/PRK1 isoform.
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PMID:Isolation and characterization of a structural homologue of human PRK2 from rat liver. Distinguishing substrate and lipid activator specificities. 909 45

Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein. The size of the protein was confirmed by in vitro coupled transcription/translation. Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase). The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring. The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase. Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner. The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases. A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis. Its expression was detected in cultures of M. bovis BCG by reverse transcriptase/PCR. Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.
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PMID:A serine/threonine protein kinase from Mycobacterium tuberculosis. 911 30

To clarify the upstream regulatory mechanism of mitogen-activated protein kinase (MAPK), we performed the reverse transcriptase-based polymerase chain reaction (RT-PCR) with degenerate primers synthesized based on sequences conserved among the kinase domains of yeast MAPK kinase kinases (MAPKKKs), Stell, Bck1, and Byr2. We isolated several mammalian cDNA fragments that encode kinase subdomains sharing significant sequence homology with yeast MAPKKKs. Subsequent screening of a HeLa cell cDNA library using one of these cDNA fragments as a probe resulted in the isolation of a full-length cDNA that encodes a novel protein kinase. The catalytic domain sequence of this gene product is closely related to those of budding yeast Sps1 and Ste20 protein kinases. Thus, we call this protein YSK1 (Yeast Sps1/Ste20-related Kinase 1). The transcript of YSK1 was detected in a wide range of tissues and cells. Immunoprecipitated YSK1 shows protein kinase activity. Although YSK1 is significantly similar in its kinase domain to kinases of the yeast and mammalian MAPK pathways, the overexpression of YSK1 did not lead to the activation of the ERK (extracellular signal-regulated kinase) pathway, JNK (c-Jun NH2-terminal kinase)/SAPK (stress-activated protein kinase) pathway, or p38/Mpk2 pathway. These results suggest that YSK1 may be involved in the regulation of a novel intracellular signaling pathway.
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PMID:YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways. 916 Aug 85

Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of functional maturity and may therefore play a role in beta-cell development.
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PMID:Expression of mixed lineage kinase-1 in pancreatic beta-cell lines at different stages of maturation and during embryonic pancreas development. 919 43

Estrogen exerts its physiological effects in the uterus by inducing a cascade of transcriptional events; however, the number of genes known to be directly activated by estrogen in the uterus is small. In this study, immature ovariectomized rats were treated with estrogen or vehicle, and 3 h later the uterine horns were flushed to extract epithelial RNA. This RNA was used in the differential display technique to search for estrogen-responsive genes. Products of reverse transcriptase-PCR, made with pairs of arbitrary and oligo-deoxythymidine primers, were separated on denaturing polyacrylamide gels; candidate bands were excised and reamplified to produce probes for use in Northern blot analysis and screening of a lambda gt10 complementary DNA library made from rat uterus. A novel estrogen-enhanced transcript, designated EET-1, was identified from a differential display band, and the estrogen sensitivity of its expression was verified in Northern analysis. Characterization of EET-1 expression in the uterus showed that estrogen treatment resulted in a rapid and transient increase in EET-1 messenger RNA; steady state levels peaked between 2-3 h, returning to basal levels by 6 h. This increase was not abolished by pretreatment with cycloheximide, indicating that induction of EET-1 is a primary response to estrogen. Induction was specific to estrogen when extracts of whole uterus were examined; in the epithelium, there was also a slight response to progesterone. Expression of the gene was found in all organs surveyed; however, hormonal regulation was observed only in tissues of the reproductive tract and in the kidney. Analysis of cloned EET-1 complementary DNA revealed a 2008-base sequence that showed 61% identity with a reported transcript that encodes a protein that plays a role in phorbol ester-induced regulation of the tumor necrosis factor-alpha gene. Potential casein kinase-2 and protein kinase C phosphorylation sites and a cysteine-rich region were identified in the amino acid sequence deduced from EET-1. Thus, it appears that EET-1 represents a primary estrogen response gene that may code for a phosphorylated protein involved in gene regulation through a protein kinase C-activated pathway.
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PMID:A novel estrogen-enhanced transcript identified in the rat uterus by differential display. 927 72


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