Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.
Mol Cell Biol 1991 Nov
PMID:Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. 192 62

Mouse lymphoma cell line W7M320b, a mutant WEH17 line, requires higher than normal concentrations of glucocorticoid to elicit the hormone responses that are characteristic of this lineage. Complementary DNA clones representing the glucocorticoid receptor (GR) mRNA were derived from the mutant cells, and the sequences coding for the hormone-binding domain were substituted for the analogous wild-type sequences in a GR cDNA expression vector. The function of the resulting GR proteins was tested by transient expression in COS-7 cells along with a glucocorticoid-inducible reporter gene in the presence of varying concentrations of glucocorticoid. From these assays and DNA sequence analyses, two independent functionally significant point mutations in the GR hormone-binding domain were identified. A mutant GR protein containing the single amino acid substitution, Pro547 to Ala, was still functional as a transcriptional activator, but only at hormone concentrations 100 times higher than those required by the wild-type receptor. A second mutant GR protein with a Cys742 to Gly substitution was unstable and almost completely nonfunctional.
Mol Endocrinol 1991 Jun
PMID:Two point mutations in the hormone-binding domain of the mouse glucocorticoid receptor that dramatically reduce its function. 192 94

Our laboratory recently isolated and sequenced cDNAs encoding the microsomal flavin-containing monooxygenases (FMOs) from rabbit liver and rabbit lung. As a first step in understanding the molecular bases for the catalytic and physical differences between these enzymes, we have expressed them in COS-1 cells and compared the properties of the recombinant and native microsomal proteins. Microsomes from transfected cells were examined immunochemically by immunoblotting and catalytically by following methimazole oxidation in the presence and absence of various modulators. The expressed and native FMOs have the same mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the same responses to pH, sodium cholate, magnesium, and temperature, all of which serve to differentiate between the lung and liver enzymes. Analysis of methimazole metabolism in microsomes isolated from rabbit liver or lung showed biphasic kinetics, indicative of two or more enzymes taking part in the reaction. In contrast, the kinetics of methimazole oxidation catalyzed by the expressed FMOs were clearly linear and matched one of the phases observed with the native preparations. Chlorpromazine and imipramine, which are not substrates for the pulmonary FMO, were found to be competitive inhibitors of the high affinity reaction in pulmonary microsomes. These results, and others, indicate that both rabbit lung and liver contain more than one form of FMO.
Mol Pharmacol 1991 Nov
PMID:Properties of expressed and native flavin-containing monooxygenases: evidence of multiple forms in rabbit liver and lung. 194 40

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3 beta-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.
Mol Cell Endocrinol 1991 Sep
PMID:3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression in rat and characterization of the testis isoform. 195 79

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.
J Steroid Biochem Mol Biol 1991
PMID:Androgen receptor abnormalities. 195 38

We report on two assays for autoantibodies to the TSH-R which have been developed using materials from mammalian cells transfected with the cDNA for the human TSH-R. In the first, a particulate fraction has been prepared from COS cells, transiently expressing the human TSH-R and used in a radioreceptor assay in conjunction with bovine 125I-TSH. Immunoglobulins (IgGs) from patients with Graves' disease (n = 11) and idiopathic myxoedema (n = 2) have been used as competitors of 125I-TSH binding to the COS TSH-R membranes and the results have been compared with those obtained with a commercially available kit for measuring TSH-R autoantibodies, which uses solubilised porcine TSH-R. Both assays showed similar performance, being particularly sensitive to antibodies from patients with idiopathic myxoedema. In the second assay system we have used a CHO cloned cell line (JP26) stably transfected with the human TSH-R. A selection of IgG preparations from patients with Graves' disease and of six normal controls was used to test the ability of this cell line to detect thyroid stimulating immunoglobulins (TSAb) by increasing its cAMP production. The assay was performed under two conditions: in standard (isotonic) medium or in hypotonic medium. Freshly thawed human thyrocytes incubated in hypotonic medium served as a reference method. Only five patients scored positive when tested in the JP26 cell line under isotonic conditions. When the assay was performed in a hypotonic medium, a significant positive correlation was observed between the results given by JP26 cells and human thyrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1990 Oct 01
PMID:Use of the recombinant human thyrotropin receptor (TSH-R) expressed in mammalian cell lines to assay TSH-R autoantibodies. 198 64

We have isolated and characterized a new human cDNA, coding for a protein kinase, related to the protein kinase C (PKC) gene family. Although this protein kinase shares some homologous sequences and structural features with the four members of the PKC family initially isolated (alpha, beta I, beta II, and gamma), it shows more homology with the recently described PKC-related subfamily, encoded by the cDNAs delta, epsilon, and zeta. The transcript for this gene product, termed PKC-L, is most abundant in lung tissue, less expressed in heart and skin tissue, and exhibited very low expression in brain tissue. Thus, its tissue distribution is different from that described for other mammalian members of the PKC gene family, their expression being enriched in brain tissues. PKC-L is also expressed in several human cell lines, including the human epidermoid carcinoma line A431. The ability of phorbol esters to bind to and stimulate the kinase activity of PKC-L was revealed by introducing the cDNA into COS cells.
Mol Cell Biol 1991 Jan
PMID:Isolation and characterization of PKC-L, a new member of the protein kinase C-related gene family specifically expressed in lung, skin, and heart. 154 21

Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.
Mol Cell Biol 1991 Jan
PMID:Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor. 198 49

A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT; EC 2.3.1.5) enzyme activity divides populations into distinguishable "slow acetylator" and "rapid acetylator" phenotypes. Two human genes, NAT1 and NAT2, encoding NAT proteins [DNA Cell Biol. 9:193-203 (1990)] were transiently expressed in cultured monkey kidney COS-1 cells, and the resulting recombinant NAT1 and NAT2 proteins were compared with N-acetyltransferase activities in human liver cytosol with respect to their stability, chromatographic behavior on anion exchange columns, electrophoretic mobility, and arylamine acceptor substrate specificity. NAT1 was far less stable in vitro than NAT2. Under conditions designed to optimize enzyme stability, anion exchange chromatography experiments revealed that enzymes corresponding to both recombinant NAT1 and NAT2 were expressed in human liver. Recombinant and human liver NAT1 enzymes showed the same characteristic selectivity (low apparent Km, high Vmax) for the "monomorphic" substrates p-aminosalicylic acid and p-aminobenzoic acid. Such substrates fail to discriminate between the acetylator phenotypes in vivo. The same criteria established that recombinant NAT2 was indistinguishable from one of two previously observed N-acetyltransferases (NAT2A and NAT2B) whose liver contents correlate with acetylator phenotype in human populations. Recombinant NAT2 and the liver NAT2 isoforms NAT2A and NAT2B selectivity N-acetylated the "polymorphic" substrates sulfamethazine and procainamide, whose disposition in vivo is affected by the acetylation polymorphism. Interestingly, the carcinogen 2-aminofluorene was very efficiently metabolized by both NAT1 and NAT2. Independent regulation of NAT1 and NAT2 genes was suggested by a lack of correlation of NAT1 and NAT2 enzyme activities in cytosols from 39 human livers. The results provide strong evidence that the NAT2 locus is the site of the human acetylation polymorphism. In addition, the use of recombinant NAT1 and NAT2 will allow us to predict whether any given arylamine will be polymorphically acetylated in humans.
Mol Pharmacol 1991 Feb
PMID:Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes. 199 83

Glucocorticoid regulation of rat growth hormone (rGH) gene expression has been investigated in a series of gene transfer studies into cells in culture. It has been established that sequences (-12 to -523) immediately flanking the start site for rGH gene transcription behave as a functional glucocorticoid inducible enhancer when associated with a heterologous promoter (RSV), displaying independence of orientation and position in mediating the glucocorticoid effect. The induction of chloramphenicol acetyl transferase (CAT) gene expression in these constructs by dexamethasone was established at the enzyme and mRNA levels and was inhibited in the presence of the antiglucocorticoid, RU 38486. The glucocorticoid inducible enhancer activity was not restricted to pituitary cells. The constructs containing the rGH-5'-flanking sequences, associated with the RSV promoter, also mediated glucocorticoid induction of CAT gene expression when transiently transfected into MH1C1 cells, a hepatoma cell line. The effect was similarly demonstrable on co-transfection of these constructs with a glucocorticoid receptor expression vector into receptor deficient COS cells. Two elements within these rGH sequences (-97 to -111 and -250 to -264) display partial homology with a consensus sequence computed for a group of glucocorticoid regulatory elements. Mutation of both of these elements or of the more proximal element alone (-97/-111) led to a complete loss of ability to mediate glucocorticoid induction of gene expression. However, the rGH sequences still mediated glucocorticoid induction of gene expression when the distal GRE-like element was mutated or deleted. Thus, the proximal rGH GRE-like element is absolutely required to mediate this glucocorticoid inducible enhancer activity.
J Steroid Biochem Mol Biol 1991 Jan
PMID:Functional glucocorticoid inducible enhancer activity in the 5'-flanking sequences of the rat growth hormone gene. 199 16


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