Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transformation of cloned rat embryo fibroblast (CREF) cells with the wild-type 5 adenovirus (wtAd5) transforming genes E1A and E1B (which extend from 0 to 11.2 map units) results in morphologically transformed cells that exhibit an increased saturation density in monolayer culture and display an anchorage-independent phenotype. WtAd5-transformed CREF (wtAd5 CREF) cells do not, however, induce tumors when injected subcutaneously into athymic nude mice or syngeneic Fischer rats. We have analyzed the effect of the ras oncogene and site-specific mutants in the ras oncogene that result in p21 proteins with altered biochemical properties on the oncogenic and metastatic properties of singly (ras) and doubly (ras + wtAd5) transformed CREF cells. Transformants expressing the wild-type ras p21 protein and ras mutants producing p21 proteins that retained GTP-binding properties grew in agar, induced tumors in nude mice and syngeneic rats, and metastasized to the lungs of rats when injected into their tail veins. In contrast, cells transformed with the ras mutant 116K (which contains a mutation at residue 116 that produces a Lys instead of an Asn and does not bind GTP or induce CREF cells to grow in agar) did not become morphologically transformed and were not oncogenic when injected subcutaneously into either nude mice or Fischer rats; further, such cells were not metastatic when injected into the tail veins of Fischer rats. When the wild-type ras or the ras mutants, including 116K, were expressed in nontumorigenic E1A-plus-E1B-expressing wtAd5 CREF cells, transformed cells induced tumors in both types of animals. The CREF cells doubly transformed with 116K + wtAd5, unlike transformants containing the wild-type ras and the other ras mutants that still retained GTP binding, were still unable to induce lung metastases. In addition, 116K + wtAd5-transformed CREF cells also did not display any alterations in morphology distinguishable from wtAd5 CREF cells and were not able to grow in agar with increased efficiency. These results indicate that the loss of GTP-binding ability by this mutant p21 ras protein eliminated the ability of these proteins to induce an oncogenic phenotype in an immortal but normal CREF cell line. However, the mutant ras could cooperate with wtAd5 transforming genes in transformed CREF cells to make these cells progress to an oncogenic (but not metastatic) phenotype.
Mol Carcinog 1992
PMID:Induction and progression of the transformed phenotype in cloned rat embryo fibroblast cells: studies employing type 5 adenovirus and wild-type and mutant Ha-ras oncogenes. 155 10

The T4-binding globulin-Gary (TBG-G) variant has severely impaired T4 binding, is unstable at 37 C, and presents an apparent anodal shift of all isoforms when submitted to isoelectric focusing. Inheritance of this abnormal TBG produces a profound decrease in the serum levels of native TBG with reciprocal changes in its denatured form, causing thyroid hormone concentrations to be as low as those found in complete TBG deficiency. The TBG-G gene possesses a single nucleotide substitution replacing the normal IIe96 (ATC) with Asn (AAC), thus creating a new site for N-linked glycosylation. In order to determine whether TBG-G contains an additional carbohydrate chain as indirectly suggested by the isoelectric focusing results, cDNAs containing the normal TBG (TBG-N), and TBG-G were inserted in the appropriate vectors to allow their expression in mammalian cells (COS-1) and in amphibian (Xenopus) oocytes. In both systems, expression of TBG-G yielded a larger molecule than TBG-N when analyzed by polyacrylamide gel electrophoresis under denaturing conditions. However, both were identical in size when synthesized in COS-1 cells in the presence of tunicamycin or when deglycosylated after their synthesis in Xenopus oocytes. Pulse chase experiments revealed impaired secretion and excessive overall intracellular degradation of TBG-G relative to TBG-N. As expected from studies on serum from affected subjects, in vitro expressed TBG-G had a 10-fold lower affinity for T4. These studies prove that the new site for potential glycosylation created by the point mutation in TBG-G is indeed glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Mar
PMID:An additional carbohydrate chain in the variant thyroxine-binding globulin-Gary (TBGAsn-96) impairs its secretion. 158 18

ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites.
Mol Cell Biol 1992 Jun
PMID:Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation. 158 70

We have mutated residues in the active site of the ribonuclease, barnase, in order to determine their effects on both enzyme activity and protein stability. Mutation of several of the positively charged residues that interact with the negatively charged RNA substrate (Lys27----Ala, Arg59----Ala and His102----Ala) causes large decreases in activity. This is accompanied, however, by an increase in stability. There is presumably electrostatic strain in the active site where positively charged side-chains are clustered. Mutation of several residues that make hydrogen bonds (Ser57----Ala, Asn58----Asp and Tyr103----Phe) causes smaller decreases in activity, but increases or has no effect on stability. Deletion of hydrogen bonding groups elsewhere in proteins has been found previously to decrease stability by 0.5 to 1.5 kcal mol-1. Conversely, we find that two mutations (Asp54----Asn and Gln104----Ala) decrease stability and increase activity. Another mutation (Glu73----Ala) decreases both activity and stability. It is clear that many residues in the active site do not contribute to stability and that for some, but not all, of the residues there is a compromise between activity and stability. This suggests that certain types of local instability may be necessary for substrate binding and catalysis by barnase. This has implications for the understanding of enzyme activity and the design of enzymes.
J Mol Biol 1992 Jun 05
PMID:Effect of active site residues in barnase on activity and stability. 160 71

A model peptide with 51 amino acid residues consisting of tandem repeats of a Lys-Lys-Leu-Leu sequence and a turn sequence of Asn-Pro-Gly at the center of the molecule has a random conformation at neutral pH but adopts an amphiphilic alpha-helical form in the presence of various salts or nucleotides [Goto, Y., & Aimoto, S. (1991) J. Mol. Biol. 218, 387-396; Goto, Y., Okamura, N., & Aimoto, S. (1991) J. Biochem. (Tokyo) 109, 746-750]. The interaction of this model peptide with liposome membranes and the resulting alpha-helical conformational transition and membrane fusion as well as the effect of the nucleotide ATP on these events were examined at neutral pH. The peptide associated stoichiometrically with liposome membranes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) in a molar ratio of 2:1, resulting in formation of an amphiphilic alpha-helix and induction of fusion of the liposomes. However, the final fusion level was not correlated with the amount of binding or the helix content and was found to increase on an increase in hydrophobicity of the peptide in the alpha-helical form by neutralization of its positive charges by the negative charges of PS. In contrast, in the presence of ATP, the peptide bound completely to the PS/PC membranes at a lower concentration of liposome and concomitantly induced membrane fusion, indicating that ATP cooperates with PS to neutralize the charges of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fusion of phospholipid vesicles induced by an amphiphilic model peptide: close correlation between fusogenicity and hydrophobicity of the peptide in an alpha-helix. 162 54

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.
Mol Cell Biol 1991 Oct
PMID:A mutation in the putative Mg(2+)-binding site of Gs alpha prevents its activation by receptors. 165 12

Insulin induces a rapid activation of p21ras in NIH 3T3 and Chinese hamster ovary cells that overexpress the insulin receptor. Previously, we suggested that p21ras may mediate insulin-induced gene expression. To test such a function of p21ras more directly, we studied the effect of different dominant inhibitory mutants of p21ras on the induction of gene expression in response to insulin. We transfected a collagenase promoter-chloramphenicol acetyltransferase (CAT) gene or a fos promoter-luciferase gene into NIH 3T3 cells that overexpressed the insulin receptor. The activities of both promoters were strongly induced after treatment with insulin. This induction could be suppressed by cotransfection of two inhibitory mutant ras genes, H-ras(Asn-17) or H-ras(Leu-61,Ser-186). In particular, insulin-induced activation of the fos promoter was inhibited completely by H-ras(Asn-17). These results show that p21ras functions as an intermediate in the insulin signal transduction route leading to the induction of gene expression.
Mol Cell Biol 1991 Dec
PMID:Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression. 165 21

Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core beta-sheet structure, connected by loops and alpha-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding site is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site into Asn. First investigations reveal that the enzymatic activity of the mutant protein is destroyed.
J Comput Aided Mol Des 1991 Oct
PMID:Computer-aided active-site-directed modeling of the herpes simplex virus 1 and human thymidine kinase. 166 55

Aminoglycoside-phosphotransferases contain several conserved amino acid sequence motifs. Using hydroxylamine we have obtained five independent missense mutations within the aphA-2 gene of transposon Tn5. Four of the mutations dramatically reduced antibiotic resistance. Two were identical and included the replacement of His-188 with Tyr. One other resulted from the replacement of Gly-189 with Asp. These three mutations map within the first of the conserved motifs. The replacement of Asp-261 with Asn maps to the third of these structural motifs. A mutation diminishing but not eliminating aminoglycoside resistance resulted from replacement of the conserved Val-36 with Met. By site-directed mutagenesis three additional mutants were obtained: His-188 was replaced with Leu and Ser, and Arg-211 within the second conserved motif was substituted by Gly. All three showed reduced levels of resistance to kanamycin. Our results show that these conserved motifs are essential for the biological activity of aminoglycoside phosphotransferases.
Mol Microbiol 1991 Jun
PMID:Mutations in the aphA-2 gene of transposon Tn5 mapping within the regions highly conserved in aminoglycoside-phosphotransferases strongly reduce aminoglycoside resistance. 166 6

Molecular cloning of the alpha 2A-adrenergic receptor has shown that this receptor is a member of the gene superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors. The alpha 2A-adrenergic receptor expressed in Chinese hamster ovary (CHO) cells attenuates and potentiates forskolin-stimulated cAMP production through independent signaling pathways. To examine the role of three conserved aspartic acid and two conserved serine residues in alpha 2A-adrenergic receptor function, we substituted asparagine for aspartic acid or alanine for serine and characterized the mutant receptors in stably transfected CHO cells. Asn113 mutant alpha 2-adrenergic receptors display no [3H] yohimbine specific binding, at concentrations up to 1000 nM. In transfected cells expressing the Asn113 mutant receptor, agonists, at concentrations up to 0.1 mM, produce small decreases (approximately 10% of wild-type values) in forskolin-stimulated cAMP and potentiate forskolin-stimulated cAMP concentrations in a dose-dependent manner, with EC50 values approximately 500-fold higher than those for the wild-type receptor. These findings suggest that Asp113 may be involved in high affinity binding of agonists and antagonists, as has been previously reported for beta 2-adrenergic and m1 muscarinic acetylcholine receptors. Asn79 mutant alpha 2-adrenergic receptors display high affinity agonist binding that is insensitive to guanine nucleotides, suggesting an altered receptor-G protein coupling. Furthermore, agonist binding to Asn79 mutant receptors elicits no change in forskolin-stimulated cAMP concentrations, similar to earlier findings that the corresponding residue in beta 2-adrenergic and muscarinic receptors is required for effector stimulation. Asp130 appears to influence receptor-G protein coupling. Mutation of this residue eliminates high affinity, guanine nucleotide-sensitive, agonist binding and produces a rightward shift in the dose-response curves for agonist-mediated inhibition of forskolin-stimulated cAMP production, compared with the wild-type receptor. Moreover, agonist potentiation of forskolin-stimulated cAMP levels is abolished if Asp130 is replaced by Asn, supporting the hypothesis that inhibition and potentiation of forskolin-stimulated cAMP production in CHO cells proceed through distinct signaling pathways. Characterization of Ala204 mutant alpha 2A-adrenergic receptors suggests a possible role for Ser204 in hydrogen bond interactions with the para-hydroxyl group of the phenyl ring of the catecholamines, as has been previously described for the corresponding serine in beta 2-adrenergic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1991 Aug
PMID:Site-directed mutagenesis of alpha 2A-adrenergic receptors: identification of amino acids involved in ligand binding and receptor activation by agonists. 167 50


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