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S G Rhee

Publications and source records attributed to S G Rhee.

At least 91 records · Page 5Linked to original sources
PubMed1995

Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells.

Differentiation of murine erythroleukaemia cells with the chemical agent DMSO leads to a cessation of proliferation and the production of a number of erythrocyte markers such as haemoglobin. We have previously demonstrated that activation of proliferation leads to an increase in the production of nuclear diacylglycerol (DAG). Here we demonstrate that differentiation leads to a decrease in the levels of nuclear DAG and the activity of the nuclear-associated phosphoinositidase C (PIC). The change in activity appears to be due to a decrease in the mass levels of the beta 1 isoform, as demonstrated by the use of isoform-specific antibodies. Moreover, the changes correlate with the cessation of proliferation and an increase in the number of cells in G1 phase of the cell cycle, rather than with the number of cells which have differentiated. Indeed, although treatment of the cells with phorbol 12-myristate 13-acetate (PMA) inhibits the differentiation programme as assessed by haemoglobin staining, it does not inhibit the number of cells blocking in G1 of the cell cycle or the changes in nuclear DAG or PIC activity. The possible involvement of this nuclear inositide cycle during progression through the cell cycle is discussed.

Animals↗
PubMed1995

Localization of TDPX1, a human homologue of the yeast thioredoxin-dependent peroxide reductase gene (TPX), to chromosome 13q12.

Reactive oxygen species and free radicals that are produced during normal metabolism can potentially damage cellular macromolecules. Defenses against such damage include a number of antioxidant enzymes that specifically target the removal or dismutation of the reactive agent. We report here the isolation and regional mapping of a human gene, TDPX1, that encodes an enzyme homologous to a yeast thioredoxin-dependent peroxide reductase (thioredoxin peroxidase, TPX). The human TDPX1 coding sequence was determined from the product of a polymerase chain reaction (PCR) amplification of human cDNA. Based on PCR analysis of DNA from a human/rodent somatic cell hybrid panel, the TDPX1 locus was assigned to chromosome 13. Further localization of the locus to 13q12 was accomplished by fluorescence in situ hybridization analysis, using as a probe DNA from a yeast artificial chromosome (YAC) that contains the TDPX1 gene. It was also determined by PCR analysis of various YACs that the TDPX1 locus is in the region of the dinucleotide repeat markers D13S289 and D13S290. This regional mapping localizes the TDPX1 gene to a genomic region recently shown to contain the breast cancer susceptibility gene BRCA2 and a gene associated with a form of muscular dystrophy. Oxygen radical metabolism has been hypothesized to be important for cancer, muscular dystrophy, and other disorders, so TDPX1 should be considered a candidate gene for these diseases.

Amino Acid Sequence↗
PubMed1995

Different ARF domains are required for the activation of cholera toxin and phospholipase D.

ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.

ADP-Ribosylation Factor 1↗
PubMed1995

Significance of PIP2 hydrolysis and regulation of phospholipase C isozymes.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important component of several intracellular signaling pathways. It serves as a substrate for phospholipase C, which produces the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. It is also a substrate for a phosphatidylinositol 3-kinase, and regulates the function of a number of actin-binding proteins. PIP2 has been shown recently to serve as a cofactor for a phosphatidylcholine-specific phospholipase D and as a membrane-attachment site for many signaling proteins containing pleckstrin homology domains. The need to stringently regulate the cellular concentration of PIP2 is reflected in part by the fact that there are at least ten distinct mammalian phospholipase C isozymes and multiple mechanisms linking these isozymes to various receptors.

Actins↗
PubMed1994

Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes.

Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-5PW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADP-ribosylation factor (ARF) 1 and guanine 5' [gamma-thio]triphosphate, required the presence of phosphatidylinositol 4,5-bisphosphate in the phosphatidylcholine substrate for demonstration of activity, as described by others. Oleate-dependent activity was unaffected by guanine 5' [gamma-thio]triphosphate, or phosphatidylinositol 4,5-bisphosphate. Both sodium oleate-and ARF-dependent activities catalyzed transphosphatidylation, thus identifying them as PLDs. ARF-dependent PLD was activated by recombinant ARF5 (class II) and ARF6 (class III), as well as ARF1 (class I). Myristoylated recombinant ARFs were more effective than their nonmyristoylated counterparts. ARFs were originally identified as activators of cholera toxin ADP-ribosyltransferase activity. The effects of recombinant ARF proteins from the three classes on cholera toxin activity (assayed under conditions identical to those used to assay PLD activity) did not, however, correlate with those on PLD, consistent with the notion that different aspects of ARF structure are involved in the two functions.

ADP-Ribosylation Factor 1↗
PubMed1994

Thioredoxin-dependent peroxide reductase from yeast.

A 25-kDa antioxidant enzyme that provides protection against oxidation systems capable of generating reactive oxygen and sulfur species has previously been identified. The nature of the oxidant eliminated by, and the physiological source of reducing equivalents for, this enzyme, however, were not known. The 25-kDa enzyme is now shown to be a peroxidase that reduces H2O2 and alkyl hydroperoxides with the use of hydrogens provided by thioredoxin, thioredoxin reductase, and NADPH. This protein is the first peroxidase to be identified that uses thioredoxin as the immediate hydrogen donor and is thus named thioredoxin peroxidase (TPx). TPx exists as a dimer of identical 25-kDa subunits that contain 2 cysteine residues, Cys47 and Cys170. Cys47-SH appears to be the site of oxidation by peroxides, and the oxidized Cys47 probably reacts with Cys170-SH of the other subunit to form an intermolecular disulfide. Mutant TPx proteins lacking either Cys47 or Cys170, therefore, do not exhibit thioredoxin-coupled peroxidase activity. The TPx disulfide is specifically reduced by thioredoxin, but can also be reduced (less effectively) by a small molecular size thiol. The Saccharomyces cerevisiae thioredoxin reductase gene was also cloned and sequenced, and the deduced amino sequence was shown to be 51% identical with that of the Escherichia coli enzyme.

Amino Acid Sequence↗
PubMed1994

Regulation of phospholipase C-beta 4 by ribonucleotides and the alpha subunit of Gq.

The fourth member of mammalian beta-type phospholipase C isozymes, PLC-beta 4, was recently purified from bovine retina, and the corresponding cDNA was cloned from rat brain and sequenced. PLC-beta 4 has now been shown to differ from the other three mammalian beta-type isozymes (PLC-beta 1, -beta 2, and -beta 3) in that it is selectively inhibited by ribonucleotides. The inhibition requires the 5'-phosphate and 2'-hydroxyl groups of ribose as well as the base moiety. Thus, deoxyribonucleotides and ribose 5-phosphate were not inhibitory. The monophosphate, diphosphate, and triphosphate nucleoside derivatives were all inhibitory, whereas cyclic nucleotides were ineffective. Purine nucleotides were more potent inhibitors than pyrimidine nucleotides; the 50% inhibitory concentrations were 20-30 microM for AMP and GMP, and 100-200 microM for UMP and CMP. Unlike the other beta-type isozymes, PLC-beta 4 contains the GX4GKS consensus sequence for the recognition of the phosphoryl group of nucleotides. In the absence of ribonucleotides, the specific activity of PLC-beta 4 toward phosphatidyl-inositol 4,5-bisphosphate was four to five times the average specific activity of PLC-beta 1 and PLC-beta 3. Thus, nucleotide-dependent inhibition may serve to reduce the activity of PLC-beta 4 in the absence of a hormonal signal. The regulation of PLC-beta 4 by G-proteins was also studied. Similar to the other three PLC-beta isozymes, PLC-beta 4 was activated by the alpha subunit of Gq but not by the transducin alpha subunit. However, unlike other PLC-beta isozymes, PLC-beta 4 was not responsive to activation by G beta gamma subunits.

Amino Acid Sequence↗
PubMed1994

Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes.

A cDNA corresponding to a thiol-specific antioxidant enzyme (TSA) was isolated from a rat brain cDNA library with the use of antibodies to bovine TSA. The cDNA clone encoded an open reading frame capable of encoding a 198-residue polypeptide. The rat and yeast TSA proteins show significant sequence homology to the 21-kDa component (AhpC) of Salmonella typhimurium alkyl hydroperoxide reductase, and we have found that AhpC exhibits TSA activity. AhpC and TSA define a family of > 25 different proteins present in organisms from all kingdoms. The similarity among the family members extends over the entire sequence and ranges between 23% and 98% identity. A majority of the members of the AhpC/TSA family contain two conserved cysteines. At least eight of the genes encoding AhpC/TSA-like polypeptides are found in proximity to genes encoding other oxidoreductase activities, and the expression of several of the homologs has been correlated with pathogenicity. We suggest that the AhpC/TSA family represents a widely distributed class of antioxidant enzymes. We also report that a second family of proteins, defined by the 57-kDa component (AhpF) of alkyl hydroperoxide reductase and by thioredoxin reductase, has expanded to include six additional members.

Amino Acid Sequence↗
PubMed1994

Dimerization of thiol-specific antioxidant and the essential role of cysteine 47.

Thiol-specific antioxidant (TSA) from yeast contains cysteine residues at amino acid positions 47 and 170 but is not associated with obvious redox cofactors. These two cysteines are highly conserved in a family of proteins that exhibit sequence identity of 23-98% with TSA. The roles of Cys-47 and Cys-170 in yeast TSA were investigated by replacing them individually with serine and expressing the mutant TSA proteins (RC47S and RC170S, respectively), as well as wild-type TSA (RWT), in Escherichia coli. Wild-type TSA purified from yeast (YWT) and RWT were both shown to exist predominantly as dimers, whereas RC47S and RC170S existed mainly as monomers under a denaturing condition. This observation suggests that the dimerization of YWT and RWT requires disulfide linkage of Cys-47 and Cys-170. The presence of the Cys-47-Cys-170 linkage in YWT was directly shown by isolation of dimeric tryptic peptides, one monomer of which contained Cys-47 and the other contained Cys-170. A small percentage of YWT, RWT, RC47S, and RC170S molecules formed dimers linked by Cys-47-Cys-47 or Cys-170-Cys-170 disulfide bonds. The antioxidant activity of the various TSA proteins was evaluated from their ability to protect glutamine synthetase against the dithiothreitol/Fe3+/O2 oxidation system. YWT, RWT, and RC170S were equally protective, whereas RC47S was completely ineffective. Thus, Cys-47, but not Cys-170, constitutes the site of oxidation by putative substrate.

Amino Acid Sequence↗
PubMed1994

Phospholipase C-gamma 1 can induce DNA synthesis by a mechanism independent of its lipase activity.

Inositol phospholipid-specific phospholipase C (PLC) is involved in several signaling pathways leading to cellular growth and differentiation. Our previous studies reported the induction of DNA synthesis in quiescent NIH 3T3 cells after microinjection of PLC and the inhibition of serum- or Ras-stimulated DNA synthesis by a mixture of monoclonal antibodies to PLC-gamma 1. In the course of our investigation of anti-PLC-gamma 1 monoclonal antibodies, we found that each antibody exerts different inhibitory effects on the phosphatidylinositol-hydrolyzing activity of PLC-gamma 1 and that the inhibition of enzymatic activity does not correlate with the inhibition of DNA synthesis observed in the microinjection assay. PLC-gamma 1 with defective enzymatic activity was synthesized by substituting phenylalanine for histidine within the PLC-gamma 1 catalytic domain at amino acids 335 and 380, and mutant enzymes were expressed using a vaccinia expression system. The mutant enzymes were purified and microinjected into quiescent NIH 3T3 cells to evaluate their mitogenic activity. A moderate induction of DNA synthesis occurred after injection of mutant PLC-gamma 1. This mitogenic activity was inhibited by an antibody (alpha E 8-4) that does not significantly inhibit PLC-gamma 1 enzyme activity, which indicates that something else has to be inhibited. Furthermore, the partial induction of DNA synthesis observed with mutant PLC-gamma 1 was increased to levels seen with wild-type PLC-gamma 1 by coinjection of mutant PLC-gamma 1 with two second messengers, diacylglycerol and inositol trisphosphate. These results suggest that the mitogenic activity of PLC-gamma 1 does not exclusively result from the enzymatic activity of the lipase and that another activity inherent to the PLC-gamma 1 molecule can also induce DNA synthesis in quiescent cells.

3T3 Cells↗
PubMed1994

Changes in IP3 3-kinase immunoreactivity following transient ischaemia in gerbil hippocampus.

The distribution of inositol 1,4,5-triphosphate 3-kinase (IP3 3-kinase) in the gerbil hippocampus was studied before and after transient ischaemia, by immunohistochemistry and immunoblot with monoclonal antibody to IP3 3-kinase. In control gerbils, intense IP3 3-kinase immunoreactivity was localized in the dendritic field of CA1 pyramidal neurones. However, after ischaemia for 5 min, the immunoreactivity decreased gradually, until after 24 h there was very little IP3 3-kinase detectable. Immunoblot study showed a similar decrease of IP3 3-kinase in ischaemic hippocampus. Infliction of 2 min ischaemia prior to the 5 min of ischaemia is known to have a protective effect on the 5 min ischaemia. With the addition of the 2 min ischaemia there was no decrease in IP3 3-kinase following the 5 min ischaemia. The results suggest that IP3 3-kinase might play a critical role in inducing the delayed neuronal death following ischaemia.

Animals↗
PubMed1994

Effect of monolayer surface pressure on the activities of phosphoinositide-specific phospholipase C-beta 1, -gamma 1, and -delta 1.

Three isoforms of phospholipase C, either PLC-beta 1, PLC-gamma 1, or PLC-delta 1, were added to the aqueous subphase beneath phospholipid monolayers formed at an air-solution interface, and the initial rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate was measured after addition of 10 microM free Ca2+. The monolayers were formed from mixtures of phosphatidylcholine (65% PC), phosphatidylserine (33% PS), and phosphatidylinositol 4,5-biphosphate (2% PIP2). Increasing the surface pressure of the monolayer, pi, from 15 to 25 mN/m decreases the rate of hydrolysis 16-, 13-, and 5-fold for PLC-beta 1, PLC-gamma 1, and PLC-delta 1, respectively. The simplest interpretation of these results is that a portion of each of the enzymes of area Ap must insert into the monolayer, doing work pi Ap, prior to hydrolysis of PIP2; binding studies with simple model compounds of known cross-sectional area are consistent with this interpretation. Removing the monovalent acidic lipid PS from the monolayer decreases the initial rates of hydrolysis of PIP2 about 3-fold for each PLC isoform, which suggests that negative electrostatic surface potentials increase the PLC activity.

Animals↗
PubMed1994

Epidermal growth factor-induced activation and translocation of phospholipase C-gamma 1 to the cytoskeleton in rat hepatocytes.

In this study, we have examined the relationship between epidermal growth factor (EGF)-induced tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and its translocation from the cytosol to the Triton X-100-insoluble cytoskeleton fraction in rat hepatocytes. The translocation of PLC-gamma 1 was specific for EGF stimulation, because a similar effect was not observed with insulin or vasopressin. EGF caused a transient increase of PLC activity in the cytoskeleton fraction which could be abolished by immunoprecipitating PLC-gamma 1. Tyrosine phosphorylated PLC-gamma 1 was seen only in the cytoskeleton fraction, suggesting that tyrosine phosphorylation is required for PLC-gamma 1 translocation to the cytoskeleton. This process may involve binding of PLC-gamma 1 to actin filaments, since actin was immunoprecipitated together with PLC-gamma 1 in the cytoskeleton after EGF treatment. EGF-induced translocation of PLC-gamma 1 to the cytoskeleton was not inhibited by pertussis toxin, but Gi alpha was translocated in an EGF-dependent manner, suggesting that the interaction of PLC-gamma 1 with its activated Gi-protein is downstream from both PLC-gamma 1 tyrosine phosphorylation and its translocation to the cytoskeleton. Taken together, the present studies indicate that EGF-induced tyrosine phosphorylation of PLC-gamma 1, its association with the cytoskeleton, and its interaction with activated Gi alpha protein are all obligatory for PLC-gamma 1 activation in hepatocytes.

Actins↗
PubMed1994

Regulation of chemotaxis by the platelet-derived growth factor receptor-beta.

Chemotaxis is an important component of wound healing, development, immunity and metastasis, yet the signalling pathways that mediate chemotaxis are poorly understood. Platelet-derived growth factor (PDGF) acts both as a mitogen and a chemoattractant. Upon stimulation, the tyrosine kinase PDGF receptor-beta (PDGFR-beta) autophosphorylates and forms a complex that includes SII2(Src homology 2)-domain-containing proteins such as the phosphatidylinositol-specific phospholipase C-gamma, Ras-GTPase-activating protein (GAP), and phosphatidylinositol-3-OH kinase. Specific tyrosine-to-phenylalanine substitutions in the PDGFR-beta can prevent binding of one SH2-domain-containing protein without affecting binding of other receptor-associated proteins. Here we use phospholipase C-gamma and PDGFR-beta mutants to map specific tyrosines involved in both positive and negative regulation of chemotaxis towards the PDGF-BB homodimer. Our results indicate that a delicate balance of migration-promoting (phospholipase C-gamma and phosphatidylinositol-3-OH kinase) and migration-suppressing (GAP) activities are recruited by the PDGFR-beta to drive chemotaxis towards PDGF-BB.

3T3 Cells↗
PubMed1994

On the protective mechanism of the thiol-specific antioxidant enzyme against the oxidative damage of biomacromolecules.

A thiol-specific antioxidant enzyme (TSA), which provides protection against the inactivation of other enzymes by the thiol/Fe(III)/oxygen system, was previously isolated and cloned. We investigated the mechanism by which TSA protects biomolecules from oxidative damage caused by the thiol-containing oxidation system using the spin trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Thiyl radicals from dithiothreitol (.DTT) were produced by horseradish peroxidase/H2O2 under aerobic and anaerobic conditions and by the Fe(III)/oxygen system. The formation of DMPO-.DTT radical adducts were inhibited by TSA regardless of the thiyl radical-generating conditions used. The active mutant C170S also quenched the signals of the radical adduct, whereas the inactive mutant C47S did not exert any effect. It was also found that C170S has a higher rate at the initial stage of the reaction than that of the native enzyme, although C170S failed to remove DMPO-.DTT radical adducts completely. These results indicate that only active TSA can catalyze the removal of thiyl radicals, and cysteine 47 is required for this activity. In addition, thiyl radicals react with oxygen to generate unidentified thiylperoxy species. Fe.EDTA reacts with this species to generate a reactive radical that can abstract hydrogen atom from ethanol to produce a hydroxyethyl radical. This reactive thiyl-oxygen radical is believed to be responsible for causing deleterious effects on biomolecules. Together, our data indicate that TSA protects biomolecules from oxidative damage by catalyzing the removal of thiyl radicals before they generate more reactive radicals. However, presently we cannot rule out the possibility that TSA can also use other thiol-containing species as substrates.

Antioxidants↗
PubMed1994

Differences in the regulation of phosphatidylinositol-specific phospholipase C in normal and neoplastic keratinocytes.

The induction of epidermal differentiation by Ca2+ in vitro is associated with enhanced activity of phosphatidylinositol-specific phospholipase C (PLC). Neoplastic keratinocyte cell lines expressing a mutant c-Ha-ras gene and normal keratinocytes transformed to the neoplastic phenotype by transduction with the v-Ha-ras gene (v-Ha-ras keratinocytes) have elevated constitutive activity of PLC that increases further in response to Ca2+, but the cells do not differentiate normally. PLC-gamma 1 (145 kDa) is the major isoform detected by immunoblotting of extracts from control, v-Ha-ras, and neoplastic keratinocyte cell lines cultured in 0.05 mM Ca2+ medium. The amount of PLC-gamma 1 protein was higher in neoplastic cell lines than in normal and v-Ha-ras keratinocytes that had similar PLC-gamma 1 protein levels. Thus, higher PLC-gamma 1 protein levels cannot account for the elevated constitutive activity PLC in v-Ha-ras keratinocytes. After induction of differentiation by Ca2+, the amount of PLC-gamma 1 protein increased in all cell types, and PLC-delta 1 (85 kDa), barely detectable in 0.05 mM Ca2+, increased. PLC-beta 1 was not detected at any Ca2+ concentration. PLC-gamma 1 and PLC-delta 1 mRNA did not increase after elevation of extracellular Ca2+, suggesting that posttranscriptional mechanisms can regulate PLC-gamma 1 and PLC-delta 1 protein levels in normal and neoplastic keratinocytes. Activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the stimulation of inositol phosphate (InsP) formation by Ca2+ but did not alter basal InsP levels in normal keratinocytes. In contrast, TPA treatment reduced both Ca(2+)-stimulated and basal InsP formation in neoplastic cells lines and v-Ha-ras keratinocytes. In both normal and v-Ha-ras keratinocytes labeled with [32P]orthophosphate, antibodies against PLC-gamma 1 immunoprecipitated a complex of 32P-labeled proteins. The relative labeling of the PLC-gamma 1 band was greater in normal than in v-Ha-ras keratinocytes. Furthermore, treatment with TPA specifically increased the relative phosphorylation of PLC-gamma 1 in v-Ha-ras keratinocytes but not in normal keratinocytes. These results suggest that the negative regulation of constitutive activity of PLC by protein kinase C differs in normal and neoplastic keratinocytes and that this could be the mechanism of increased PLC activity produced by an oncogenic ras gene in keratinocytes.

Animals↗