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S Nada

Publications and source records attributed to S Nada.

At least 19 recordsLinked to original sources
PubMed2006

The interferon-induced GTPase, mGBP-2, confers resistance to paclitaxel-induced cytotoxicity without inhibiting multinucleation.

Interferon (IFN) exposure promotes a wide variety of cellular changes, many of which are still poorly characterized. Many of these changes are initiated through the induction or repression of hundreds of genes. One multigene family of GTPases induced by both type I and type II IFNs is the Guanylate Binding Protein (GBP) family. In this study we show that a murine GBP, mGBP-2, confers resistance to a microtubule-stabilizing drug used in cancer therapy, paclitaxel. Paclitaxel-treated, mGBP-2-expressing NIH 3T3 cells showed less rounding and substrate detachment than treated control cells. mGBP-2 also conferred resistance to paclitaxel-induced cytotoxicity at paclitaxel concentrations from 0.005 to 15 microM. mGBP-2-mediated paclitaxel resistance did not protect against the generation of multinucleate cells but fewer of these cells progressed to apoptosis.

Animals↗
PubMed2001

Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter.

Bacterial Tn10-encoded metal-tetracycline/H(+) antiporter was the first found drug exporter and has been studied as a paradigm of antiporter-type major facilitator superfamily transporters. Here the 400 amino acid residues of this protein were individually replaced by cysteine except for the initial methionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the precise boundaries of all the transmembrane segments on the basis of the reactivity with N-ethylmaleimide (NEM). The NEM binding results indicated the presence of a transmembrane water-filled channel in the transporter. The twelve transmembrane segments can be divided into three groups; four are totally embedded in the hydrophobic interior, four face a putative water-filled channel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic interior. These three types of transmembrane segments are mutually arranged with a 4-fold symmetry. The competitive binding of membrane-permeable and -impermeable SH reagents in intact cells indicates that the transmembrane water-filled channel has a thin barrier against hydrophilic molecules in the middle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection or conformational change of the Tn10-encoded metal-tetracycline/H(+) antiporter. The mutations protected from NEM binding by tetracycline were mainly located around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.

Amino Acid Sequence↗
PubMed2001

Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli.

Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)His] as an antigen. From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1. Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein. First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed. Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C. Then, we constructed 13 cysteine-scanning mutants, F179C to T191C. Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding. These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B). TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.

Amino Acid Sequence↗
PubMed2000

Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2.

Multidrurg resistance-associated protein 2 (MRP2)/canalicular multispecific organic anion transporter (cMOAT) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane. To identify functional amino acid residues that play essential roles in the substrate transport, each of 13 basic residues around transmembrane regions (TMs) 6-17 were replaced with alanine. Wild type and mutant proteins were expressed in COS-7 cells, and the transport activity was measured as the excretion of glutathione-methylfluorescein. Four mutants, K324A (TM6), K483A (TM9), R1210A (TM16), and R1257A (TM17), showed decreased transport activity, and another mutant, K578A (TM11), showed decreased protein expression. These five mutants were normally delivered to the cell surface similar to the other fully active mutants and wild type MRP2. The importance of TM6, TM16, and TM17 in the transport function of MRP2 is consistent with the previous observation indicating the importance of the corresponding TM1, TM11, and TM12 on P-glycoprotein (Loo, T. W., and Clarke, D. M. (1999) J. Biol. Chem. 274, 35388-35392). Another observation that MRP2 inhibitor, cyclosporine A, failed to inhibit R1230A specifically, indicated the existence of its binding site within TM16.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel.

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Amino Acid Sequence↗
PubMed2000

Transmembrane phosphoprotein Cbp regulates the activities of Src-family tyrosine kinases.

The Src family of protein tyrosine kinases (Src-PTKs) is important in the regulation of growth and differentiation of eukaryotic cells. The activity of Src-PTKs in cells of different types is negatively controlled by Csk, which specifically phosphorylates a conserved regulatory tyrosine residue at the carboxy-terminal tail of the Src-PTKs. Csk is mainly cytoplasmic and Src-PTKs are predominantly membrane-associated. This raises a question about the mechanism of interaction between these enzymes. Here we present Cbp--a transmembrane phosphoprotein that is ubiquitously expressed and binds specifically to the SH2 domain of Csk. Cbp is involved in the membrane localization of Csk and in the Csk-mediated inhibition of c-Src. In the plasma membrane Cbp is exclusively localized in the GM1 ganglioside-enriched detergent-insoluble membrane domain, which is important in receptor-mediated signalling. These findings reveal Cbp as a new component of the regulatory mechanism controlling the activity of membrane-associated Src-PTKs.

Amino Acid Sequence↗
PubMed2000

Proximity of periplasmic loops in the metal-Tetracycline/H(+) antiporter of Escherichia coli observed on site-directed chemical cross-linking.

Our previous study on second-site suppressor mutations of the Tn10-encoded metal-tetracycline/H(+) antiporter suggested that Leu(30) and Ala(354), located in periplasmic loop 1-2 and 11-12, respectively, are conformationally linked to each other (Kawabe, T., and Yamaguchi, A. (1999) FEBS Lett. 457, 169-173). To determine the spatial proximity of these two residues, cross-linking gel-shift assays of the L30C/A354C double mutant were performed after the mutant had been oxidized with Cu(2+)/o-phenanthroline. The results indicated that Leu(30) and Ala(354) are close to each other but that Gly(62), which is located in cytoplasmic loop 2-3, and Ala(354) are distant from each other, as a negative control. Then, a single Cys residue was introduced into each of the six periplasmic loop regions (P1-P6), and eleven double mutants were constructed. Of these eleven double Cys mutants, the L30C/A354C and L30C/T235C mutants showed a mobility shift on oxidation, indicating that P1 is spatially close to P4 as well as P6. In contrast, the other nine mutants, L30C/S92C, L30C/S156C, L30C/S296C, S92C/S296C, S92C/T235C, S92C/A354C, S156C/T235C, S156C/S296C, and S156C/A354C, showed no mobility shift under oxidized conditions on intramolecular cross-linking. The S92C and S296C mutants showed dimerization on intermolecular cross-linking, indicating that P2 and P5 are located at the periphery of the helix bundle.

Amino Acid Sequence↗
PubMed1998

Essential roles of Lyn in fibronectin-mediated filamentous actin assembly and cell motility in mast cells.

Although the requirement for c-Src in extracellular matrix (ECM)-mediated fibroblast motility has been well established, the roles of hemopoietic Src family protein tyrosine kinases in leukocyte migration have not been fully elucidated. To address the issue, we analyzed fibronectin (Fn)-mediated adhesion signaling in rat basophilic leukemia (RBL) 2H3 cells overexpressing 1) Csk, 2) a membrane-anchored, gain-of-function Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(-)). Parent RBL2H3 cells, expressing autoactivated c-kit, readily adhered to Fn-coated surface, developed typical leukocyte adhesion machinery (podosome), and migrated toward Fn without cytokine priming, thus provided a simple experimental system to analyze Fn-mediated outside-in signaling. While overexpression of Csk or the Csk mutants did not significantly affect cell adhesion to the Fn surface or alpha5 integrin recruitment to the attachment sites, Csk suppressed and mCsk almost abolished Fn-mediated tyrosine phosphorylation of paxillin, filamentous actin assembly to podosomes, and cell migration, but mCsk(-) did not. Coexpression of LynA devoid of C-terminal negative regulatory tyrosine in mCsk cells successfully restored Fn-mediated podosome formation and cell migration. Coexpression of c-Src lacking the C-terminal tyrosine reconstructed podosomes, but could not restore the cell migration regardless of its expression level. Collectively, these observations provide evidence that Src family protein tyrosine kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated cytoskeletal changes leading to cell locomotion in RBL2H3 cells, and they suggest that Lyn and c-Src are differentially involved in cell motility.

Actins↗
PubMed1997

Roles of C-terminal Src kinase in the initiation and the termination of the high affinity IgE receptor-mediated signaling.

As an attempt to analyze the roles of C-terminal Src kinase (Csk) in the high affinity IgE receptor (FcepsilonRI)-mediated signaling, we overexpressed Csk, a membrane-targeted form of Csk (mCsk), and a kinase-defective, membrane-targeted form of Csk (mCsk(-)) in rat basophil leukemia (RBL) 2H3 cells. Specific activity of Lyn at the basal state was decreased in Csk-expressing cells, and further decreased in mCsk-expressing cells. In mCsk(-)-expressing cells, basal specific activity of Lyn was increased, thereby indicating that mCsk(-) functioned as a dominant negative molecule. The onset of FcepsilonRI-mediated Lyn activation was delayed in Csk-expressing cells, and further delayed in mCsk-expressing cells. In mCsk(-)-expressing cells, Lyn activation was rapid and quite long lasting. These findings indicate (i) Csk negatively regulates rapid FcepsilonRI/Lyn coupling, and (ii) Csk activity is potentially required for its termination. The onsets of the series of events including tyrosyl phosphorylation of Syk, mitogen-activated protein (MAP) kinase activation, elevation of intracellular calcium concentration ([Ca2+]i), and histamine release were all stepwisely delayed in Csk-expressing cells and in mCsk-expressing cells. The durations of Syk phosphorylation and MAP kinase activation also closely correlated with those of Lyn activation, but [Ca2+]i elevation and histamine release followed different temporal patterns: the delayed responses in Csk-expressing cells and in mCsk-expressing cells led to sustained [Ca2+]i oscillation and histamine release, while the prompt responses in parent cells and mCsk(-)-expressing cells rapidly subsided. These findings provide further evidence that the initiations of the FcepsilonRI-mediated signals are upstreamly regulated by Src family protein tyrosine kinases and revealed that their terminations are regulated by Lyn-dependent (Syk and MAP kinase) and -independent ([Ca2+]i elevation and histamine release) mechanisms.

Amino Acid Sequence↗
PubMed1997

Role of Csk in neural differentiation of the embryonic carcinoma cell line P19.

To examine the neural function of Csk (C-terminal Src kinase), a membrane-targeted form of Csk (Src/Csk) and its kinase-defective variant (DK-Src/Csk) were expressed in the embryonic carcinoma cell line P19. Expression of Src/Csk, but not DK-Src/Csk, caused reduction of the specific activities of Src and Fyn in the differentiated P19 cells. During neural differentiation, the specific activity of Src was elevated in the control P19 cells, whereas the activation was completely eliminated in the Src/Csk transfectant. In normally differentiated P19 cells, cross-linking of a cell adhesion molecule, L1, induced a short-term activation of Src and Fyn. In the Src/Csk transfectant, L1 stimulation induced delayed activation of Src and Fyn peaking at much lower levels than in the control cells. Src/Csk transfectants developed normally in the initial stages of neural differentiation, but exhibited an apparent defect in cell-to-cell interaction, i.e. neurite fasciculation and aggregation of cell bodies, in the latter stages. These findings imply that Csk is involved in the regulation of Src family kinases that play roles in cell-to-cell interaction mediated by cell adhesion molecules.

Animals↗
PubMed1995

Analysis of truncated forms of Bombyx mori glycyl-tRNA synthetase: function of an N-terminal structure in RNA binding.

Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl-tRNA synthetase activity compared to wild type enzyme. Active site titration indicated that this difference in activity was not the result of production of inactive enzyme. Sedimentation and gel filtration experiments indicate that the N-terminally deleted forms and the wild type enzyme were dimers. Deletion of 55 N-terminal residues did not result in significant effects on the Michaelis constants for ATP, glycine, or tRNA, while deletion of 108 N-terminal residues and two internal 64- and 200-residue deletions generated inactive forms. Five forms with C-terminal deletions of 24, 37, 59, 162, and 327 amino acid residues were soluble and intact but lacked detectable pyrophosphate exchange activity or aminoacyl-tRNA synthetase activity. The C-terminal sequence may be required for catalysis or to maintain a stable structure. Zone electrophoresis demonstrated the wild type enzyme bound both tRNA(Gly) and noncognate tRNA(Ala). Deletion of 55 N-terminal residues resulted in altered binding of tRNA(Gly) and eliminated binding of tRNA(Ala). The first 55 N-terminal residues are not essential for catalysis, dimerization, or substrate binding in aminoacylation but are required for RNA binding not associated with aminoacylation.

Adenosine Triphosphate↗
PubMed1995

Control of lymphopoiesis by p50csk, a regulatory protein tyrosine kinase.

The csk gene encodes a nonreceptor protein tyrosine kinase that acts in part by regulating the activity of src-family protein tyrosine kinases. Since the src-family kinases p56lck and p59fyn play pivotal roles during lymphocyte development, it seemed plausible that p50csk might contribute to these regulatory circuits. Using a gene targeting approach, mouse embryonic stem cell lines lacking functional csk genes were generated. These csknull embryonic stem cells proved capable of contributing to many adult tissues, notably heart and brain. However, although csknull progenitors colonized the developing thymus, T and B cell differentiation were both blocked at very early stages. This represented a relatively selective interdiction of lymphocyte maturation, since csknull hematopoietic progenitors supported the development of normal-appearing MAC-1+ blood leukocytes, and the successful maturation of granulocyte/macrophage-colony-forming units from fetal liver progenitors. We conclude that p50csk regulates normal lymphocyte differentiation, but that it almost certainly does so by acting on targets other than p56lck and p59fyn.

Alleles↗
PubMed1994

Regulation of Src family kinases in the developing rat brain: correlation with their regulator kinase, Csk.

We have so far suggested that the functions of Src family protein-tyrosine kinases are under the control of their regulator kinase, Csk. To evaluate the role of Csk-mediated regulation in neural tissues, we examined the correlation between the activities of Src family kinases and the expression level of Csk during development of the rat brain. Csk was expressed at high levels in the developing embryonic brain and then rapidly decreased as the brain matured. Consistent with the decrease in the Csk level, the kinase activity of a member of the Src family, Fyn, was greatly enhanced, but that of Src was not correlated inversely with the level of Csk expression. Src exhibited elevated activity in the developing brain, in which a neuronal form of Src (N-Src) is expressed as the dominant form of Src. Although N-Src was readily down-regulated by Csk when coexpressed in yeast, it showed much higher specific activity than c-Src, even in the repressed form. These findings suggest that neural tissues acquire high activities of Src family kinases, which might be important for differentiation and development of the nervous system, through induction of the active form of Src (N-Src) and down-regulation of their suppresser, Csk.

Amino Acid Sequence↗
PubMed1994

Mitotic activation of c-Src is suppressed by Csk.

The kinase activity of the proto-oncogene product, c-Src, increases during mitosis through partial dephosphorylation of Tyr527, the negative regulatory site of c-Src. To examine whether or not Csk, a candidate kinase specific for Tyr527, is involved in this regulation, we developed a Balb/c 3T3 cell line overexpressing Csk and a Csk-deficient cell line. The overexpression of wild-type Csk caused significant suppression of the c-Src activity during mitosis. A membrane-targeted Csk, which has an amino-terminal myristylation signal of c-Src, exhibited an effective suppression of the c-Src activity, even though its expression level was lower than that of endogenous Csk. Concomitant with the suppression of the c-Src activation, the level of tyrosine phosphorylation of a cortactin-related protein, a potential substrate of c-Src in vivo, was reduced. In contrast, the Csk-deficient cells exhibited constitutive activation of c-Src, which showed no significant change in its activity during mitosis. These results suggest that Csk indeed participates in the regulation of the c-Src activity during mitosis.

3T3 Cells↗
PubMed1994

Identification of major tyrosine-phosphorylated proteins in Csk-deficient cells.

Csk is a non-receptor protein-tyrosine kinase that acts as a negative regulator of Src family tyrosine kinases. Csk-deficient mouse embryos exhibited developmental defects including inability to turn and impaired formation of neural tube. In these embryos, an accumulation of tyrosine phosphorylated proteins was observed as a consequence of constitutive activation of Src family kinases. In order to identify those tyrosine phosphorylated proteins, we established a Csk-deficient cell line from embryos lacking both Csk and the anti-oncogene product p53. On surveying several proteins known as Src substrates, we found that phosphorylation level of p80/85 (cortactin) was markedly elevated in the Csk-deficient cells. Enhancement of cortactin phosphorylation was also seen in Csk-deficient embryos. Furthermore, immunoprecipitated Src was able to directly phosphorylate cortactin in vitro. Thus, we suggest that cortactin is a good substrate of activated Src family kinases in vivo and may play important roles in signaling pathways mediated by Src family kinases.

Animals↗
PubMed1994

Fyn expression during early neurogenesis in mouse embryos.

Fyn is a member of the Src family of tyrosine kinases which are thought to play important roles in cell to cell interactions during morphogenesis. The developmental profile of Fyn expression was examined using mutant mice in which lacZ gene was introduced into this locus. The expression was characteristic in the neural system. Though at low levels, it was detected in the headfold at embryonic day (E) 7.5 and in the luminal surface of neuroectoderm along the entire neural groove at E8.5. The expression appeared regional in rhombomeres at E8.5 and E9.5. Consistent expression was also found at a low level in the notochord. The expression was high in later stages of the neural tube which consists of three layers; it was in the marginal layer but not in the germinal layer. High expression was also found in developing dorsal root filaments of neural crest origin. Non-expression in dividing neuroepithelial cells and expression in developing neural fibers appeared ubiquitous features of Fyn expression throughout the entire brain.

Animals↗