PubMed Health⌕ Search

Biomedical subjects

N Morii

Publications and source records attributed to N Morii.

At least 19 recordsLinked to original sources
PubMed1998

Intracellular Signal-transducing elements involved in transendothelial migration of lymphoma cells.

To investigate the molecular mechanisms underlying transendothelial migration of tumor cells, an essential process for their hematogenous dissemination, we developed an in vitro model system that allows the separate monitoring of cell adhesion and transmigration processes. This system uses a human pre-B lymphoma cell line, Nalm-6, and a cultured mouse endothelial cell line, KOP2.16. Nalm-6 cells rapidly adhered to KOP2.16 and subsequently transmigrated underneath them. Using this model, we examined the effects on transendothelial migration, of various reagents which specifically interfere with the function of intracellular signal transduction molecules. Treatment of Nalm-6 cells with wortmannin (WMN), herbimycin A, pertussis toxin, or C3 exoenzyme of Clostridium botulinum, which specifically inhibit P13 kinase and/or myosin light chain kinase, herbimycin-sensitive tyrosine kinases, heterotrimeric G proteins, and the small G proteins, and the small G proteins rho/rac, respectively, reduced transmigration in a dose-dependent manner, Pretreatment of KOP2.16 endothelial cells with WMN also reduced transmigration in a dose-dependent manner. Binding of Nalm-6 binding to KOp2.16 was not affected, even when Nalm-6 or KOP2.16 cells were pretreated with these inhibitors, indicating that the reduction of transmigration was not due to a reduction of Nalm-6 to KOP2.16. These results also indicate that the signal transduction pathway(s) involved in transmigration can be dissociated from that of adhesion. Our results support the notion that endothelial cells are not a passive barrier in lymphoma extravasation, but that they assist lymphoma cell extravasation.

Androstadienes↗
PubMed1996

Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain.

Using a mouse embryo cDNA library, we conducted a two-hybrid screening to identify new partners for the small GTPase Rho. One clone obtained by this procedure contained a novel cDNA of 291 base pairs and interacted strongly with RhoA and RhoC, weakly with RhoB, and not at all with Rac1 and Cdc42Hs. Full-length cDNAs were then isolated from a mouse brain library. While multiple splicing variants were common, we identified three cDNAs with an identical open reading frame encoding a 61-kDa protein that we named rhotekin (from the Japanese "teki," meaning target). The N-terminal part of rhotekin, encoded by the initial cDNA and produced in bacteria as a glutathione S-transferase fusion protein, exhibited in vitro binding to 35S-labeled guanosine 5'-3-O-(thio)triphosphate-bound Rho, but not to Rac1 or Cdc42Hs in ligand overlay assays. In addition, this peptide inhibited both endogenous and GTPase-activating protein-stimulated Rho GTPase activity. The amino acid sequence of this region shares approximately 30% identity with the Rho-binding domains of rhophilin and a serine/threonine kinase, PKN, two other Rho target proteins that we recently identified (Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y., Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). Thus, not only is rhotekin a novel partner for Rho, but it also belongs to a wide family of proteins that bear a consensus Rho-binding sequence at the N terminus. To our knowledge, this is the first conserved sequence for Rho effectors, and we have termed this region Rho effector motif class 1.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Roles of insulin, guanosine 5'-[gamma-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation.

Insulin, guanosine 5'-[gamma-thio]triphosphate (GTP[S] and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of Gl UT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rass17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways.

3T3 Cells↗
PubMed1996

The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1996

Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho.

The Rho guanosine 5'-triphosphatase (GTPase) cycles between the active guanosine triphosphate (GTP)-bound form and the inactive guanosine diphosphate-bound form and regulates cell adhesion and cytokinesis, but how it exerts these actions is unknown. The yeast two-hybrid system was used to clone a complementary DNA for a protein (designated Rhophilin) that specifically bound to GTP-Rho. The Rho-binding domain of this protein has 40 percent identity with a putative regulatory domain of a protein kinase, PKN. PKN itself bound to GTP-Rho and was activated by this binding both in vitro and in vivo. This study indicates that a serine-threonine protein kinase is a Rho effector and presents an amino acid sequence motif for binding to GTP-Rho that may be shared by a family of Rho target proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton.

Hypo-osmotic stimulation of human Intestine 407 cells rapidly activated compensatory CL- and K+ conductances that limited excessive cell swelling and, finally, restored the original cell volume. Osmotic cell swelling was accompanied by a rapid and transient reorganization of the F-actin cytoskeleton, affecting both stress fibers as well as apical ruffles. In addition, an increase in total cellular F-actin was observed. Pretreatment of the cells with recombinant Clostridium botulinum C3 exoenzyme, but not with mutant enzyme (C3-E173Q) devoid of ADP-ribosyltransferase activity, greatly reduced the activation of the osmo-sensitive anion efflux, suggesting a role for the ras-related GTPase p21rho. In contrast, introducing dominant negative N17-p21rac into the cells did not affect the volume-sensitive efflux. Cell swelling-induced reorganization of F-actin coincided with a transient, C3 exoenzyme-sensitive tyrosine phosphorylation of p125 focal adhesion kinase (p125FAK) as well as with an increase in phosphatidylinositol-3-kinase (PtdIns-3-kinase) activity. Pretreatment of the cells with wortmannin, a specific inhibitor of PtdIns-3-kinase, largely inhibited the volume-sensitive ion efflux. Taken together, our results indicate the involvement of a p21rho signaling cascade and actin filaments in the activation of volume-sensitive chloride channels.

ADP Ribose Transferases↗
PubMed1996

The involvement of the rho gene product, a small molecular weight GTP-binding protein, in polyploidization of a human megakaryocytic cell line, CMK.

The role of rho proteins, which are ras p21-related small GTP-binding proteins, in megakaryocyte endomitosis was examined using a botulinum C3 exoenzyme (C3), a rho inactivating enzyme. The megakaryocytic leukemia cell line CMK expressed high levels of rhoA and rhoC mRNAs, whereas rhoB mRNA was expressed at a very low level. The addition of C3 to the culture medium caused ADP-ribosylation of the rho proteins in CMK cells in a dose- and time-dependent manner. This procedure also induced a higher frequency of polyploid cells with increased glycoprotein (GP) IIb/IIIa antigens on the cells. This effect of C3 on both ploidy and the antigen expression was abolished by prior incubation of C3 with an anti-C3 monoclonal antibody. Cytochalasin B, an actin polymerization inhibitor, also induced polyploid cells; however, it did not stimulate the expression of GP IIb/IIIa antigens in CMK cells. This finding suggests that C3-induced increase in the expression of GP IIb/IIIa antigens was not through the actin microfilament disassembly. The present study suggests that the rho p21 is a partly regulatory component in polyploidization and GP IIb/IIIa antigen expression of a human megakaryocytic cell line, CMK.

ADP Ribose Transferases↗
PubMed1995

A novel partner for the GTP-bound forms of rho and rac.

Using the yeast two hybrid system and overlay assays we identified a putative rholrac effector, citron, which interacts with the GTP-bound forms of rho and rac1, but not with cdc42. Extensive homologies to known proteins were not observed. This 183 kDa protein contains a C6H2 zinc finger, a PH domain, and a long coiled-coil forming region including 4 leucine zippers and the rholrac binding site. We recently identified three others putative rho effectors characterized by a common rho binding motif. Citron does not share this motif and displays a distinctive protein organization, thus defining a separate class of rho partners.

Amino Acid Sequence↗
PubMed1995

Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21, and its inhibition by Clostridium botulinum C3 exoenzyme.

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.

ADP Ribose Transferases↗
PubMed1995

Identification of Glu173 as the critical amino acid residue for the ADP-ribosyltransferase activity of Clostridium botulinum C3 exoenzyme.

Clostridium botulinum C3 exoenzyme specifically ADP-ribosylates rho-p21 in eukaryotic cells. Trp18 and Glu173 of this enzyme were substituted with other amino acids via site-directed mutagenesis. All substitutions at Glu173 caused a significant reduction in affinity for NAD and diminished ADP-ribosyltransferase activity. On the other hand, the activity of enzymes with the substitution at Trp18 remained intact. Swiss 3T3 cells treated with the enzyme with the Trp18 substitution showed the typical morphologic changes of the C3 exoenzyme phenotype. In contrast, no changes were found in cells incubated with the Glu173-substituted enzyme. These results indicate that the Glu173 residue of the C3 exoenzyme plays a key role in interacting with NAD and in expression of ADP-ribosyltransferase activity, which is essential for the phenotypic change by C3 exoenzyme treatment.

3T3 Cells↗
PubMed1995

Lysophosphatidic acid-induced activation of protein Ser/Thr kinases in cultured rat 3Y1 fibroblasts. Possible involvement in rho p21-mediated signalling.

Renaturation kinase assay was used to detect protein kinases activated by lysophosphatidic acid (LPA) in cultured rat 3Y1 fibroblasts. LPA activated several Ser/Thr protein kinases with apparent molecular weights of 145K, 85K, 64-65K (a doublet), and 60K (each named p145, p85, p64165 and p60, respectively) in addition to p43 mitogen activated protein (MAP)-kinase. Experiments using pertussis toxin and botulinum C3 exoenzyme showed that p145, p85, and p64165 kinases were activated by a pertussis toxin-insensitive rho p21-dependent pathway and that the activation of MAP-kinase was mediated by both the pertussis toxin-sensitive rho p21-independent and the pertussis toxin-insensitive rho p21-dependent pathways.

ADP Ribose Transferases↗
PubMed1995

Guanosine 5'-3-O-(thio)triphosphate stimulates tyrosine phosphorylation of p125FAK and paxillin in permeabilized Swiss 3T3 cells. Role of p21rho.

Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands. Specifically, GTP gamma S stimulated tyrosine phosphorylation of both focal adhesion kinase (p125FAK) and paxillin. GTP gamma S induced tyrosine phosphorylation was dose-dependent (EC50 of 2.5 microM) and reached maximum levels after 1.5 min for the M(r) 110,000-130,000 band and 2 min for the M(r) 70,000-80,000 paxillin band. Guanosine 5'-O-(2-thiodiphosphate) inhibited GTP gamma S-induced tyrosine phosphorylation with an IC50 of 100 microM. Protein kinase C did not mediate GTP gamma S-induced tyrosine phosphorylation. Varying the Ca2+ concentration from 0 to 6 microM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTP gamma S to induce tyrosine phosphorylation. GTP gamma S was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg2+. Furthermore, 30 microM AlF4- only weakly induced tyrosine phosphorylation in permeabilized cells. Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21rho, markedly reduced the ability of GTP gamma S to stimulate tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands including p125FAK and paxillin in permeabilized Swiss 3T3 cells. Furthermore, a peptide of p21rho (p21rho17-44) inhibited GTP gamma S-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 microM). This peptide also inhibited tyrosine phosphorylation of p125FAK and paxillin. In contrast, 20 microM p21ras17-44 peptide failed to inhibit GTP gamma S-induced tyrosine phosphorylation. Using permeabilized cells, our findings demonstrate that GTP gamma S stimulates tyrosine phosphorylation of p125FAK and paxillin and that a functional p21rho is implicated in this process.

3T3 Cells↗
PubMed1995

The small GTP-binding protein, rho p21, is involved in bone resorption by regulating cytoskeletal organization in osteoclasts.

Rho protein (rho p21), a p21ras-related small guanine nucleotide binding protein, regulates cytoskeletal organization in a number of different types of cells. Evidence has indicated that Clostridium botulinum-derived ADP-ribosyltransferase (C3 exoenzyme) specifically ADP-ribosylates rho p21 at Asn41 and renders it functionally inactive. In this study, we examined the involvement of rho p21 in osteoclastic bone resorption using the C3 exoenzyme. When osteoclast-like multinucleated cells obtained from cocultures of mouse osteoblastic cells and bone marrow cells were placed on dentine slices, they formed ringed structures of podosomes containing F-actin (corresponding to the clear zone) within 8 hours. Many resorption pits were formed on dentine slices after culture for 24 hours. The C3 exoenzyme at 0.15-10 micrograms/ml added to the culture medium disrupted the ringed structure of podosomes in osteoclast-like cells in a dose-dependent manner. Correspondingly, pit formation by osteoclast-like cells on dentine slices was dose-dependently inhibited also by adding the C3 exoenzyme. Microinjection of the C3 exoenzyme into osteoclast-like cells placed on culture dishes completely disrupted the ringed podosome structure within 20 minutes. The amount of the rho p21 which was ADP-ribosylated by the C3 exoenzyme in vitro was much greater in purified osteoclast-like cells than in osteoblastic cells. Prior exposure of the purified osteoclast-like cell preparation to the C3 exoenzyme in vivo markedly decreased the amount of unribosylated rho p21. This indicated that the C3 exoenzyme incorporated into osteoclast-like cells effectively ADP-ribosylates rho p21 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1994

Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin.

In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation. Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21. C3 exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin. Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.

3T3 Cells↗
PubMed1994

Inhibitory effects of botulinum toxin on 5-HT1C receptor-induced Cl- current in Xenopus oocytes.

Several low molecular weight G proteins have been identified, but their functional roles remain unclear. To clarify the involvement of low molecular weight G protein in receptor-stimulated turnover of polyphosphoinositide (PI) turnover, influences of botulinum toxins on serotonin (5-HT)-stimulated Cl- current mediated by PI turnover were investigated using Xenopus oocytes injected with rat brain mRNA. Treatment with botulinum toxin C, D or purified ADP-ribosyltransferase of botulinum toxin (botulinum toxin C3 enzyme) inhibited the 5-HT-induced Cl- current in oocytes, and ADP-ribosylated 23 kDa proteins. Both botulinum toxin C3 enzyme-induced inhibition of the current and ADP-ribosylation were suppressed by pretreatment with antibotulinum toxin C3 enzyme antibody. Botulinum toxin D treatment of oocytes was ineffective in the response of Cl- current induced by injection of 50 pmol inositol 1,4,5-trisphosphate and 50 pmol Ca2+. It is suggested that low molecular weight G proteins ADP-ribosylated by botulinum toxin C3 enzyme are involved in phospholipase C activation in Xenopus oocytes.

ADP Ribose Transferases↗
PubMed1994

Effect of botulinum C3 exoenzyme on cell growth and cytoskeleton organization in transformed human epidermal cells in culture: a possible role for rho protein in epidermal cells.

We examined the role of rho gene products (rho proteins) on cell growth and cytoskeleton organization in transformed human epidermal cells in culture (HSC-1), using recombinant botulinum C3 exoenzyme which specifically ADP-ribosylates rho proteins. Incubation of HSC-1 cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with a molecular weight of 23,000. This protein was identified as rhoA protein by isoelectric focusing (pI 6.0). Addition of C3 exoenzyme to the culture medium of HSC-1 cells changed the shape of HSC-1 cells to a round form with beaded processes in a time- and dose-dependent manner. Moreover, C3 treatment reduced the cell growth rate; 72-h treatment with C3 exoenzyme at 1, 3, 10, 30 and 60 micrograms/ml culture medium resulted in 9.0 +/- 1.8%, 20 +/- 2.9%, 26 +/- 2.3%, 50 +/- 1.4% and 40 +/- 2.0% inhibition of the growth rate relative to controls, respectively. Under this condition, actin stress fibers were disassembled, as revealed using fluorescent-labeled phallacidin, whereas keratin intermediate filaments were not affected, visualized by immunofluorescence using anti-keratin antibody. These results suggest that rho proteins are closely related to cell growth and that these proteins regulate, at least in part, the assembly of actin stress fibers in transformed human epidermal cells.

ADP Ribose Transferases↗
PubMed1994

Inhibition of lysophosphatidate- and thrombin-induced neurite retraction and neuronal cell rounding by ADP ribosylation of the small GTP-binding protein Rho.

Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.

ADP Ribose Transferases↗