Biomedical subjects
Publications and source records attributed to Y Takai.
We experienced a rare case of lactating adenoma in the mammary gland of a pregnant woman. A 21-year-old pregnant woman visited our clinic with a chief complaint of left mammary gland tumor. Based on palpation, mammograms and echograms, fibroadenoma or phyllodes tumor was diagnosed and enucleation was performed at the time of biopsy. The histopathological diagnosis was lactating adenoma.
We have recently purified a Ki-Ras- and Ha-Ras-dependent extracellular signal-regulated kinase kinase from bovine brain and identified it as B-Raf protein kinase complexed with 14-3-3 proteins (Yamamori, B., Kuroda, S., Shimizu, K., Fukui, K., Ohtsuka, T., and Takai, Y. (1995) J. Biol. Chem. 270, 11723-11726). Moreover, we found that Rap1B as well as Ki-Ras and Ha-Ras stimulate the B-Raf activity. Since B-Raf contains a cysteine-rich domain originally found in protein kinase C as a domain responsible for interaction with phosphatidylserine (PS) and diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate, we have examined here the effect of these compounds on the Ki-Ras-, Ha-Ras-, and Rap1B-induced activation of bovine brain B-Raf. Bovine brain PS enhanced Ki-Ras-stimulated B-Raf activity. Phosphatidic acid was slightly active, but other phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol (PI), PI-4-monophosphate, PI-4,5-bisphosphate, and PI-3,4,5-trisphosphate, were inactive. However, none of the above phospholipids affected the Ha-Ras-stimulated B-Raf activity, whereas PI, PS, phosphatidylethanolamine, and phosphatidic acid inhibited the Rap1B-stimulated B-Raf activity. Phosphatidylcholine or PI-4-monophosphate did not show any effect on the Rap1B-stimulated B-Raf activity. Synthetic PS with two unsaturated fatty acids, such as 1,2-dioleoyl-PS or 1,2-dilinoleoyl-PS, showed the same effect toward the Ki-Ras- and Rap1B-stimulated B-Raf activities, but synthetic PS with two saturated fatty acids, such as 1, 2-distearoyl-PS, was inactive. 12-O-Tetradecanoylphorbol-13-acetate did not affect the stimulatory or inhibitory effect of PS on the Ki-Ras- and Rap1B-stimulated B-Raf activities, respectively. PS did not affect the Ki-Ras-, Ha-Ras-, or Rap1B-independent basal B-Raf activity or the mitogen-activated protein kinase kinase or extracellular signal-regulated kinase activity. These results indicate that various phospholipids differently affect Ki-Ras-, Ha-Ras, and Rap1B-induced B-Raf activation.
The RHO1 gene encodes a homolog of the mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. Multicopy suppressors of a temperature-sensitive, dominant negative mutant allele of RHO1, RHO1(G22S, D125N), were isolated and named ROM (RHO1 multicopy suppressor). Rom1p and Rom2p were found to contain a DH (Dbl homologous) domain and a PH (pleckstrin homologous) domain, both of which are conserved among the GDP/GTP exchange proteins (GEPs) for the Rho family small GTP binding proteins. Disruption of ROM2 resulted in a temperature-sensitive growth phenotype, whereas disruption of both ROM1 and ROM2 resulted in lethality. The phenotypes of deltarom1deltarom2 cells were similar to those of deltarho1 cells, including growth arrest with a small bud and cell lysis. Moreover, the temperature-sensitive growth phenotype of deltarom2 was suppressed by overexpression of RHO1 or RHO2, but not of CDC42. The glutathione-S-transferase (GST) fusion protein containing the DH domain of Rom2p showed the lipid-modified Rholp-specific GDP/GTP exchange activity which was sensitive to Rho GDP dissociation inhibitor. These results indicate that Rom1p and Rom2p are GEPs that activate Rho1p in S.cerevisiae.
The enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall, beta(1-->3)-D-glucan synthase (also known as 1,3-beta-glucan synthase), requires a guanosine triphosphate (GTP) binding protein for activity. The GTP binding protein was identified as Rho1p. The rho1 mutants were defective in GTP stimulation of glucan synthase, and the defect was corrected by addition of purified or recombinant Rho1p. A protein missing in purified preparations from a rho1 strain was identified as Rho1p. Rho1p also regulates protein kinase C, which controls a mitogen-activated protein kinase cascade. Experiments with a dominant positive PKC1 gene showed that the two effects of Rho1p are independent of each other. The colocalization of Rho1p with actin patches at the site of bud emergence and the role of Rho1p in cell wall synthesis emphasize the importance of Rho1p in polarized growth and morphogenesis.
We previously isolated a new protein having two C2-like domains which interacted with Ca2+ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca2+-dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K+ in an extracellular Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged Doc2 stimulated the Ca2+-dependent, high K+-induced release of co-expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with the coding sequence of Doc2 in the antisense orientation, the high K+-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal fragment containing two C2-like domains inhibited the high K+-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca2+-dependent exocytosis of dense core vesicles from PC12 cells.
Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca2+-dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser306 and Ser313. The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane.
Specific receptors for brain-gut peptide hormones, cholecystokinin (CCK) and gastrin, are expressed in a variety of human tumor cells. CCK and gastrin promote the growth of NIH3T3 cells into which the CCK-B/gastrin receptor had been introduced via a eukaryotic expression vector. In this study, we have examined the effect of CCK-8 on the actin cytoskeleton by using two mouse fibroblast cell lines expressing human CCK-B/gastrin receptors. Treatment with very low concentration of CCK-8 (10(-10) M) induced the formation of actin stress fibers within one minute. Stress fiber formation increased for 30 min. In contrast, a potent mitogen for fibroblasts, platelet-derived growth factor (PDGF), initially induced membrane ruffling and, later, a weak formation of stress fibers. Microinjection of rho GDP dissociation inhibitor or Clostridium botulinum ADP-ribosyltransferase C3 which is known to impair the function of a small GTP-binding protein, rho p21, inhibited the stress fiber formation by CCK-8 as well as by PDGF. These results indicate that CCK-B/gastrin receptor could regulate stress fiber formation in a rho p21-dependent manner. The signals from CCK-B/gastrin receptor might affect cell growth as well as cell motility or adhesion by regulating the actin cytoskeleton.
Rabphilin-3A, a putative target molecule of Rab3A small GTP-binding protein implicated in Ca2+-dependent exocytosis, consists of two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal two C2-like domains (C2A and C2B domains) interacting with Ca2+ and phospholipid. Here, we used the growth hormone (GH) co-expression assay system of PC12 cells in which expressed GH is released in response to high K+. Reduction of endogenous rabphilin-3A inhibited the Ca2+-dependent, high K+-induced GH release. Various rabphilin-3A mutants expressing an N-terminal, C-terminal, or C2B fragment, but not the rabphilin-3A mutant expressing a C2A fragment, inhibited the high K+-induced GH release. These results indicate that rabphilin-3A is involved at least in Ca2+-dependent exocytosis from PC12 cells and that the C2A and C2B domains have different functions.
The Rho subfamily, consisting of three members (RhoA, -B and -C), belongs to the small GTP-binding protein superfamily. The Rho subfamily is implicated in regulation of various actin filament-dependent cell functions, such as cell aggregation, cell motility and cytokinesis. The Rho subfamily receives an upstream signal and is converted from the GDP-bound inactive form to the GTP-bound active form which transduces a signal to a downstream pathway. This conversion is regulated by GDP/GTP exchange proteins (GEPs) and several GEPs for the Rho subfamily have been identified. The GEPs thus far reported are mainly isolated from the cytosol fraction of various tissues and are not specific for the Rho subfamily. Here we have partially purified a membrane-associated GEP specific for the Rho subfamily (mRho GEP). mRho GEP was extracted from the crude synaptic membrane fraction of rat brain by a combination of detergent and NaCl, and partially purified by several column chromatographies. The partially purified mRho GEP was active on RhoA but was inactive on other small GTP-binding proteins including at least Rac1, Ki-Ras and Rab3A. RhoA undergoes post-translational lipid modifications and mRho GEP required these lipid modifications for its GEP activity. mRho GEP was not active in the presence of Rho GDI, an inhibitory Rho GEP. These results indicate that there is a membrane-associated GEP specific for Rho and suggest that Rho is activated by this GEP on the membranes.
Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.
Rab11 p24 is a Ras-like small guanosine triphosphate (GTP)-binding protein, and specific antibodies against it were newly developed to explore its function. Using the antibody, Rab11 p24 was shown to be abundant in rat pancreas as well as in most rat tissues. To explore the involvement of Rab11 p24 into the exocytotic process, the subcellular distribution of Rab11 p24 in rat pancreatic acini was evaluated also by use of the antibody. When the isolated acini were incubated with 1 x 10(-10) M cholecystokinin octapeptide (CCK-8) that induced the maximal stimulation, the amount of Rab11 p24 increased in the fractions of plasma membrane and zymogen granules, but decreased in the cytosol fraction. This redistribution was time-dependent and occurred within 1 min after the CCK-8 stimulation and reached a maximal level within 2 min after the stimulation. Moreover, a light microscopic immunolabeling technique on the isolated rat pancreatic acini also revealed that higher immunoreactivity with Rab11 p24 was observed over the zymogen granule membrane under CCK-8 stimulation. The present results indicate that Rab11 p24 is translocated from cytosol to the membrane fraction during stimulation with CCK-8 and suggest that Rab11 p24 is involved in the intracellular vesicular transport of isolated acini.
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There exists a small GTP-binding protein (G protein) superfamily, consisting of more than 50 members, from yeast to mammal. The Rab family belongs to this superfamily and is implicated in intracellular vesicle trafficking. Rab3A small G protein is a member of the Rab3 subfamily which belongs to this Rab family. The regulators and downstream targets of Rab3A have been isolated, and evidence is accumulating that Rab3A and these Rab3A-interacting proteins are involved in Ca(2+)-dependent exocytosis, particularly in neurotransmitter release from nerve terminals.
BACKGROUND: The Rho small G protein family, which includes the Rho, Rac and Cdc42 subfamilies, is implicated in various cell functions such as cell shape change, cell motility and cytokinesis, through the reorganization of actin filaments. Rho GDI is an inhibitory regulator of the Rho small G protein family and inhibits the Rho family dependent cell functions. Reorganization of actin filaments is also known to regulate Ca2+-dependent exocytosis. RESULTS: We have examined here whether the Rho family members are also involved in Ca2+-dependent exocytosis. We have found, by the use of the human growth hormone (GH) co-expression assay system on PC12 cells, that overexpression of Rho GDI inhibits high K+-induced, Ca2+-dependent GH release. This inhibitory action of Rho GDI is restored by co-expression of a dominant active mutant of RhoA or Rac1, but not of a dominant active mutant of Cdc42. C3 transferase, known to ADP-ribosylate Rho and to inhibit its function, also inhibits this GH release. Overexpression of a dominant active mutant of RhoA or Rac1 alone shows only a small effect on GH release. Moreover, immunocytochemical studies show that the overexpression of Rho GDI prevents a partial disruption of the cortical actin network which accompanies exocytosis. CONCLUSIONS: These results suggest that RhoA, Rac1 and Rho GDI are involved in Ca2+-dependent exocytosis at least partly through the reorganization of actin filaments, and that the activation of RhoA or Rac1 alone is not sufficient for this reaction.
Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.
The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5-PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism.
Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.
The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.