Biomedical subjects

Y Takai

Publications and source records attributed to Y Takai.

At least 271 records · Page 15Linked to original sources

PubMed1995

Identification of Rap1 as a target for the Crk SH3 domain-binding guanine nucleotide-releasing factor C3G.

C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.

Animals↗

PubMed1995

A patch technique for reconstruction of stenotic pulmonary bifurcation in a Rastelli-type operation after palliations for cyanotic congenital heart disease. A report of two cases.

A Rastelli-type operation was successfully performed on two patients with cyanotic congenital heart disease with pulmonary artesia (PA) and severe stenosis or obstruction of the pulmonary bifurcation, following the improvement of a pulmonary artery index (PA index) after palliative systemic pulmonary shunts. It is a point that stenotic or obstructive pulmonary bifurcation was reconstructed by a patch technique. An extracardiac conduit was used a combination of a bicuspid valved pericardial roll and a prosthetic vessel graft. In a 5-year-old male with an extreme type of tetralogy of Fallot associated with PA and patent ductus arteriosus (PDA), the enlargement of stenotic pulmonary bifurcation was attempted using a brimshaped equine pericardial patch in order to get sufficient suture margin and be safely anastomosed with a conduit. In a 6-year-old female with complete transposition of the great arteries, PA, PDA and right aortic arch (RAA), an obstructive pulmonary bifurcation behind the RAA was enlarged using a brimshaped equine pericardial patch. The attachment of the conduit was shifted to the left pulmonary artery to avoid compression of an extracardiac conduit and pulmonary artery behind RAA. Reconstruction of the pulmonary bifurcation using an equine pericardial patch is useful to anastomose an extracardiac conduit and the stenotic or obstructive pulmonary bifurcation.

Cardiac Surgical Procedures↗

PubMed1994

Phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. We have examined here whether Rabphilin-3A is phosphorylated by calmodulin-dependent protein kinase II (CaMKII). Recombinant Rabphilin-3A was phosphorylated by CaMKII purified from rat brain. Two moles of phosphate were maximally incorporated into one mole of Rabphilin-3A. The phosphorylation sites were Ser34, Thr205, Thr209, and Thr537. These results suggest that the CaMKII-catalyzed phosphorylation of Rabphilin-3A may modulate neurotransmitter release.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Rabphilin-3A is associated with synaptic vesicles through a vesicle protein in a manner independent of Rab3A.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein, which is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A is associated with synaptic vesicles, although it has no transmembrane segment. Here we have studied how rabphilin-3A is associated with synaptic vesicles. Treatment of the synaptic vesicles isolated from rat brain with 1 M NaCl completely solubilized rabphilin-3A from the vesicles. These vesicles deprived of rabphilin-3A still contained Rab3A and synaptophysin. Exogenous rabphilin-3A bound to the vesicles deprived of endogenous rabphilin-3A in dose-dependent and saturable manners. The concentration of exogenous rabphilin-3A giving a half-maximal binding was about 50 nM and maximally 5 +/- 1 molecules of exogenous rabphilin-3A bound to one vesicle. Addition of exogenous Rab3A bound to one vesicle. Addition of exogenous Rab3A or removal of endogenous Rab3A by the action of Rab GDI did not affect the binding of exogenous rabphilin-3A to the vesicles. However, treatment of the vesicles with trypsin completely abolished the binding of exogenous rabphilin-3A. These results suggest that rabphilin-3A is associated with synaptic vesicles at least through a vesicle protein in a manner independent of Rab3A.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Identification of a rabphilin-3A-interacting protein as GTP cyclohydrolase I in PC12 cells.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. To identify a Rabphilin-3A-interacting protein, proteins were immunoprecipitated by an anti-Rabphilin-3A polyclonal antibody from the lysate of PC12 cells and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by protein staining. Several proteins were coimmunoprecipitated with Rabphilin-3A and one of these proteins with a M(r) of about 30 KDa was phosphorylated in intact PC12 cells stimulated by high KCl. The amino acid sequence analysis of this 30 KDa protein revealed that it is GTP cyclohydrolase I.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Identification as beta-adducin of a protein interacting with rabphilin-3A in the presence of Ca2+ and phosphatidylserine.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein implicated in neurotransmitter release. We have previously identified a Rabphilin-3A-interacting protein with a Mr of about 115 kDa in bovine brain. We have attempted here to purify this protein and to determine its primary structure. Amino acid sequence analysis has revealed that this protein is a bovine counterpart of human beta-adducin which is known to be a good substrate for protein kinase C. The Rabphilin-3A-interacting protein also binds to protein kinase C in the presence of Ca2+ and phosphatidylserine. These results indicate that Rabphilin-3A binds to beta-adducin in the presence of Ca2+ and phosphatidylserine.

Adaptor Proteins, Signal Transducing↗

PubMed1994

GDP/GTP exchange reaction-stimulating activity of Rabphilin-3A for Rab3A small GTP-binding protein.

Rabphilin-3A is a putative target protein for Rab3A, a small GTP-binding protein particularly implicated in neurotransmitter release. Rabphilin-3A interacts more preferentially with GTP-Rab3A than with GDP-Rab3A. Moreover, Rabphilin-3A shows a weak activity to stimulate the GTPase activity of Rab3A and a strong activity to inhibit the Rab3A GTPase-activating protein (GAP)-stimulated GTPase activity of Rab3A. Here, we show that Rabphilin-3A has another activity to stimulate the GDP/GTP exchange reaction of Rab3A. Rabphilin-3A may keep Rab3A continuously in the GTP-bound form by converting again GDP-Rab3A, which may be converted from GTP-Rab3A by Rab3A GAP, to GTP-Rab3A, until the function of Rab3A is accomplished.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Phosphorylation of Rabphilin-3A, a putative target protein for Rab3A, by cyclic AMP-dependent protein kinase.

Rab3A small GTP-binding protein and its associated proteins, such as Rabphilin-3A, a putative target protein for Rab3A, MSS4, a stimulatory GDP/GTP exchange protein for Rab3A, and Rab GDI, a translocator for the Rab family members including Rab3A, are implicated in neurotransmitter release. We have investigated here the phosphorylation of these proteins by cyclic AMP-dependent protein kinase (protein kinase A). Neither Rab3A, MSS4, nor Rab GDI was phosphorylated, but Rabphilin-3A was phosphorylated Rab GDI was phosphorylated, but Rabphilin-3A was phosphorylated at its N-terminal region. About 0.8 mol of phosphate was maximally incorporated into one mol of Rabphilin-3A. These results suggest that Rabphilin-3A is one of the targets for protein kinase A which modulates neurotransmitter release.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Synergistic activation by Ras and 14-3-3 protein of a mitogen-activated protein kinase kinase kinase named Ras-dependent extracellular signal-regulated kinase kinase stimulator.

We have identified, in Xenopus oocyte cytosol, a protein kinase named REKS (Ras-dependent extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) stimulator), which phosphorylates and activates recombinant ERK2 through recombinant MEK in a recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ras-dependent manner. We show here that this REKS activity is synergistically enhanced by a combination of mammalian recombinant GTP gamma S-KiRas and 14-3-3 protein purified from rat brain. 14-3-3 protein is known to activate tyrosine and tryptophan hydroxylases, to modulate the protein kinase C activity, to stimulate secretion, and to show phospholipase A2 activity per se. 14-3-3 protein did not affect the MEK activity. 14-3-3 protein neither interacted with Ki-Ras nor affected the neurofibromin activity to stimulate the GTPase activity of Ki-Ras under the conditions where the recombinant N-terminal fragment of c-Raf-1 inhibited it. These results suggest that 14-3-3 protein has an additional function in the regulation of the Ras-MEK-ERK cascade pathway through the activation of REKS.

14-3-3 Proteins↗

PubMed1994

Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins.

The kinetic properties of MSS4 are studied in comparison with those of Rab3A GRF. MSS4 stimulates the dissociation of [3H]GDP from the lipid-modified and lipid-unmodified forms of Rab3A to the same extent, although Rab3A GRF is more effective on the lipid-modified form than on the lipid-unmodified form. Both MSS4 and Rab3A GRF are inactive on other Rab/Sec/Ypt family members including at least Rab2, Rab5, and Rab11. Rab GDI inhibits the MSS4 and Rab3A GRF effects on the lipid-modified form of Rab3A, but the doses of Rab GDI necessary for this inhibitory effect on Rab3A GRF are lower than those on MSS4. Moreover, Rab GDI slightly inhibits the Rab3A GRF effect on the lipid-unmodified form of Rab3A, but does not affect the MSS4 effect on the lipid-unmodified form of Rab3A. These results suggest that MSS4 and Rab3A GRF are different GDP/GTP exchange proteins for Rab3A.

GTP-Binding Proteins↗

PubMed1994

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis.

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.

Animals↗

PubMed1994

Localization of Rabphilin-3A on the synaptic vesicle.

Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. Rabphilin-3A is expressed mainly in brain, but its subcellular localization remains to be clarified. Immunohistochemical analysis has revealed that Rabphilin-3A is most abundant in the synaptic area of the rat cerebellum, retina, and neuromuscular junction. Ultrastructural analysis of the neuromuscular junction using the immunogold method indicates that Rabphilin-3A is localized on the synaptic vesicle. Subcellular fractionation analysis of rat brain has shown that Rabphilin-3A is most highly concentrated in the purified synaptic vesicle fraction. These results indicate that Rabphilin-3A is localized on the synaptic vesicle in the presynapse.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Molecular cloning and characterization of yeast rho GDP dissociation inhibitor.

We have previously isolated rho GDP dissociation inhibitor (rho DGI) from bovine brain and characterized it. Bovine rho GDI is a protein of a M(r) of 23,421 with 204 amino acids. rho GDI inhibits the GDP/GTP exchange reaction of post-translationally lipid-modified small GTP-binding proteins (G proteins) of the rho family, including the rho, rac, and cdc42 subfamilies, and keeps them in the GDP-bound inactive form. In the present study, we first purified rho GDI from the cytosol fraction of the yeast Saccharomyces cerevisiae and isolated its gene. Yeast rho GDI gene had an open reading frame without introns encoding a protein of a M(r) of 23,138 with 202 amino acids. Yeast rho GDI protein was 36% identical with bovine rho GDI. Yeast rho GDI expressed in Escherichia coli was active not only on yeast rho1 but also on mammalian rho family members which were post-translationally modified. Disruption of rho GDI did not induce apparent phenotypes, whereas overexpression of yeast or bovine rho GDI resulted in the inhibition of cell growth. These results indicate that rho GDI exists and regulates the function of the rho family members in yeast.

Amino Acid Sequence↗

PubMed1994

Phosphorylation and activation of mitogen-activated protein kinase by kainic acid-induced seizure in rat hippocampus.

Injection of kainic acid into rat induced a limbic seizure and increased the activities of two protein kinases with Mrs of 42 kDa and 44 kDa in the hippocampus. These two protein kinases were identified as MAP kinases by an anti-MAP kinase antibody. These MAP kinases were phosphorylated at least at a tyrosine residue. The time course of the MAP kinase activation was roughly parallel with that of the seizure. These results indicate that the kainic acid-induced seizure induces MAP kinase activation in rat hippocampus.

Animals↗

PubMed1994

Different functions of Smg GDP dissociation stimulator and mammalian counterpart of yeast Cdc25.

We have previously shown that both Smg GDP dissociation stimulator (GDS) and mammalian Cdc25 (mCdc25) stimulate the GDP/GTP exchange reaction of Ki-Ras and that Smg GDS is active only on the post-translationally lipid-modified form of Ki-Ras, whereas mCdc25 is active on both the lipid-modified and unmodified forms but is more active on the lipid-modified form. In the present study, we compared more detailed kinetic properties of Smg GDS and mCdc25 by use of the lipid-modified form of Ki-Ras as a common substrate. Both Smg GDS and mCdc25 stimulated the dissociation of GDP from Ki-Ras and formed the stable binary complex with Ki-Ras. In the presence of guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), the stable ternary complex of Smg GDS-GTP gamma S-Ki-Ras was produced, whereas GTP gamma S induced the dissociation of mCdc25 from mCdc25-Ki-Ras complex, yielding GTP gamma S-Ki-Ras. mCdc25 stimulated the dissociation of GDP from both the membrane-bound and soluble forms of Ki-Ras, whereas Smg GDS was far less active on the membrane-bound form than on the soluble form. Moreover, Smg GDS translocated the GTP gamma S-bound form of membrane-bound Ki-Ras to the soluble fraction as the stable ternary complex of Smg GDS-GTP gamma S-Ki-Ras, whereas mCdc25 did not show this activity. These results suggest that Smg GDS and mCdc25 play different roles in the regulation of Ki-Ras.

Animals↗

PubMed1994

Molecular cloning and characterization of two rab GDI species from rat brain: brain-specific and ubiquitous types.

Rab GDP dissociation inhibitor (GDI) is a regulatory protein for Rab proteins that controls not only the GDP/GTP exchange reaction of Rab proteins but also their translocation between the cytosol and cell membranes. Recently, rab GDI cDNAs have been isolated from human, bovine, rat, and Drosophila, and these Rab GDI proteins indicated an important role in the regulation of intracellular vesicle traffic. In this study, we have isolated two different rab GDI cDNAs, designated rat rab GDI alpha and beta, from a rat brain cDNA library using bovine rab GDI cDNA as a probe. Rat rab GDI alpha and beta show 99 and 86% amino acid identity with bovine rab GDI, respectively, indicating that rat rab GDI alpha is a rat counterpart of bovine rab GDI. Both rat Rab GDI alpha and beta proteins expressed in Escherichia coli showed a similar degree of activity of regulating the GDP/GTP exchange reaction to that of bovine Rab GDI using Rab3A and Rab11 as substrates. Northern blot analysis revealed that rab GDI alpha mRNA was expressed abundantly in brain but much less in other tissues, whereas rat rab GDI beta mRNA was ubiquitously expressed. Reverse transcription-polymerase chain reaction analysis revealed that astrocytes expressed rat rab GDI beta gene but not rat rab GDI alpha gene. These results indicate that Rab GDI alpha is involved in sorting of highly specialized vesicles in brain such as neurosecretory vesicles, whereas Rab GDI beta plays a general role in vesicular trafficking in diverse types of cells.

Amino Acid Sequence↗

PubMed1994

Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.

Ash/Grb-2 is an adaptor protein composed only of Src homology (SH) 2 and SH3 domains that is considered to be essential for Ras activation. To clarify the downstream of Ash signaling, we investigated Ash-bound proteins. Ash-glutathione S-transferase (GST) fusion proteins were used to affinity-purify proteins bound to Ash. We found 180-, 150-, 100-, and 70-kDa proteins bound to GST-Ash, among which the 100 kDa protein was found to be dynamin by amino acid sequencing and Western blot with anti-dynamin antibody. Next, the in vitro and in vivo associations between Ash and dynamin were examined using PC12 cells. Dynamin in PC12 cell lysates bound to GST-Ash independent of NGF treatment. Also, Ash and dynamin co-precipitated when cell lysates of PC12 were immunoprecipitated with anti-Ash antibody or anti-dynamin antibody. Using various GST-Ash constructs, we studied the importance of the individual domains in binding and found that the SH3 domain is necessary for binding. This binding was inhibited by a synthetic peptide (GPPQVPSRPNRC, amino acids 827-838 in dynamin). These data show that Ash SH3 domains bind to the proline-rich region of dynamin. Considering the function of dynamin in membrane trafficking, Ash may regulate endocytosis in addition to Ras activation.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Kinetic properties of Ash/Grb2-interacting GDP/GTP exchange protein.

Ash/Grb2 is a protein having one SH2 domain flanked by two SH3 domains and is implicated to serve as an adaptor protein which links the EGF receptor to mammalian Sos (mSos), a GDP/GTP exchange protein for Ras. We isolated here several Ash-interacting proteins from bovine brain cytosol by use of glutathione-S-transferase-Ash-linked agarose column chromatography. The Ash-interacting proteins stimulated the GDP/GTP exchange reaction of Ki-Ras and Ha-Ras but not that of other small GTP-binding proteins including at least Rap1, RhoA, Rac1, and Rab3A. The Ash-interacting proteins were much more active on the post-translationally lipid-modified form of Ki-Ras than on the unmodified form. At least one of them was identified as mSos by Western blot analysis using a specific anti-mSos antibody.

Adaptor Proteins, Signal Transducing↗