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Koh-ichi Nagata

Publications and source records attributed to Koh-ichi Nagata.

10 recordsLinked to original sources
PubMed2006

Identification of a cell polarity-related protein, Lin-7B, as a binding partner for a Rho effector, Rhotekin, and their possible interaction in neurons.

Rhotekin, an effector of Rho, is highly expressed in the brain but its function(s) in neurons is almost unknown. In an attempt to define the properties of Rhotekin in neuronal cells, we focused on its interaction with polarity-related molecules. In the present study, we identified a PDZ protein, Lin-7B, as a binding partner for Rhotekin by yeast two-hybrid screening of human brain cDNA library. We then found that Rhotekin interacts with Lin-7B in in vitro pull-down assays, and forms an immunocomplex in COS7 cells and the rat brain. The C-terminal three amino acids of Rhotekin were essential for the interaction with Lin-7B. Their binding affinity became increased in the presence of active RhoA in the COS7 cell expression system. In addition, immunohistochemical analyses demonstrated that Lin-7 as well as Rhotekin is enriched in neurons. These results suggest that Lin-7 plays some role in neuronal functions in concert with Rho/Rhotekin signals.

Animals↗
PubMed2006

Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction.

Among various effector proteins for the small GTPase Rho, the function(s) of Rhotekin is (are) almost unknown. We have identified PIST [PDZ (PSD-95, Discs-large and ZO-1) domain protein interacting specifically with TC10 (a Rho-family small GTPase)] as a binding partner for Rhotekin, using yeast two-hybrid screening. Rhotekin was found to associate with PIST in vitro and in both polarized and non-polarized MDCK (Madin-Darby canine kidney) cells. The C-terminal SPV (Ser-Pro-Val) motif of Rhotekin exhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated version of Rho and partially by that of Rac or Cdc42 in COS7 cells. In contrast, TC10 had no effects on the binding. Immunofluorescence analyses revealed the co-localization of PIST and Rhotekin at the Golgi apparatus in non-polarized fibroblast-like MDCK cells and AJs (adherens junctions) in the fully polarized cells. PIST and Rhotekin are recruited from the cytosol to AJs as the cell becomes polarized. Expression of constitutively active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in polarized MDCK cells. These results suggest that there is (1) Rho-dependent regulation of Rhotekin-PIST interaction, (2) involvement of PIST in the recruitment of Rhotekin to AJs and (3) a possible role(s) for these two proteins in cell-polarity development and/or maintenance.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27.

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like Gö6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.

Animals↗
PubMed2005

Identification of trichoplein, a novel keratin filament-binding protein.

Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

Amino Acid Sequence↗
PubMed2004

Biochemical and cell biological analyses of a mammalian septin complex, Sept7/9b/11.

Septins are members of a conserved family of cytoskeletal GTPases present in organisms as diverse as yeast and mammals. Unlike lower eukaryotic cells, the physiological significance of mammalian septin complexes is largely unknown. Using specific antibodies, we found at least five septins, Sept2, Sept7, Sept8, Sept9b, and Sept11, in septin complexes affinity-purified with anti-Sept7 antibody-conjugated column from rat embryonic fibroblast REF52 cells. Immunofluorescence studies revealed co-localization of Sept7, Sept9b, and Sept11 along stress fibers in REF52 cells. Biochemical and immunoprecipitation analyses revealed that the three septins directly bind with each other through their N- or C-terminal divergent regions. These septins per se formed distinct and characteristic filament structures when transiently expressed in COS7 cells. When two of the three septins were co-expressed in COS7 cells, combination-dependent filament elongation, bundling, or disruption was observed. Taken together, our results suggest that septin filament structures may be affected by interactions with other septins included in the complex.

Animals↗
PubMed2004

Biochemical and cell biological characterization of a mammalian septin, Sept11.

Septins are a family of conserved cytoskeletal GTPases implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking. Here, we report identification of an yet uncharacterized septin, Sept11, in septin complexes purified from porcine brain. The transcripts were detected in all tested tissues except leukocytes. A Sept11 mutant with apparently reduced GTPase activity did not form filaments in the transient expression system using COS7 cells. By Western blot analysis using a specific antibody, Sept11 was detected in various cell lines as well as brain tissues. Septin complexes immunoisolated from porcine brain with anti-Sept9 and anti-Sept11 antibodies were found to contain different Sept9 isoforms based on SDS-PAGE analyses followed by silver-staining and Western blotting. Immunofluorescent study revealed cell type-dependent intracellular localization of the protein; Sept11 was colocalized dominantly with microtubules and actin stress fibers in HMEC cells and REF52 cells, respectively, and their filamentous distribution was dependent on the cytoskeleton structures with which the protein is colocalized. Sept11 partially colocalized with stress fibers and microtubules in HeLa cells.

Amino Acid Sequence↗
PubMed2004

Autophosphorylation of a newly identified site of Aurora-B is indispensable for cytokinesis.

Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.

Alanine↗
PubMed2002

Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process.

Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.

Amino Acid Sequence↗
PubMed2002

B plexins activate Rho through PDZ-RhoGEF.

Plexins are receptors for the repulsive axon guidance molecules semaphorins. Previously, we have shown that plexin-B1 binds activated Rac, but that clustering of plexin-B1 causes Rho activation, resulting in stress fiber formation. Using the yeast two-hybrid system, we found that the C-terminus of B plexins interacted directly with Rho-specific exchange factors, via their PDZ domain. Mutation of the carboxy-terminal amino acids of plexin-B1 or coexpression of a dominant negative PDZ-RhoGEF abrogated the ability of plexin-B1 to cause stress fiber formation. Our results demonstrate a role for PDZ-RhoGEF in B plexin-mediated activation of Rho/Rho kinase signaling, implicated in the regulation of axon guidance and cell migration.

3T3 Cells↗