Tomiko Asano

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.

Possible role of Rho/Rhotekin signaling in mammalian septin organization

There is growing evidence for crosstalk between septin filaments and actin cytoskeleton which is regulated by Rho family of GTPases. Here we show that active Rho disrupts septin filament structures in rat embryonic fibroblast REF52 cells. Among Rho effector molecules tested, Rhotekin induced morphological changes of septin filaments similar to those by activated Rho. The center region of Rhotekin was sufficient for the septin reorganization in the cells, and likely to interact indirectly with the C-terminal half of a septin Sept9b, where a GTPase domain is located. Rhotekin and Sept9b are colocalized mainly in perinuclear regions in serum-starved REF52 cells. Upon stimulation with lysophosphatidic acid, they translocated to actin stress fibers in 10u2009min and then redistributed again to cytoplasm after 90u2009min treatment. In neuroblastoma Neuro2a cells, Rhotekin and Sept9b were enriched in the tip of neurites, a location where cortical actin reorganization is induced upon stimulation with lysophosphatidic acid. Taken together, we propose that Rhotekin is a novel regulator organizing mammalian septin structures and provide a new link between the septin and Rho-signaling.

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 αB‐crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of αB‐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.

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.

Phosphorylation by extracellular signal‐regulated kinase of a multidomain adaptor protein, vinexin, at synapses

Vinexin is an adaptor protein that is supposed to play pivotal roles in cell adhesion, cytoskeletal organization and signaling. At least three splice variants, vinexinα, β and γ, have so far been reported. In spite of the possible importance of vinexin, the properties and functions of vinexin in neuronal cells are almost unknown. Here we show that vinexin isoforms are expressed in rat brain in a developmental stage‐dependent manner, and that vinexinα is relatively abundant in the telencephalon regions of the adult rat brain. An immunohistochemical study showed the localization of vinexinα in neurons and glia in the rat brain. In primary cultured rat hippocampal neurons, vinexin was found to be present at synapses and filopodia in growth cones by immunofluorescent analyses. Biochemical fractionation revealed the distribution of vinexin in synaptosomes. Nerve terminal localization of vinexin was confirmed by electron microscopy. Vinexinβ is reported to be phosphorylated by extracellular signal‐regulated kinase (ERK) at Ser189, which is equivalent to Ser593 of vinexinα. We thus constructed a site‐ and phosphorylation state‐specific antibody to monitor the ERK‐mediated phosphorylation of vinexin. In immunofluorescent analyses, the phosphorylation was observed at synapses formed among cultured rat hippocampal neurons and it was reduced by treatment of the cells with PD98059. In an immunoelectron microscopic examination, the phosphorylation signal was mainly detected on the postsynaptic side of synapses in the rat hippocampal neurons. As active ERK was co‐localized with vinexin in synapses, the ERK signal is likely to be involved in the regulation of vinexin‐dependent cellular processes in synapses. On the other hand, the phosphorylation was hardly detected in neurons cultured for 3u2003days, suggesting the presence of a yet unidentified regulatory mechanism of vinexin at the growth cone.

Essential roles of ERK-mediated phosphorylation of vinexin in cell spreading, migration and anchorage-independent growth

Vinexin is an adaptor protein supposed to play pivotal roles in various cellular events such as cell adhesion, cytoskeletal organization, signaling and gene expression. Despite the possible importance, physiological functions and regulatory mechanisms of vinexin are largely unknown. In addition, although vinexin was reported to be phosphorylated by extracellular signal-regulated kinase (ERK), physiological significance of the phosphorylation remains to be elucidated. Here we carried out characterization of endogenous vinexin and found that it was enriched at the leading edge of migrating cells and focal adhesions of spread cells. In the analyses using ERK-phosphorylated vinexin-specific antibody, the phosphorylation signal was also detected at the leading edges of migrating cells and at cell periphery of spreading cells, whereas only faint signal was observed at focal adhesions of well-spread cells. We then established LNCaP cell lines stably expressing GFP-fused vinexinβ or two mutants at Ser189 that mimic the ERK-phosphorylated or -unphosphorylated vinexinβ. Based on the analyses using the lines, the phosphorylation was likely to inhibit the cell spreading and migration. On the other hand, anchorage-independent cell growth was inhibited by unphosphorylated vinexinβ. Taken together, ERK-mediated phosphorylation of vinexinβ is strongly suggested to occur in a spatio-temporally regulated manner and play important roles in cell spreading, migration and anchorage-independent growth.

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.

Expression of smooth muscle cell-specific proteins in neural progenitor cells induced by agonists of G protein-coupled receptors and transforming growth factor-β

Neural progenitor cells isolated from the embryonic cerebral cortex are well known to differentiate into neurons and glial cells, but recent reports have demonstrated differentiation into smooth muscle cells (SMCs) under the influence of fetal bovine serum. In this study, we report that agonists for G protein‐coupled receptors (GPCRs), including endothelin, lysophosphatidic acid and carbachol, effectively promote the expression of SMC‐specific proteins in the presence of transforming growth factor‐β (TGF‐β). Incubation of neural progenitor cells with agonists for GPCRs or TGF‐β alone induced the expression of an SMC‐specific protein, α‐smooth muscle actin (SMA), and their combination resulted in incremental increase. Stimulation with combinations of each GPCR agonist and TGF‐β increased the numbers of large, flat cells with thick actin fibers and also caused expression of other SMC marker proteins. Endothelin and TGF‐β enhanced SMA promoter‐luciferase reporter activity at different times after stimulation. The mutation of TGF‐β control element of SMA promoter constructs decreased TGF‐β‐enhanced luciferase activity but not endothelin‐stimulated activity. Transfection of active forms of RhoA and its effector, mDia, strongly enhanced SMA promoter activity, and a dominant negative form of RhoA inhibited endothelin‐stimulated promoter activity but not TGF‐β‐stimulated activity. Whereas endothelin consistently activated RhoA, TGF‐β did not, and a specific inhibitor of TGF‐β type I receptor blocked TGF‐β‐enhanced SMA promoter activity, suggesting involvement of Smad phosphorylation. These results suggest that separate signaling pathways of G protein and TGF‐β cooperatively promote the expression of SMC‐specific proteins in neural progenitor cells.

Possible interaction of a Rho effector, Rhotekin, with a PDZ-protein, PIST, at synapses of hippocampal neurons.

Rhotekin, an effector of Rho, is highly expressed in the brain but its function 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 raised the possibility that Rhotekin interacts with a PDZ-protein, PIST (PDZ domain protein interacting specifically with TC10) in vitro, and found that these proteins form complex in the rat brain tissues. We then demonstrated that Rhotekin and PIST are expressed in developmental stage-specific manners in the rat brain. In immunofluorescence analyses, Rhotekin and PIST were suggested to co-localize at synapses in rat primary cultured hippocampal neurons. On the other hand, PIST was found to form immunocomplex with another PDZ-binding protein, beta-catenin, in HEK293 cells and in the rat brain, and co-localized with this protein at dendritic filopods. The interaction of PIST with beta-catenin was inhibited by the presence of Rhotekin. These results suggest a possible yet unidentified role of Rhotekin, in harmony with PIST and beta-catenin, in neuronal cells.

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation‐dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin‐V‐FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation‐dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK‐886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.