Rika Morishita

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.

G Protein βγ Subunits Induce Stress Fiber Formation and Focal Adhesion Assembly in a Rho-dependent Manner in HeLa Cells

In fibroblasts, the G protein α subunits Gα12 and Gα13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly, whereas G protein βγ subunits instead exert a disruptive influence. We show here that the latter can, however, stimulate the formation of stress fibers and focal adhesions in epithelial-like HeLa cells. Transient expression of β1 with γ2, γ5, γ7, and γ12 in quiescent HeLa cells induced stress fiber formation and focal adhesion assembly as did expression of the constitutively active Gα12. Co-expression of βγ with Gαi2 and the C-terminal fragment of the β-adrenergic receptor kinase, both of which are known to bind and sequester free βγ, blocked βγ-induced stress fiber and focal adhesion formation. Inhibition was also noted with co-expression of a dominant negative mutant of Rho. Botulinum C3 exoenzyme, which ADP-ribosylates and inactivates Rho, and a Rho-associated protein kinase inhibitor, Y-27632, similarly inhibited βγ-induced stress fiber and focal adhesion assembly. These results indicate that G protein βγ subunits regulate Rho-dependent actin polymerization in HeLa cells.

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 3 days, 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.

Two Forms of GO Type G Proteins: Identification and Distribution in Various Rat Tissues and Cloned Cells

Abstract: A Go type G protein distinct from the major species of Go was recently isolated from bovine brain and designated Go*. The cDNAs encoding two forms of mammalian Goα were also isolated and designated GoAα and GoBα. To recognize two forms of Go type G proteins, we raised antibodies in rabbits against two peptides with sequences found only in the respective proteins of murine GoAα (SNTYE‐DAAAYIQTQF) and GoBα (TEAVAHIQGQYESK). Purified anti‐GoAα antibodies reacted with the major species of Goα purified from bovine and rat brain, whereas anti‐GoBα antibodies reacted only with rat Go*α, but not with the major species of Goα or bovine Go*α. These results indicate that the major species of Goα is encoded by GoAα cDNA and Go*α is encoded by GoBα cDNA. Using these antibodies, the distribution of GoA and GoB was studied in various rat tissues and cloned cells. Both GoA and GoB were present in many tissues, but their distribution in peripheral tissues was distinct. GoAα seemed to associate mainly with neural tissues. On the other hand, relatively high concentrations of GoBα were present in the brain, pituitary gland, adipose tissue, lung, and testis. The concentrations of both GoA and GoB in the brain increased during ontogenic development, but the increase in GoB was observed at a later age. Both GoA and GoB were found in such cloned cells as PC 12, NG108‐15, C6, GA‐1, G8, and 3T3‐L1 cells. Treatment of PC 12 cells with nerve growth factor caused the extension of neuron‐like processes and the increase in the level of GoA, but not in the level of GoB.

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.

Phosphorylation of F-actin-Associating G Protein γ12 Subunit Enhances Fibroblast Motility

Eleven isoforms of G protein γ subunit have been found thus far, but the precise roles of individual γ subunits are not known. The γ12 subunit has two unique properties: phosphorylation by protein kinase C and association with F-actin. To elucidate the role of γ12, we overexpressed γ12 and other γ subunits in NIH 3T3 cells together with the β1 subunit. The overexpressed γ12 as well as endogenous γ12, but not γ2, γ5, and γ7 subunits, associated with cytoskeletal components. Expression of γ12 induced remarkable changes including cell rounding, disruption of stress fibers, and enhancement of cell migration, but expression of other γ subunits did not induce significant changes. Deletion of the N-terminal region of γ12 decreased the abilities of γ12 to associate with cytoskeletal fractions, to induce cell rounding, and to increase cell motility. Replacement by alanine of Ser2 of γ12 (Ser1 of a mature γ12 protein), a phosphorylation site for protein kinase C, eliminated these effects of γ12, whereas a mutant in which Ser2 was replaced with glutamic acid showed effects equivalent to wild-type γ12. These results indicate that phosphorylation of γ12 at Ser2 enhances the motility of cells.