Faculty Profiles - Nakayama Masanori

Papers - Nakayama Masanori

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Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor-specific angiogenic signaling. Reviewed International journal

Swetha S D Peddibhotla, Benjamin F Brinkmann, Daniel Kummer, Hüseyin Tuncay, Masanori Nakayama, Ralf H Adams, Volker Gerke, Klaus Ebnet

Molecular biology of the cell 24 ( 7 ) 933 - 44 2013.4

Language：English Publishing type：Research paper (scientific journal)

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin family with diverse functions in epithelial cells, including cell migration, cell contact maturation, and tight junction formation. In endothelial cells, JAM-A has been implicated in basic fibroblast growth factor (bFGF)-regulated angiogenesis through incompletely understood mechanisms. In this paper, we identify tetraspanin CD9 as novel binding partner for JAM-A in endothelial cells. CD9 acts as scaffold and assembles a ternary JAM-A-CD9-αvβ3 integrin complex from which JAM-A is released upon bFGF stimulation. CD9 interacts predominantly with monomeric JAM-A, which suggests that bFGF induces signaling by triggering JAM-A dimerization. Among the two vitronectin receptors, αvβ3 and αvβ5 integrin, which have been shown to cooperate during angiogenic signaling with bFGF and vascular endothelial growth factor (VEGF), respectively, CD9 links JAM-A specifically to αvβ3 integrin. In line with this, knockdown of CD9 blocks bFGF- but not VEGF-induced ERK1/2 activation. JAM-A or CD9 knockdown impairs endothelial cell migration and tube formation. Our findings indicate that CD9 incorporates monomeric JAM-A into a complex with αvβ3 integrin, which responds to bFGF stimulation by JAM-A release to regulate mitogen-activated protein kinase (MAPK) activation, endothelial cell migration, and angiogenesis. The data also provide new mechanistic insights into the cooperativity between bFGF and αvβ3 integrin during angiogenic signaling.

DOI： 10.1091/mbc.E12-06-0481

Spatial regulation of VEGF receptor endocytosis in angiogenesis. Reviewed International journal

Masanori Nakayama, Akiko Nakayama, Max van Lessen, Hiroyuki Yamamoto, Sarah Hoffmann, Hannes C A Drexler, Norimichi Itoh, Tomonori Hirose, Georg Breier, Dietmar Vestweber, Jonathan A Cooper, Shigeo Ohno, Kozo Kaibuchi, Ralf H Adams

Nature cell biology 15 ( 3 ) 249 - 60 2013.3

Language：English Publishing type：Research paper (scientific journal)

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.

DOI： 10.1038/ncb2679

Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography. Reviewed

Tomoki Nishioka, Masanori Nakayama, Mutsuki Amano, Kozo Kaibuchi

Cell structure and function 37 ( 1 ) 39 - 48 2012

Language：English Publishing type：Research paper (scientific journal)

The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.

Involvement of Girdin in the determination of cell polarity during cell migration. Reviewed International journal

Kei Ohara, Atsushi Enomoto, Takuya Kato, Takahiko Hashimoto, Mayu Isotani-Sakakibara, Naoya Asai, Maki Ishida-Takagishi, Liang Weng, Masanori Nakayama, Takashi Watanabe, Katsuhiro Kato, Kozo Kaibuchi, Yoshiki Murakumo, Yoshiki Hirooka, Hidemi Goto, Masahide Takahashi

PloS one 7 ( 5 ) e36681 2012

Language：English Publishing type：Research paper (scientific journal)

Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement.

DOI： 10.1371/journal.pone.0036681

Screening of Rho kinase substrates by combining inhibitors of protein kinase and phosphatase

Nishioka Tomoki, Nakayama Masanori, Amano Mutsuki, Kaibuchi Kozo

Abstracts for Annual Meeting of Japanese Proteomics Society 2011 131 - 131 2011

EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Reviewed International journal

Simona Parrinello, Ilaria Napoli, Sara Ribeiro, Patrick Wingfield Digby, Marina Fedorova, David B Parkinson, Robin D S Doddrell, Masanori Nakayama, Ralf H Adams, Alison C Lloyd

Cell 143 ( 1 ) 145 - 55 2010.10

Language：English Publishing type：Research paper (scientific journal)

The peripheral nervous system has astonishing regenerative capabilities in that cut nerves are able to reconnect and re-establish their function. Schwann cells are important players in this process, during which they dedifferentiate to a progenitor/stem cell and promote axonal regrowth. Here, we report that fibroblasts also play a key role. Upon nerve cut, ephrin-B/EphB2 signaling between fibroblasts and Schwann cells results in cell sorting, followed by directional collective cell migration of Schwann cells out of the nerve stumps to guide regrowing axons across the wound. Mechanistically, we find that cell-sorting downstream of EphB2 is mediated by the stemness factor Sox2 through N-cadherin relocalization to Schwann cell-cell contacts. In vivo, loss of EphB2 signaling impaired organized migration of Schwann cells, resulting in misdirected axonal regrowth. Our results identify a link between Ephs and Sox proteins, providing a mechanism by which progenitor cells can translate environmental cues to orchestrate the formation of new tissue.

DOI： 10.1016/j.cell.2010.08.039

Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Reviewed International journal

Mutsuki Amano, Masanori Nakayama, Kozo Kaibuchi

Cytoskeleton (Hoboken, N.J.) 67 ( 9 ) 545 - 54 2010.9

Language：English Publishing type：Research paper (scientific journal)

Rho-associated kinase (Rho-kinase/ROCK/ROK) is an effector of the small GTPase Rho and belongs to the AGC family of kinases. Rho-kinase has pleiotropic functions including the regulation of cellular contraction, motility, morphology, polarity, cell division, and gene expression. Pharmacological analyses have revealed that Rho-kinase is involved in a wide range of diseases such as vasospasm, pulmonary hypertension, nerve injury, and glaucoma, and is therefore considered to be a potential therapeutic target. This review focuses on the structure, function, and modes of activation and action of Rho-kinase.

DOI： 10.1002/cm.20472

Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Reviewed International journal

Yingdi Wang, Masanori Nakayama, Mara E Pitulescu, Tim S Schmidt, Magdalena L Bochenek, Akira Sakakibara, Susanne Adams, Alice Davy, Urban Deutsch, Urs Lüthi, Alcide Barberis, Laura E Benjamin, Taija Mäkinen, Catherine D Nobes, Ralf H Adams

Nature 465 ( 7297 ) 483 - 6 2010.5

Language：English Publishing type：Research paper (scientific journal)

In development, tissue regeneration or certain diseases, angiogenic growth leads to the expansion of blood vessels and the lymphatic vasculature. This involves endothelial cell proliferation as well as angiogenic sprouting, in which a subset of cells, termed tip cells, acquires motile, invasive behaviour and extends filopodial protrusions. Although it is already appreciated that angiogenesis is triggered by tissue-derived signals, such as vascular endothelial growth factor (VEGF) family growth factors, the resulting signalling processes in endothelial cells are only partly understood. Here we show with genetic experiments in mouse and zebrafish that ephrin-B2, a transmembrane ligand for Eph receptor tyrosine kinases, promotes sprouting behaviour and motility in the angiogenic endothelium. We link this pro-angiogenic function to a crucial role of ephrin-B2 in the VEGF signalling pathway, which we have studied in detail for VEGFR3, the receptor for VEGF-C. In the absence of ephrin-B2, the internalization of VEGFR3 in cultured cells and mutant mice is defective, which compromises downstream signal transduction by the small GTPase Rac1, Akt and the mitogen-activated protein kinase Erk. Our results show that full VEGFR3 signalling is coupled to receptor internalization. Ephrin-B2 is a key regulator of this process and thereby controls angiogenic and lymphangiogenic growth.

DOI： 10.1038/nature09002

Identification of focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3-kinase) as Par3 partners by proteomic analysis. Reviewed International journal

Norimichi Itoh, Masanori Nakayama, Takashi Nishimura, Shin Fujisue, Tomoki Nishioka, Takashi Watanabe, Kozo Kaibuchi

Cytoskeleton (Hoboken, N.J.) 67 ( 5 ) 297 - 308 2010.5

Language：English Publishing type：Research paper (scientific journal)

Partition defective 3 (Par3) is involved in a variety of polarity events including establishment of apico-basal polarity of epithelial cell, axon/dendrite specification of neurons and directional migration of cells with front-rear polarity. Par3 is thought to regulate cell polarity as a scaffold protein by interacting with various partner proteins such as Par6, aPKC, Tiam1/2 and Numb. However, the mode of actions of Par3 in polarized migration remains largely unknown. To explore Par3 functions, we screened Par3-interacting proteins by combining Par3 affinity column chromatography and shotgun analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We obtained about two hundred Par3-interacting proteins from the rat brain cytosol fraction. Among them, we focused on FAK and PI3-kinase, as both of them participate in directional cell migration. FAK associated with the PDZ domain and the coiled-coil region of Par3 and p110 of PI3-kinase associated with the coiled-coil region of Par3. Par3 was partially colocalized with FAK in spreading cells. Depletion of Par3 by RNA interference inhibited adhesion-induced activation of FAK and PI3-kinase, and RNA interference-resistant Par3 restored the inhibitory effects. In addition, Par3 was required for the adhesion-induced cell spreading as well as for directional cell migration toward collagen. These results suggest that Par3 directly interacts with FAK and PI3-kinase, enhancing their activities for polarized cell migration.

DOI： 10.1002/cm.20444

ケラチン結合蛋白質アルバトロスは上皮細胞極性を制御する(The keratin-binding protein Albatross regulates polarization of epithelial cells)

猪子誠人, 杉本昌彦, 白水崇, 中山雅敬, 鄒鵬, 米村重信, 林裕子, 井澤一郎, 佐宗幹夫, 宇治幸隆, 貝淵弘三, 清野透, 稲垣昌樹

日本細胞生物学会大会講演要旨集 61回 176 - 176 2009.5

The keratin-binding protein Albatross regulates polarization of epithelial cells. Reviewed International journal

Masahiko Sugimoto, Akihito Inoko, Takashi Shiromizu, Masanori Nakayama, Peng Zou, Shigenobu Yonemura, Yuko Hayashi, Ichiro Izawa, Mikio Sasoh, Yukitaka Uji, Kozo Kaibuchi, Tohru Kiyono, Masaki Inagaki

The Journal of cell biology 183 ( 1 ) 19 - 28 2008.10

Language：English Publishing type：Research paper (scientific journal)

The keratin intermediate filament network is abundant in epithelial cells, but its function in the establishment and maintenance of cell polarity is unclear. Here, we show that Albatross complexes with Par3 to regulate formation of the apical junctional complex (AJC) and maintain lateral membrane identity. In nonpolarized epithelial cells, Albatross localizes with keratin filaments, whereas in polarized epithelial cells, Albatross is primarily localized in the vicinity of the AJC. Knockdown of Albatross in polarized cells causes a disappearance of key components of the AJC at cell-cell borders and keratin filament reorganization. Lateral proteins E-cadherin and desmoglein 2 were mislocalized even on the apical side. Although Albatross promotes localization of Par3 to the AJC, Par3 and ezrin are still retained at the apical surface in Albatross knockdown cells, which retain intact microvilli. Analysis of keratin-deficient epithelial cells revealed that keratins are required to stabilize the Albatross protein, thus promoting the formation of AJC. We propose that keratins and the keratin-binding protein Albatross are important for epithelial cell polarization.

DOI： 10.1083/jcb.200803133

ケラチン結合蛋白質Albatrossは上皮細胞の極性化を制御する(The keratin-binding protein Albatross regulates the polarization of epithelial cells)

猪子誠人, 杉本昌彦, 白水崇, 中山雅敬, 鄒鵬, 米村重信, 林裕子, 井澤一郎, 佐宗幹夫, 宇治幸隆, 貝淵弘三, 清野透, 稲垣昌樹

日本細胞生物学会大会講演要旨集 60回 123 - 123 2008.6

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation. Reviewed International journal

Masanori Nakayama, Takaaki M Goto, Masayuki Sugimoto, Takashi Nishimura, Takafumi Shinagawa, Sigeo Ohno, Mutsuki Amano, Kozo Kaibuchi

Developmental cell 14 ( 2 ) 205 - 15 2008.2

Language：English Publishing type：Research paper (scientific journal)

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.

DOI： 10.1016/j.devcel.2007.11.021

Rho-kinase phosphorylates eNOS at threonine 495 in endothelial cells. Reviewed International journal

Masayuki Sugimoto, Masanori Nakayama, Takaaki M Goto, Mutsuki Amano, Kimihiro Komori, Kozo Kaibuchi

Biochemical and biophysical research communications 361 ( 2 ) 462 - 7 2007.9

Language：English Publishing type：Research paper (scientific journal)

Endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which is involved in various physiological functions of the cardiovascular system. eNOS is activated by dephosphorylation at Thr495 and phosphorylation at Ser1177. Inhibition of Rho-kinase, an effector of the small GTPase RhoA, leads to activation of Akt/PKB, which phosphorylates eNOS at Ser1177 and thereby promotes NO production. However, little is known about the effects of Rho-kinase on phosphorylation of Thr495. We here found that the constitutively active form of Rho-kinase phosphorylated eNOS at Thr495 in vitro. Expression of the constitutively active form of RhoA or Rho-kinase increased this phosphorylation in COS-7 cells. Addition of thrombin to cultured human umbilical vein endothelial cells induced phosphorylation of eNOS at Thr495. Treatment with Y27632, a Rho-kinase inhibitor, suppressed thrombin-induced phosphorylation at Thr495. These results indicate that Rho-kinase can directly phosphorylate eNOS at Thr495 to suppress NO production in endothelium.

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation. Reviewed International journal

Mikito Takefuji, Kazutaka Mori, Yasuhiro Morita, Nariko Arimura, Takashi Nishimura, Masanori Nakayama, Mikio Hoshino, Akihiro Iwamatsu, Toyoaki Murohara, Kozo Kaibuchi, Mutsuki Amano

Biochemical and biophysical research communications 355 ( 3 ) 788 - 94 2007.4

Language：English Publishing type：Research paper (scientific journal)

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.

Hydrophilic statin suppresses vein graft intimal hyperplasia via endothelial cell-tropic Rho-kinase inhibition. Reviewed International journal

Dai Yamanouchi, Hiroshi Banno, Masanori Nakayama, Masayuki Sugimoto, Hiromine Fujita, Masayoshi Kobayashi, Hiroyuki Kuwano, Kimihiro Komori

Journal of vascular surgery 42 ( 4 ) 757 - 64 2005.10

Language：English Publishing type：Research paper (scientific journal)

BACKGROUND: Recent studies suggest that statins can protect the vasculature in a manner that is independent of their lipid-lowering activity through inhibition of the small guanosine triphosphate-binding protein, Rho, and Rho-associated kinase. Little information is available on the inhibitory effect of statins on vein graft intimal hyperplasia, the main cause of late graft failure after bypass grafting. We therefore examined the effects of a hydrophilic statin on vein graft intimal hyperplasia in vivo and Rho-kinase activity in vitro. METHODS: In the first experiment, rabbits were randomized to a control group (n = 7) that was fed regular rabbit chow or to a pravastatin group (n = 7) that was fed regular rabbit chow supplemented with 10 mg/kg pravastatin sodium. The branches of the jugular vein were ligated and an approximately 3-cm segment of the jugular vein was taken for an autologous reversed-vein graft. The carotid artery was cut and replaced with the harvested autologous jugular vein. At 2 and 4 weeks after the operation, vein grafts in both groups were harvested, and intimal hyperplasia of the vein grafts was assessed. In the second experiment, human umbilical vein endothelial cells and vascular smooth muscle cells were cultured and then treated with 1 micromol/L and 30 micromol/L pravastatin for 24 hours and harvested. Immunoblotting was performed on the resulting precipitates. Quantitative evaluation of phosphorylated myosin binding subunit and endothelial nitric oxide synthase was performed by densitometric analysis. RESULTS: We demonstrated that oral administration of the hydrophilic statin pravastatin to normocholesterolemic rabbits inhibited intimal hyperplasia of carotid interposition-reversed jugular vein grafts 4 weeks after implantation (pravastatin group, 39.5 +/- 3.5 microm vs control group, 64.0 +/- 7.1 microm; n = 7; P < .05) and suppressed cell proliferation and apoptosis in the neointima 2 weeks after implantation. In addition, we found that pravastatin inhibited Rho-kinase activity and accelerated endothelial nitric oxide synthase expression in human umbilical vein endothelial cells but did not inhibit Rho-kinase activity in vascular smooth muscle cells. CONCLUSIONS: These novel findings clearly demonstrate that a hydrophilic statin can suppress intimal hyperplasia of the vein graft in vivo and also show endothelial cell-tropic inhibition of Rho-kinase in vitro. Furthermore, these results strongly support the clinical use of hydrophilic statins to prevent intimal hyperplasia of the vein graft after bypass grafting. CLINICAL RELEVANCE: Late graft failure caused by neointimal hyperplasia limits the efficacy of vein grafting. Various treatments were examined to reduce neointimal hyperplasia, but a standard clinical treatment has not yet been established. We report here the inhibitory effect of pravastatin on the development of vein graft intimal hyperplasia. In addition, we demonstrate that pravastatin showed endothelial cell-tropic benefits through both the inhibition of Rho-kinase activity and acceleration of eNOS expression in vitro. Because the clinical benefits and safety of pravastatin have been established to a certain extent through long-term clinical usage, pravastatin may soon become standard treatment after vein bypass grafting.

Rho mediates endocytosis of epidermal growth factor receptor through phosphorylation of endophilin A1 by Rho-kinase. Reviewed International journal

Takako Kaneko, Akio Maeda, Mikito Takefuji, Hiroki Aoyama, Masanori Nakayama, Saeko Kawabata, Yoji Kawano, Akihiro Iwamatsu, Mutsuki Amano, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms 10 ( 10 ) 973 - 87 2005.10

Language：English Publishing type：Research paper (scientific journal)

After binding of epidermal growth factor (EGF), the EGF receptor is activated, internalized by endocytosis, and subsequently degraded in the lysosomal pathway. Endocytotic trafficking of the activated EGF receptor is essential for controlling EGF signaling. Upon ligand-induced activation of EGF receptors, Cbl (ubiquitin ligase) binds to the activated receptor and leads to translocation of the CIN85 (Cbl-interacting protein of 85 kDa)/endophilin complex in the vicinity of the activated EGF receptors. Endophilin is known as a key regulator of clathrin-mediated endocytosis, and the translocation of endophilin in the vicinity of active EGF receptor is thought to promote receptor internalization. The constitutively active mutant of small GTPase Rho inhibits EGF receptor endocytosis. In this study, we found that this inhibitory effect was canceled by the dominant negative form of Rho-associated kinase (Rho-kinase), which is an effector of Rho. To clarify the molecular mechanisms of endocytosis downstream of Rho/Rho-kinase signal, we searched for and identified endophilin A1 as a novel substrate of Rho-kinase. We identified the phosphorylation site of endophilin A1 at Thr-14 and made endophilin T14D (substitution of Thr-14 by Asp), which is expected to mimic the phosphorylation state of endophilin A1. Endophilin T14D inhibited EGF receptor internalization. Furthermore, phosphorylation of endophilin by Rho-kinase inhibited the binding to CIN85. Taken together, these results suggest that Rho-kinase phosphorylates endophilin downstream of Rho and regulates EGF receptor endocytosis through the inhibition of binding between endophilin and CIN85.

Rho-kinase and myosin II activities are required for cell type and environment specific migration. Reviewed International journal

Masanori Nakayama, Mutsuki Amano, Akira Katsumi, Takako Kaneko, Saeko Kawabata, Mikito Takefuji, Kozo Kaibuchi

Genes to cells : devoted to molecular & cellular mechanisms 10 ( 2 ) 107 - 17 2005.2

Language：English Publishing type：Research paper (scientific journal)

Cell migration is important in the development of atherosclerotic lesions. Macrophages and smooth muscle cells migrate into the subendothelial space of arteries, leading to plaque formation. Long-term inhibition of the activity of Rho-kinase induces a regression of atherosclerotic coronary lesions, probably by preventing migration of macrophages and smooth muscle cells. Previous reports concerning the effect of Rho-kinase inhibitors on cell migration are contradictory, however. We examined here the cell type specificity of Rho-kinase inhibitors and found that migration of endothelial cells, macrophages, and smooth muscle cells was inhibited by treatment with Rho-kinase inhibitors in a dose-dependent fashion in a three-dimensional migration assay, whereas that of fibroblasts and epithelial cells was not inhibited. Myosin II inhibitor prevented cell migration in a manner similar to Rho-kinase inhibitors. In contrast, in a two-dimensional migration assay, cell migration was not inhibited by Rho-kinase or myosin II inhibitors for any of the cell types examined. Taken together, these results indicate that Rho-kinase inhibitors suppress migration of specific cell types under specific conditions through the regulation of myosin II activity. Our findings suggest that Rho-kinase is the therapeutic target of atherosclerosis accompanied with invasion by leukocytes and smooth muscle cells.

[Involvement of small GTPase Rho and Rho-kinase in the pathogenesis of hypertension and hypertensive target organ damage]. Reviewed

Masanori Nakayama, Mutsuki Amano, Saeko Kawabata, Kozo Kaibuchi, Mikihito Takefuji

Nihon rinsho. Japanese journal of clinical medicine 62 Suppl 3 202 - 6 2004.3

Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase. Reviewed International journal

Mutsuki Amano, Takako Kaneko, Akio Maeda, Masanori Nakayama, Masaaki Ito, Takashi Yamauchi, Hideyuki Goto, Yuko Fukata, Noriko Oshiro, Azusa Shinohara, Akihiro Iwamatsu, Kozo Kaibuchi

Journal of neurochemistry 87 ( 3 ) 780 - 90 2003.11

Language：English Publishing type：Research paper (scientific journal)

Rho-kinase and myosin phosphatase are implicated in the phosphorylation-state of myosin light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fibre formation in non-muscle cells. Here, we found that microtubule-associated proteins, Tau and MAP2, interacted with the myosin-binding subunit (MBS) of myosin phosphatase, and were the possible substrates of both Rho-kinase and myosin phosphatase. We determined the phosphorylation sites of Tau (Thr245, Thr377, Ser409) and MAP2 (Ser1796) by Rho-kinase. We also found that Rho-kinase phosphorylated Tau at Ser262 to some extent. Phosphorylation by Rho-kinase decreased the activity of Tau to promote microtubule assembly in vitro. Substitutions of Ala for Ser/Thr at the phosphorylation sites of Tau (Tau-AAA) did not affect the activity to promote microtubule assembly, while substitutions of Asp for Ser/Thr (Tau-DDD), which are expected to mimic the phosphorylation-state of Tau, slightly reduced the activity. When Tau, or mutated forms of Tau, were expressed in PC12 cells, followed by treatment with cytochalasin D, they promoted extension of the cell process in a cytochalasin-dependent manner. However, Tau-DDD showed the weaker activity in this capacity than wild-type Tau or Tau-AAA. These results suggest that the phosphorylation-state of these residues of Tau affects its activity both in vitro and in vivo. Thus, it is likely that the Rho-kinase/MBS pathway regulates not only the actin-myosin system but also microtubule dynamics.

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