Roles played by Arf6 in cell migration and invasion

• Project/Area Number: 16370090
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Cell biology
• Research Institution: OSAKA BIOSCIENCE INSTITUTE
• Principal Investigator: SABE Hisataka OSAKA BIOSCIENCE INSTITUTE, Dept.of Molecular Biology, Head, 分子生物学部門, 研究部長 (40187282)
• Co-Investigator(Kenkyū-buntansha): HASHIMOTO Shigeru OSAKA BIOSCIENCE INSTITUTE, Dept.of Molecular Biology, Research Associate, 分子生物学部門, 研究員 (50311303) ; MIURA Koichi OSAKA BIOSCIENCE INSTITUTE, Dept.of Molecular Biology, Research Associate, 分子生物学部門, 研究員 (20360349) ; HASHIMOTO Ari OSAKA BIOSCIENCE INSTITUTE, Dept.of Molecular Biology, Research Associate, 分子生物学部門, 研究員 (60390803) ; 真崎 雄一 日本学術振興会, 特別研究員
• Project Period (FY): 2004 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥15,700,000 (Direct Cost: ¥15,700,000); Fiscal Year 2005: ¥5,300,000 (Direct Cost: ¥5,300,000); Fiscal Year 2004: ¥10,400,000 (Direct Cost: ¥10,400,000)
• Keywords: Arf6 / Cell motility / Cell invasion / ArfGEF / breast cancer / non-ubiquitination / E3 ligase / ArfGAP / AMAP1 / AMAP2 / endocytosis

Research Abstract

Cell migration is a multifactorial process in which a number of distinct events occur simultaneously. The major purpose of this study is to understand basic molecules and mechanisms coordinately regulating cell migration and invasion.
Arf6 plays essential roles in recycling of plasma membrane component, as well as both membrane and cytoskeletal remodeling at cell peripheries. We have previously identified several proteins bearing ArfGAP domains as binding proteins to paxillin, an integrin signaling adaptor/scaffolding protein. These ArfGAPs include AMAP1 and AMAP2. We have also shown that AMAP2 has a role in recruiting paxillin to sites of focal adhesions in epithelial cells. We have further demonstrated that AMAP1 is crucial for invasive activities of different breast cancer cells, in which AMAP1 functions by forming a trimeric protein complex with paxillin and cortactin. siRNA-mediated knockdown of AMAP1 effectively block invasive and metastatic activities of breast cancer cells.
Then, there was an enigma why GAP protein like AMAP1 can act as a necessary factor for tumor invasion, in spite of the simple idea that it may rather act to inhibit the invasion. Indeed, biochemical assays have shown a lack of efficient GAPing activity of AMAP1 against Arf6. We thus tested a mode of interaction between AMAP1 and Arf6, and found that AMAP1 via its ArfGAP domain binds to GTP-Arf6 stably, even in the presence of divalent cations. Binding of AMAP1 to other GTP bound Arfs, like Arf1 and Arf5, was negligible. We also conducted several cell biological assays, and concluded that AMAP1, and also AMAP2, act as effectors for GTP-Arf6. Both paxillin and cortactin are known to be essential for invadopodia formation, that are sites of tumor cell invasion into basement membranes. We showed that by binding to GTP-Arf6, AMAP1 has a role in recruiting paxillin and cortactin to invadopodia in breast cancer cells. Next we addressed to identify a GEF that is responsible for Arf6 activation in migration and invasion. We have succeeded in this identification, and also identified signaling pathways as to how this Arf6GEF becomes activated by extracellular stimuli. Besides these, we also showed that Arf6 can be ubiquitinated. We identified a E3 ligase involved in this ubiquitination, and shown that this is a non-canonical ubiquitination and that loss of the E3 ligase expression may contribute to invasive phenotypes tumor cells.

Research Products

• [Journal Article] Expression of AMAP1, an ArfGAP, provides novel targets to Inhibit breast cancer invasive activities. (2005)
  • Author(s): Onodera, Y., Hashimoto, S., Hashimoto, A., Morishige, M., Mazaki, Y., Yamada, A., Ogawa, E., Adachi, M., Sakurai, T., Manabe, T., Wada, H., Matsuura, N., Sabe, H.
  • Journal Title: EMBO J. 24・5
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Interaction of paxillin with poly(A)-binding protein 1 and its role in focal adhesion turnover and cell migration. (2005)
  • Author(s): Woods, A.J., Kantidakis, T., Sabe, H., Critchley, D.R., Norman, J.C.
  • Journal Title: MOl. Cell Biol. 25・9
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Assays and properties of ArfGAPs, AMAPI and AMAP2 in Arf6 function. Methods Enzymol. (2005)
  • Author(s): Hashimoto, S., Hashimoto, A., Yamada, A., Onodera, Y., Sabe, H.
  • Journal Title: Methods Enzymol. 404 Pages: 216-231
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Interaction of paxillin with poly(A)-binding protein 1: a role in focal adhesion turnover and cell migration (2005)
  • Author(s): Woods, A.J., Kandidakis, T., Sabe, H., Critchley, D.R., Norman, J.C.
  • Journal Title: Mol.Cell Biol. 25 Pages: 3763-3773
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression of AMAP1, an ArfGAP, provides novel targets to inhibit breast cancer invasive activities. (2005)
  • Author(s): Onodera, Y., Hashimoto, S., Hashimoto, A., Morishige, M., Mazaki, Y., Yamada, A., Ogawa, E., Adachi, M., Sakurai, T., Manabe, T., Wada, H., Matsuura, N., Sabe, H.
  • Journal Title: EMBO J 24 Pages: 963-973
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Interaction of paxillin with poly(A)-binding protein 1 and its role in focal adhesion turnover and cell migration. (2005)
  • Author(s): Woods, A.J., Kantidakis, T., Sabe, H., Critchley, D.R., Norman, J.C.
  • Journal Title: Mol.Cell Biol. 25・9 Pages: 3763-3773
• [Journal Article] Assays and properties of ArfGAPs, AMAP1 and AMAP2, in Arf6 function. (2005)
  • Author(s): Hashimoto, S., Hashimoto, A., Yamada, A., Onodera, Y., Sabe, H.
  • Journal Title: Methods Enzymol. 404 Pages: 213-231
• [Journal Article] Expression of AMAP1, an ArfGAP, Provides novel targets to inhibit breast cancer invasive activities. (2005)
  • Author(s): Y.Onodera, S.Hashimoto, A.Hashimoto, M.Morishige, Y.Mazaki, A.Yamada, E.Ogawa, M.Adachi, T.Sakurai, T.Manabe, H.Wada, N.Matsuura, H.Sabe
  • Journal Title: EMBO J. in press
• [Journal Article] A novel mode of action of an ArfGAP, PAG3/AMAP2/Papα, in Arf6 function. (2004)
  • Author(s): Hashimoto, S., Hashimoto, A., Yamada, A., Kojima, C., Yamamoto, H., Tsutsumi, T., Higashi, M., Mizoguchi, A., Yagi, R., Sabe, H.
  • Journal Title: J. Biol. Chem. 279・36 Pages: 37677-37684
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive. (2004)
  • Author(s): Romanova, L.Y., Hashimoto, S., Chay, K.O., Blagosklonny, M.V., Sabe, H., Mushinski, J.F.
  • Journal Title: J. Cell Sci. 117・17 Pages: 3759-3768
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Requirement for Arf6 in breast cancer invasive activities. (2004)
  • Author(s): Hashimoto, S., Onodera, Y., Hashimoto, A., Tanaka, M., Hamaguchi, M., Yamad, A., Sabe, H.
  • Journal Title: Proc. Natl. Acad. Sci. USA 101・17 Pages: 6647-6652
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A role played by a subset of integrin-signaling molecules in cadherin-based cell-cell adhesions. (2004)
  • Author(s): Yano, H., Mazaki, Y., Kurokawa, K., Hanks.K.S., Matsuda, M., Sabe, H.
  • Journal Title: J. Cell Biol. 101・17 Pages: 6647-6652
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Regulation of Binl SH3 domain binding by phosphoinositides. (2004)
  • Author(s): Kojima, C., Hashimoto, A., Yabuuta, I., Hirose, M., Hashimoto, S., Kanaho, Y., Sumimoto, H., Ikegawa, T.
  • Journal Title: EMBO J. 166・2 Pages: 283-295
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Requirement for Arf6 in breast cancer invasive activities. (2004)
  • Author(s): Hashimoto, S., Onodera, Y, Hashimoto, A., Tanaka, M., Hamaguchi, M., Yamada, A., Sabe, H.
  • Journal Title: Proc Natl Acad Sci USA 101 Pages: 6647-6652
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Roles played by a subset of integrin-signaling molecules in cadherin-based cell-cell adhesion. (2004)
  • Author(s): Yano, H., Mazaki, Y., Kurokawa, K., Hanks, K.S., Matsuda, M., Sabe, H
  • Journal Title: J.Cell Biol. 166 Pages: 283-295
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Regulation of Bin1 SH3 domain binding by phosphoinositides (2004)
  • Author(s): Kojima, C., Hashimoto, A., Yabuta, I., Hirose, M., Hashimoto, S., Kanaho, Y., Sumimoto, H., Ikegami, T., Sabe, H.
  • Journal Title: EMBO J. 23 Pages: 4413-4422
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A novel mode of action of an ArfGAP, AMAP2/PAG3/PAPalpha in Arf6 function. (2004)
  • Author(s): Hashimoto, S., Hashimoto, A., Yamada, A., Kojima, C., Yamamoto, H., Tsutsumi, T., Higashi, M., Mizoguchi, A., Yagi, R., Sabe, H.
  • Journal Title: J.Biol.Chem. 279 Pages: 37677-37684
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] IL-3-induced phosphorylation of paxillin tyrosine residues 31 and 118 is required for polarization andmotility of pre-B lymphoid cells : TPA abolishes the effect of IL-3. (2004)
  • Author(s): Romanova, L.Y., Hashimoto, S., Chay, K.O., Blagosklonny, M.V., Sabe, H., Mushinski, J.F.
  • Journal Title: J.Cell Sci. 117 Pages: 3759-3768
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts. (2004)
  • Author(s): Watanabe, H., Shimizu, T., Nishihira, J., Abe, R., Nakayama, T., Taniguchi, M., Sabe, H., Ishibashi, T., Shimizu H.
  • Journal Title: J.Biol.Chem. 279 Pages: 1676-1683
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A novel mode of action of an ArfGAP,PAG3/AMAP2/Papα, in Arf6 function. (2004)
  • Author(s): S.Hashimoto, A.Hashimoto, A.Yamada, C.Kojima, H.Yamamoto, T.Tsutsumi, M.Higashi, A.Mizoguchi, R.Yagi, H.Sabe
  • Journal Title: J.Biol.Chem. 279・36 Pages: 37677-37684
• [Journal Article] Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive. (2004)
  • Author(s): L.Y.Romanova, S.Hashimoto, K.-O.Chay, M.V.Blagosklonny, H.Sabe, J.F.Mushinski
  • Journal Title: J.Cell.Sci. 117・17 Pages: 3759-3768
• [Journal Article] Requirement for Arf6 in breast cancer invasive activities. (2004)
  • Author(s): S.Hashimoto, Y.Onodera, A.Hashimoto, M.Tanaka, M.Hamaguchi, A.Yamada, H.Sabe
  • Journal Title: Proc.Natl.Acad.Sci.USA 101・17 Pages: 6647-6652
• [Journal Article] A role played by a subset of integrin-signaling molecules in cadherin-based cell-cell adhesions. (2004)
  • Author(s): H.Yano, Y.Mazaki, K.Kurokawa, S.K.Hanks, M.Matsuda, H.Sabe
  • Journal Title: J.Cell.Biol. 166・2 Pages: 283-295
• [Journal Article] Regulation of Bin1 SH3 domain binding by phosphoinositides. (2004)
  • Author(s): C.Kojima, A.Hashimoto, I.Yabuta, M.Hirose, S.Hashimoto, Y.Kanaho, H.Sumimoto, T.Ikegami, H.Sabe
  • Journal Title: EMBO J. 23・22 Pages: 4413-4422
• [Journal Article] Assays and properties of ArfGAPs, AMAP1 and AMAP2, in Arf6 function.
  • Author(s): Hashimoto, S., Hashimoto, A., Yamada, A., Onodera Y., Sabe, H.
  • Journal Title: Methods Enzymol. 404 Pages: 216-238
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] 発生分化再生研究2005 実験医学増刊 『上皮細胞における接着分子機能の動的制御』 (2005)
  • Author(s): 三浦浩一, 佐邊壽孝
  • Publisher: 羊土社
  • Description: 「研究成果報告書概要(和文)」より
• [Book] 発生分化再生研究2005 実験医学増刊『上皮細胞における接着分子機能の動的制御』 (2005)
  • Author(s): 三浦浩一, 佐邊壽孝
  • Publisher: 羊土社
• [Patent(Industrial Property Rights)] ヒトがん細胞の浸潤および転移を阻害するベプチド、それをコードするポリヌクレオチドおよびそれらを含有する薬剤 (2005)
  • Inventor(s): 佐邊壽孝, 橋本 茂
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2005-09-14
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] 非典型的なプロリンリッチ配列結合阻害剤 (2005)
  • Inventor(s): 佐邊壽孝, 橋本 茂, 中野洋文, 我妻 勉
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2005-09-29
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] ヒト癌細胞の侵潤活性を阻害するオリゴリボヌクレオチド (2005)
  • Inventor(s): 佐邊壽孝, 橋本 茂, 古林秀則, 森重真毅
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2005
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] ヒトがん細胞の浸潤および転移を阻害するペプチド、それをコードするポリヌクレオチドおよびそれらを含有する薬剤 (2005)
  • Inventor(s): 佐邊 壽孝, 橋本 茂
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2005-09-14
• [Patent(Industrial Property Rights)] 非典型的なプロリンリッチ配列結合阻害剤 (2005)
  • Inventor(s): 佐邊 壽孝, 橋本 茂, 中野 洋文, 我妻 勉
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2005-09-29
• [Patent(Industrial Property Rights)] ヒトがん細胞の浸潤活性を阻害するオリゴリボヌクレオチド (2005)
  • Inventor(s): 佐邊 壽孝, 橋本 茂, 古林 秀則, 森重 真毅
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2005-09-30
• [Patent(Industrial Property Rights)] ヒト癌細胞の運動と浸潤を阻害する物質およびそのスクリーニング方法 (2004)
  • Inventor(s): 佐邊壽孝, 橋本 茂
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2004-09-06
  • Description: 「研究成果報告書概要(和文)」より
• [Patent(Industrial Property Rights)] ヒト癌細胞の運動と浸潤を阻害する物質およびそのスクリーニング方法 (2004)
  • Inventor(s): 佐邊 壽孝, 橋本 茂
  • Industrial Property Rights Holder: (財)大阪バイオサイエンス研究所
  • Filing Date: 2004-09-06