| Project/Area Number |
16370090
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | OSAKA BIOSCIENCE INSTITUTE |
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Principal Investigator |
SABE Hisataka OSAKA BIOSCIENCE INSTITUTE, Dept.of Molecular Biology, Head, 分子生物学部門, 研究部長 (40187282)
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| 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
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥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)
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| Keywords | Arf6 / Cell motility / Cell invasion / ArfGEF / breast cancer / non-ubiquitination / E3 ligase / ArfGAP / AMAP1 / AMAP2 / endocytosis |
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| 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,
… More
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. Less
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