| Co-Investigator(Kenkyū-buntansha) |
YAGI Ryohei Osaka Bioscience Institute, Dept.of Molecuar Biology, Research Fellow, 分子生物学部門, 日本学術振興会特別研究員
YANO Hajime Osaka Bioscience Institute, Dept.of Molecuar Biology, Research Associate, 分子生物学部門, 研究員 (00284414)
HASHIMOTO Shigeru Osaka Bioscience Institute, Dept.of Molecuar Biology, Research Associate, 分子生物学部門, 研究員 (50311303)
HASHIMOTO Ari Osaka Bioscience Institute, Dept.of Molecuar Biology, Research Fellow, 分子生物学部門, 日本学術振興会特別研究員
近藤 明子 (財)大阪バイオサイエンス研究所, 分子生物学部門, 日本学術振興会特別研究員
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| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2003: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2002: ¥10,900,000 (Direct Cost: ¥10,900,000)
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| Research Abstract |
Previously we reported that AMAP2/PAG3/Papα/KIAAO400, a GTPase-activating protein (GAP), acts to antagonize Arf6 function when overexpressed, while it was shown to exhibit efficient GAPing activities for other Arf isoforms in vitro. During this period of the two fiscal years we first found that AMAP2, through its ArfGAP domain, binds to GTP-Arf6, but not to GDP-Arf6 nor other Arfs irrespective of nucleotide status. The majority of AMAP2 was localized to intracellular tubulovesicular structures, and redistributed to Arf6-enriched membrane areas upon Arf6 activation. In HeLa cells, Arf6 has been shown to be involved in the clathrin-independent endocytosis of Tac, but not the clathrin-dependent endocytosis of transferrin. We found that Arf6 silencing inhibited the internalization of Tac, but not transferrin, in HeLa cells. Internalization of Tac, but not transferrin, was also significantly inhibited by AMAP2 silencing and overexpression. AMAP2 was moreover found to bind to amphiphysin-IIm, a component of the endocytic machinery, via its proline-rich domain. We propose that AMAP2 has dual mechanisms for its function ; it exhibits efficient catalytic GAP activity for the class I and III Arfs, and yet is involved in the cellular function of the class III Arf without immediate GAPing activity. These dual mechanisms of AMAP2 may be important for the cellular function of GTP-Arf6.
During analyzing physiological roles of AMAP1, another paxillin-binding ArfGAP we have previously isolated, we found that this protein is localized to invadopodia of breast cancer cells and plays an essential role for the invasion. Since AMAP1 acts to antagonize Arf6, we also analyzed possible role of Arf6 in cancer invasion, and found that Arf6 is also essential for breast cancer invasion. We have proposed that Arf6, and the intracellular machinery regulating Arf6 during invasion, should be considered as therapeutic targets for the prevention of breast cancer invasion.
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