| Project/Area Number |
12480219
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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(OBI) |
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Principal Investigator |
SABE Hisataka OBI,FIRST DEPARTMENT,HEAD, 第1研究部, 研究部長 (40187282)
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| Co-Investigator(Kenkyū-buntansha) |
YAGI Ryohei OBI, FIRST DEPARTMENT, RESERCH ASSOSIATE, 第1研究部, 研究員 (00321703)
YANO Hajime OBI, FIRST DEPARTMENT, RESERCH ASSOSIATE, 第1研究部, 研究員 (00284414)
HASIMOTO Sigeru OBI, FIRST DEPARTMENT, RESERCH ASSOSIATE, 第1研究部, 研究員 (50311303)
真崎 雄一 (財)大阪バイオサイエンス研究所, 第1研究部, 研究員 (60311304)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥14,300,000 (Direct Cost: ¥14,300,000)
Fiscal Year 2001: ¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 2000: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Keywords | PAG3 / paxillin / ARFGAP / ARF6 / endocytosis / endosomal recycling / cell migration / PAG / integrin / パキシリン / ARF / アクチン細胞骨格 / 形質膜 / マクロファージ / 貪食作用 |
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| Research Abstract |
ARF6 regulates endosomal recycling. We have shown that PAG3/Papα/KIAA0400 acts as a GTPase-activation protein (GAP) specific for ARF6.We study here molecular mechanims how PAG3 is involved in endosomal recycling to be an ARF6GAP. We found that PAG3, via its proline-rich region, binds to the src homology 3 (SH3) domain of several components of the endocytic machinery, and analysed its interaction with amphiphysin IIa. PAG3 existed at ARF6(Q67L)-positive membrane ruffles colocalized with amphyphysin Ha, but the majority exists at intracellular tubulovesicular structure. Overexpression of the amphiphysin ha SH3 domain is known to block endocytosis. Likewise, overexpression of the proline-rich region of PAG3 blocked both clathrin-dependent and independent endocytosis, while mutations of amino acids essential for the binding abolished such blockage. The SH3 domain of amphiphysin IIa. also binds to dynamin, a mechano-enzyme essential for the late step of endocytosis. We found that PAG3 exhibits almost one order of magnitude higher affinity than that of dynamin towards amphiphysin ha. We also demonstrated that PAG3 can be phosphorylated by a protein tyrosine kinase, Pyk2, but not by its close relative Fak ; and this phosphorylation inhibits the association with amphiphysin ha. With further results, we propose that PAG3 recruits amphiphysin ha to the plasma membrane, probably through interaction with the activity of GTP-bound Arf6 ; and external stimuli triggering endocytosis evoke tyrosine phosphorylation of PAG3, the phosphorylated PAG3 then releases amphiphysin ha to associate with components of endocytic machinery such as dynamin.
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