Role of paxillin-associatcd ARFGAPs in cell migration.

桩蛋白相关的 ARFGAP 在细胞迁移中的作用。

• 批准号: 12480219
• 负责人: SABE Hisataka
• 金额: $ 9.15万
• 依托单位: OSAKA BIOSCIENCE INSTITUTE(OBI)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480219/
• 关键词: 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

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.

ARF 6调节内体再循环。我们已经证明PAG 3/Papα/KIAA 0400是一种特异性的ARF 6 GTP酶激活蛋白（GAP）。我们发现，PAG 3，通过其富含脯氨酸的区域，结合到src同源3（SH 3）结构域的几个组成部分的内吞机制，并分析了其相互作用与两性霉素IIa。PAG 3存在于与Amphyphysin Ha共定位的ARF 6（Q67 L）阳性膜皱褶处，但大多数存在于细胞内管泡结构处。已知双端生理蛋白ha SH 3结构域的过表达阻断内吞作用。同样地，PAG 3的富含脯氨酸的区域的过表达阻断了网格蛋白依赖性和独立性的内吞作用，而对结合必需的氨基酸的突变废除了这种阻断。两性生理素IIa的SH 3结构域。还结合发动蛋白，一种内吞作用后期必需的机械酶。我们发现，PAG 3表现出几乎一个数量级更高的亲和力比发动蛋白对两性霉素ha。我们还证明，PAG 3可以被磷酸化的蛋白酪氨酸激酶，Pyk 2，但不是由其近亲Fak，这种磷酸化抑制与两性霉素ha的协会。进一步的结果，我们提出，PAG 3招聘两栖physin ha的质膜，可能通过与GTP结合的Arf 6的活性的相互作用;和外部刺激触发内吞引起的PAG 3的酪氨酸磷酸化，磷酸化的PAG 3然后释放两栖physin ha与组件的内吞机制，如发动蛋白。

项目成果

H.Yano,H.Uchida,T.Iwasaki,M.Mukai,H.Akedo,K.Nakamura,S.Hashimoto & H.Sabe.: "Paxillin αand Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation."Proc.Natl.Acad.Sci.USA. 97・16.

H. Yano、H. Uchida、T. Iwasaki、M. Mukai、H. Akedo、K. Nakamura、S. Hashimoto 和 H. Sabe.：“Pacillin α 和 Crk 相关底物对细胞迁移和接触抑制发挥相反作用通过酪氨酸磷酸化生长。“Proc.Natl.Acad.Sci.USA. 97・16。

Yagi, R., Ishimaru, S., Yano, H., Gaul, U., Hanafusa, H. and Sabe, H.: "A novel muscle LIM-only protein is generated from paxillin gene locus in Drosophila."EMBO J.. 2(9). 814-820 (2001)

Yagi, R.、Ishimaru, S.、Yano, H.、Gaul, U.、Hanafusa, H. 和 Sabe, H.：“果蝇中的桩蛋白基因座产生了一种新型肌肉 LIM 蛋白。”EMBO J

Woods, AJ., Roberts, MS., Choudhary, J., Barry, ST., Mazaki, Y., Sabe, H., Morley, SJ., Critchley, DR., Norman, JC.: "Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells"J Biol Chem

Woods, AJ.、Roberts, MS.、Choudhary, J.、Barry, ST.、Mazaki, Y.、Sabe, H.、Morley, SJ.、Critchley, DR.、Norman, JC.：“Paxillin 与聚

Hashimoto, S., Tsubouchi, A., Mazaki, Y., Sabe, H.: "Interaction of paxillin with p21-activated kinase(PAK)Association of paxillin alpha with the kinase-inactive and the Cdc42-activated forms of PAK3"J.Biol.Chem.. 276・8. 6037-6045 (2001)

Hashimoto, S.、Tsbouchi, A.、Mazaki, Y.、Sabe, H.：“桩蛋白与 p21 激活激酶 (PAK) 的相互作用、桩蛋白 α 与激酶失活和 Cdc42 激活形式的 PAK3 的关联”生物化学杂志 276・8. (2001)

A.Kondo,S.Hashimoto,H.Yano,K,Nagayama,Y.Mazaki & H.Sabe.: "A new paxillin-binding protein, PAG3/Papα/KIAA0400, bearing an Arf GTPaseactivating protein activity is involved in paxillin recruitment to focal adhesions and cell migration."Mol.Biol.Cell. 11・4.

A. Kondo、S. Hashimoto、H. Yano、K、Nagayama、Y. Mazaki 和 H. Sabe.：“一种新的桩蛋白结合蛋白 PAG3/Papα/KIAA0400，具有 Arf GTP 酶激活蛋白活性，参与桩蛋白招募粘着斑和细胞迁移。“Mol.Biol.Cell.11・4。