Cell motility and the backward bulk flow of the plasma membrane components

以前我们报道过，AMAP 2/PAG 3/Papα/KIAAO 400，一种GTP酶激活蛋白（GAP），当过表达时，其作用是拮抗Arf 6的功能，而在体外，它被证明对其他Arf亚型表现出有效的GAP活性。在这两个财政年度期间，我们首次发现AMAP 2通过其ArfGAP结构域与GTP-Arf 6结合，但不与GDP-Arf 6或其他Arf结合，无论核苷酸状态如何。大多数AMAP 2定位于细胞内的管泡状结构，并在Arf 6活化后重新分布到Arf 6富集的膜区域。在HeLa细胞中，Arf 6已被证明参与Tac的网格蛋白非依赖性内吞，但不参与转铁蛋白的网格蛋白依赖性内吞。我们发现，Arf 6沉默抑制了HeLa细胞中Tac的内化，但不抑制转铁蛋白。Tac的内化，而不是转铁蛋白，也被AMAP 2沉默和过表达显著抑制。此外，AMAP 2被发现通过其富含脯氨酸的结构域与内吞机制的一种组分--两性蛋白酶-IIm结合。我们建议，AMAP 2具有双重机制，其功能，它表现出有效的催化GAP活性的I类和III类Arf，但参与的III类Arf的细胞功能没有立即GAP活性。AMAP 2的这种双重机制可能对GTP-Arf 6的细胞功能起重要作用。在分析AMAP 1的生理作用时，我们发现该蛋白定位于乳腺癌细胞的侵袭伪足，并在侵袭中起重要作用。由于AMAP 1的作用是拮抗Arf 6，我们还分析了Arf 6在癌症侵袭中的可能作用，发现Arf 6也是乳腺癌侵袭所必需的。我们已经提出，Arf 6，和细胞内机制调节Arf 6在入侵过程中，应被视为预防乳腺癌侵袭的治疗靶点。

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.