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

先前我们报道了gtpase激活蛋白（GAP） AMAP2/PAG3/Papα/KIAAO400在过表达时可以拮抗Arf6的功能，同时在体外也显示出对其他Arf亚型具有有效的GAPing活性。在这两个财政年度期间，我们首次发现AMAP2通过其ArfGAP结构域与GTP-Arf6结合，但不与GDP-Arf6或其他Arfs结合，无论核苷酸状态如何。AMAP2大部分定位于细胞内管泡结构，在Arf6激活后重新分布到富含Arf6的膜区。在HeLa细胞中，Arf6已被证明参与Tac的不依赖网格蛋白的内吞作用，但不参与转铁蛋白依赖网格蛋白的内吞作用。我们发现，在HeLa细胞中，Arf6沉默抑制Tac的内化，而不抑制转铁蛋白的内化。AMAP2的沉默和过表达也显著抑制了Tac的内化，而转铁蛋白不受影响。此外，AMAP2还通过富含脯氨酸的结构域与内吞机制的组成部分amphiphysin-IIm结合。我们认为AMAP2具有双重功能机制；它对I类和III类Arf具有有效的催化GAP活性，但参与III类Arf的细胞功能而没有立即的GAP活性。AMAP2的这些双重机制可能对GTP-Arf6的细胞功能很重要。在分析AMAP1的生理作用时，我们发现该蛋白定位于乳腺癌细胞的侵袭过继体，并在侵袭中发挥重要作用。由于AMAP1有拮抗Arf6的作用，我们也分析了Arf6在肿瘤侵袭中的可能作用，发现Arf6在乳腺癌侵袭中也是必不可少的。我们提出Arf6及侵袭过程中调控Arf6的细胞内机制可作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.