Role of paxillin-associatcd ARFGAPs in cell migration.

ARF6调节内体循环。我们已经证明PAG3/Papα/KIAA0400是ARF6特异性的gtpase激活蛋白（GAP）。我们在此研究PAG3如何参与内体循环成为ARF6GAP的分子机制。我们发现PAG3通过其富含脯氨酸的区域，与内吞机制的几个组分的src同源3 （SH3）结构域结合，并分析了它与amphiphysin IIa的相互作用。PAG3存在于ARF6（Q67L）阳性的膜褶上，但大部分存在于细胞内的管泡结构上。众所周知，过表达amphiphysin SH3结构域可阻断内吞作用。同样，PAG3富含脯氨酸区域的过表达阻断了网格蛋白依赖和独立的内吞作用，而结合所必需的氨基酸突变消除了这种阻断。两性蛋白IIa的SH3结构域。也与动力蛋白结合，动力蛋白是内吞作用后期必需的机械酶。我们发现PAG3对两性蛋白ha的亲和力几乎比dynamin高一个数量级。我们还证明PAG3可以被蛋白酪氨酸激酶Pyk2磷酸化，但不能被其近亲Fak磷酸化；这种磷酸化抑制了与两栖素ha的联系。根据进一步的研究结果，我们认为PAG3可能通过与gtp结合的Arf6活性相互作用将两性肽招募到质膜上；和触发内吞作用的外部刺激引起PAG3的酪氨酸磷酸化，磷酸化的PAG3随后释放出与内吞机制成分（如动力蛋白）相关的amphiphysin ha。

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.