【作者】 方志友；

【导师】 姚雪彪；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2006， 博士

【摘要】 ADP核糖基化因子（Arfs）是真核细胞中广泛表达的且是高度保守的GTP结合蛋白家族，是Ras超家族成员之一。从生化特性和序列的相似性上，Arfs蛋白分为Arf蛋白和Arf相似蛋白。Arf蛋白可再被划分为三类，第一类包括Arf1，Arf2和Arf3；第二类包括Arf4和Arf5；第三类只有Arf6。与其它的GTP结合蛋白一样，Arfs蛋白在功能上是一个分子开关，在GTP结合状态和GDP结合状态之间循环，它与许多不同的靶蛋白结合影响一系列细胞事件。Arf6是唯一在细胞质膜上发挥功能的Arf蛋白，GTP结合的Arf6调节内吞膜的运输，同时调节在细胞外周的细胞骨架变化。对Arf6的功能来说，关键性因素是GTP结合状态的时期，细胞内Arf6与GTP结合或与GDP结合是分别由鸟苷交换因子和GTP酶激活蛋白（GAPs）协调来实现的。GAPs是Arf功能关键性调节子，控制Arf回到失活的GDP结合状态。据报道Atf6的周期性变化是调节乳腺癌细胞侵袭的必需条件，然而，Arf6介导的细胞迁移的分子机制了解依然很少。 为了系统地分析在细胞迁移过程中，参与调节Arf6活性的蛋白，利用功能蛋白质组学的方法，通过质谱鉴定筛选特异地与激活态的Arf6结合的蛋白。我们鉴定了一个Arf蛋白新的GTP酶激活蛋白，根据它包含的保守的结构特征，命名为ACAP4。和以前描述的Arf激活蛋白ASAP1，PAP／PAG3及ACAP1／2一样，ACAP4被划入AZAP蛋白家族，它们拥有许多共同的结构基序，包括两个coiled-coil结构，一个PH结构，一个GAP结构以及两个ANK重复结构。 我们生化结果证明ACAP4有磷酸肌醇依赖的GAP活性，ACAP4是一个Arf6特异的GTP酶激活蛋白。体外活性检测结果显示Arf6是ACAP4的偏好底物，而不是Arf1和Arf5蛋白。在HeLa细胞中，过表达ACAP4能抑制AlF₄诱导的Arf6依赖的膜突起的形成。另外，ACAP4被募集到细胞外周，管状的膜结构上，Arf6的激活引起内吞膜重新回到细胞质膜。在非EGF的刺激下，ACAP4的过表达抑制Arf6被募集到膜皱褶处，但是在EGF的刺激下，ACAP4与Arf6共定位于actin丰富的膜皱褶上。ACAP4和更多还原

【Abstract】 The ADP-ribosylation factors (Arfs) belong to the Ras superfamily members and are a family of ubiquitous and highly conserved GTP-binding proteins in eukaryotes. Based on biochemical activities and sequence similarity, proteins within the Arf family can be divided into Arf and Arf-like proteins. The Arf proteins have been further subdivided into class I (Arf1, Arf2, and Arf3), class II (Arf4 and Arf5), and class III (Arf6) Arfs. Similar to other GTP binding proteins, Arfs function as molecular switches, cycling between GTP-bound state and GDP-bound state, with multiple targets affecting a variety of cellular events. Arf6 is the sole Arf protein function on plasma membrane, the GTP-binding protein Arf6 regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. A key determinant of Arf6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTP-GDP exchanging factors and GTPase-activating proteins. GTPase-activating proteins(GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. It has reported that Arf6 is essential to regulate breast cancer cell invasion dependent on Arf6 GTPase cycle. However, very little is known about the molecular mechanisms underlying Arf6-mediated cell migration.To systematically analyze proteins that regulate Arf6 activity during cell migration, we perform a proteomic analysis of proteins selectively interacted with active Arf6 using mass Spectrometry. Here, we report the identification and characterization of a novel Arf GAPs. We named it as ACAP4 according to its conservedly structual characterization. Together with previously described Arf GAPs, ASAP1, PAP/PAG3 and ACAP1/2, ACAP4 can be grouped into AZAP protein family defined by several common structural motifs including two coiled coils, one PH domain, one GAP motif, and two ankyrin repeats.Our biochemical characterization demonstrates that ACAP4 has a phosphoinositide dependent GAP activity, ACAP4 functioned as Arf6 GAPs. In vitro, ACAP4 preferred Arf6 as a substrate, rather than Arf1 and Arf5. In HeLa cells, overexpression of ACAP4更多还原