Proteomic identification and functional characterization of a novel ARF6 GTPase-activating protein, ACAP4
Proteomic identification and functional characterization of a novel ARF6 GTPase-activating protein, ACAP4
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新型 ARF6 GTP 酶激活蛋白 ACAP4 的蛋白质组学鉴定和功能表征
DOI：
10.1074/mcp.m600050-mcp200
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发表时间：
2006-08-01
影响因子：
7
通讯作者：
Yao, Xuebiao
中科院分区：
文献类型：
--
作者：
Fang, Zhiyou;Miao, Yong;Yao, Xuebiao
ARF 6 GTdR是一种保守的调节膜运输和肌动蛋白为基础的细胞骨架动力学的前沿迁移细胞。ARF 6功能的一个关键决定因素是GTP结合活性状态的寿命，这是由GTP酶激活蛋白（GAP）和GTP-GDP交换因子协调的。然而，对ARF 6介导的细胞迁移的分子机制知之甚少。为了系统地分析细胞迁移过程中调节ARF 6活性的蛋白质，我们使用质谱法对选择性结合活性ARF 6的蛋白质进行了蛋白质组学分析，并鉴定了一种新型ARF 6特异性GAP（ACAP 4）。ACAP 4编码903个氨基酸，包含两个卷曲螺旋，一个普列克底物蛋白同源结构域，一个GAP基序和两个锚蛋白重复序列。我们的生物化学表征表明，ACAP 4具有对ARF 6特异的磷脂酰肌醇4，5-二磷酸依赖性GAP活性。ACAP 4与ARF 6的共定位发生在AIF（4）和表皮生长因子刺激后形成的皱褶膜中。在没有表皮生长因子刺激的情况下，ACAP 4过表达限制了ARF 6向膜皱褶的募集。GTP水解抗性的ARF 6（Q67 L）的表达导致ACAP 4和ARF 6在细胞质膜中积累，表明GTP水解是ARF 6依赖性膜重塑所必需的。显著地，通过小干扰RNA消耗ACAP 4或通过过表达GAP缺陷型ACAP 4抑制ARF 6 GTP水解抑制了伤口愈合中的ARF 6依赖性细胞迁移，证明了ACAP 4在细胞迁移中的重要性。因此，我们的研究揭示了ARF 6介导的细胞迁移的生物学功能。
ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. 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 performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF(4) and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis- resistant ARF6(Q67L) resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.