Fang Zhiyou, Miao Yong, Ding Xia, Deng Hui, Liu Siqi, Wang Fengsong, Zhou Rihong, Watson Charles, Fu Chuanhai, Hu Qicong, Lillard James W, Powell Michael, Chen Yong, Forte John G, Yao Xuebiao
Laboratory of Cellular Dynamics, Hefei National Laboratory for Physical Sciences at Microscale, Hefei 230027, China.
Mol Cell Proteomics. 2006 Aug;5(8):1437-49. doi: 10.1074/mcp.M600050-MCP200. Epub 2006 May 30.
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.
ARF6 GTP酶是迁移细胞前沿膜运输和基于肌动蛋白的细胞骨架动力学的保守调节因子。ARF6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交换因子精心调控的。然而，关于ARF6介导的细胞迁移的分子机制知之甚少。为了系统分析在细胞迁移过程中调节ARF6活性的蛋白质，我们使用质谱对与活性ARF6选择性结合的蛋白质进行了蛋白质组学分析，并鉴定出一种新型的ARF6特异性GAP，即ACAP4。ACAP4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。我们的生化特性表明，ACAP4具有对ARF6特异的磷脂酰肌醇4,5-二磷酸依赖性GAP活性。ACAP4与ARF6的共定位发生在AIF(4)和表皮生长因子刺激后形成的褶皱膜中。在没有表皮生长因子刺激的情况下，ACAP4的过表达限制了ARF6向膜褶皱的募集。抗GTP水解的ARF6（Q67L）的表达导致ACAP4和ARF6在细胞质膜中积累，这表明GTP水解是ARF6依赖性膜重塑所必需的。重要的是，通过小干扰RNA耗尽ACAP4或通过过表达缺乏GAP的ACAP4抑制ARF6 GTP水解，均抑制了伤口愈合中ARF6依赖性细胞迁移，证明了ACAP4在细胞迁移中的重要性。因此，我们的研究为ARF6介导的细胞迁移的生物学功能提供了新的线索。
