Venkateswarlu Kanamarlapudi, Brandom Kevin G, Lawrence Joanna L
Department of Pharmacology, School of Medical Sciences, The University of Bristol, Bristol BS8 1TD, UK.
J Biol Chem. 2004 Feb 20;279(8):6205-8. doi: 10.1074/jbc.C300482200. Epub 2003 Nov 18.
The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6GDP. Centaurin-alpha1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here, we show that centaurin-alpha1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-alpha1 also inhibited cortical actin formation in a PIP3-dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-alpha1. An artificially plasma membrane-targeted centaurin-alpha1 bypasses the requirement of PIP3 for its involvement in ARF6 inactivation, suggesting that PIP3 is required for recruitment of centaurin-alpha1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-alpha1 negatively regulates ARF6 activity by functioning as an in vivo PIP3-dependent ARF6 GAP.
ADP核糖基化因子（ARF）6小GTP酶调节囊泡运输和细胞骨架肌动蛋白重组。GTP酶激活蛋白（GAPs）催化无活性的ARF6GDP的形成。Centaurin-α1包含一个ARF GAP和两个普列克底物蛋白同源（PH）结构域，它们与第二信使磷脂酰肌醇3,4,5-三磷酸（PIP3）结合。在此，我们表明Centaurin-α1特异性抑制ARF6在体内的GTP加载以及ARF6从内体区室到质膜的重新分布，这些都是其激活的指征。Centaurin-α1还以PIP3依赖的方式抑制皮质肌动蛋白的形成。此外，ARF6的组成型活性突变体而非ARF1的突变体可逆转Centaurin-α1对皮质肌动蛋白形成的抑制作用。一种人工靶向质膜的Centaurin-α1绕过了PIP3对其参与ARF6失活的需求，这表明PIP3是将Centaurin-α1募集到质膜所必需的，但不是其活性所必需的。总之，这些数据表明Centaurin-α1作为一种体内PIP3依赖的ARF6 GAP对ARF6活性起负调节作用。
