Pan Shujuan, Wang Ruoning, Zhou Xi, He Guangan, Koomen John, Kobayashi Ryuji, Sun Le, Corvera Joe, Gallick Gary E, Kuang Jian
Department of Experimental Therapeutics, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 2006 Nov 10;281(45):34640-50. doi: 10.1074/jbc.M602263200. Epub 2006 Sep 10.
The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with alpha-actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of alpha-actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.
保守的衔接蛋白Alix，也称为AIP1或Hp95，可促进培养的哺乳动物成纤维细胞的扁平化和排列；然而，Alix调节成纤维细胞形态的机制尚不清楚。在这里，我们证明在WI38细胞中，Alix与丝状肌动蛋白（F-肌动蛋白）以及基于F-肌动蛋白的结构片状伪足和应力纤维相关联，WI38细胞需要Alix表达来维持典型的成纤维细胞形态。通过小干扰RNA（siRNA）降低Alix表达会降低F-肌动蛋白含量并抑制应力纤维组装。在无细胞系统中，Alix在N端Bro1结构域和C端富含脯氨酸的结构域均直接与F-肌动蛋白相互作用。在从WI38细胞裂解物中免疫沉淀的Alix中，肌动蛋白是Alix最丰富的伴侣蛋白。此外，Alix中间区域的N端一半与cortactin结合，cortactin是ARP2/3复合物介导的肌动蛋白聚合起始的激活剂。Alix是cortactin在片状伪足定位所必需的。Alix中间区域的C端一半与α-辅肌动蛋白相互作用，α-辅肌动蛋白是在应力纤维中捆绑F-肌动蛋白的关键因子。敲低Alix会减少与F-肌动蛋白相关的α-辅肌动蛋白的量。这些发现证实了Alix在肌动蛋白细胞骨架组装中的关键作用。
