Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO
Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO
复制标题
DOI：
10.1091/mbc.9.11.3133
复制
发表时间：
1998-11-01
影响因子：
3.3
通讯作者：
Casanova, JE
中科院分区：
文献类型：
--
作者：
Frank, SR;Hatfield, JC;Casanova, JE
ARNO 是鸟嘌呤核苷酸交换因子家族的成员，对 ADP-核糖基化因子 (ARF) GTP 酶具有特异性。 ARNO 拥有一个与酵母 Sec7p 同源的中心催化结构域和一个相邻的 C 端 pleckstrin 同源 (PH) 结构域。我们之前已经证明，ARNO 在体内定位于质膜，并在体外有效催化 ARF6 核苷酸交换。除了在内吞作用中发挥作用外，ARF6 还被证明可以调节肌动蛋白细胞骨架的组装。为了确定 ARNO 是否是体内 ARF6 的上游调节因子，我们检查了过度表达 ARNO 的 HeLa 细胞中肌动蛋白的分布。我们发现，虽然 ARNO 的表达导致肌动蛋白应力纤维解体，但它不会导致细胞形态的明显变化。然而，用 PKC 激动剂佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理 ARNO 转染子会导致 ARNO、ARF6 和肌动蛋白显着重新分布成类似片状伪足的膜突起。该过程需要 ARF 激活，因为表达催化失活 ARNO 突变体的细胞中不会发生肌动蛋白重排。 PKC 在紧邻其 PH 结构域 C 端的位点磷酸化 ARNO。然而，该位点的突变对 ARNO 调节肌动蛋白重排的能力没有影响，表明 PKC 磷酸化 ARNO 并不能正向调节其活性。最后，我们证明缺乏 C 端 PH 结构域的 ARNO 突变体不再介导细胞骨架重组，表明该结构域在适当的膜定位中的作用。总而言之，这些数据表明 ARNO 代表细胞表面受体、ARF6 和肌动蛋白细胞骨架之间的重要联系。
ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.