Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domain
Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domain
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DOI：
10.1091/mbc.e06-11-0998
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发表时间：
2007-06-01
影响因子：
3.3
通讯作者：
Donaldson, Julie G.
中科院分区：
文献类型：
--
作者：
Cohen, Lee Ann;Honda, Akira;Donaldson, Julie G.
ARNO是GTP酶的Arf家族的可溶性鸟嘌呤核苷酸交换因子（GEF）。虽然在生物化学测定中，ARNO更喜欢Arf 1而不是Arf 6作为底物，但其在细胞质膜（PM）上的定位表明与Arf 6相互作用。在这项研究中，我们发现，ARNO激活Arf 1在HeLa和COS-7细胞导致招聘的Arf 1上的动态PM皱褶。相比之下，Arf 6被ARNO激活的程度低于EFA 6，EFA 6是一种典型的Arf 6格尔。值得注意的是，Arf 6以其GTP结合形式将ARNO募集到PM，并且这两种蛋白质可以免疫沉淀。ARNO与Arf 6的结合不是通过催化Sec 7结构域介导的，而是通过普列克底物蛋白同源（PH）结构域介导的。活性Arf 6还结合了另一个ARNO家族成员Grp 1的PH结构域。这种相互作用是直接的，需要肌醇磷脂和GTP。我们提出了一个模型的顺序Arf激活在PM Arf 6-GTP招聘ARNO家族GEFs进一步激活其他Arf亚型。
ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GER Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.