ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles
ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles
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DOI：
10.1242/jcs.186817
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发表时间：
2016-10
影响因子：
4
通讯作者：
S. Shibata;Tsubasa Kawanai;T. Hara;A. Yamamoto;T. Chaya;Yasunori Tokuhara;Chinami Tsuji;Manabu Sakai;T. Tachibana;S. Inagaki
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文献类型：
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作者：
S. Shibata;Tsubasa Kawanai;T. Hara;A. Yamamoto;T. Chaya;Yasunori Tokuhara;Chinami Tsuji;Manabu Sakai;T. Tachibana;S. Inagaki
摘要ARHGEF 10是一种已知的RhoA的鸟嘌呤核苷酸交换因子（GEF），在各种疾病中发挥作用，但其功能尚不清楚。为了了解这种蛋白的确切功能，我们提出了针对ARHGEF 10的单克隆抗体，并确定其在HeLa细胞中的定位。发现ARHGEF 10以Rab 6依赖性方式定位于含有Rab 6（其中有三种同种型Rab 6a、Rab 6 b和Rab 6c）、Rab 8（其中有两种同种型Rab 8a和Rab 8b）和/或分泌标志物神经肽Y（NPY）-Venus的囊泡。已知这些囊泡起源于高尔基体，并含有分泌蛋白或膜蛋白。N-末端截短的ARHGEF 10突变体的异位表达导致产生含有Rab 6和Rab 8的大囊泡样结构。此外，小干扰（si）RNA介导的敲低ARHGEF 10损害Rab 8定位到这些胞吐囊泡。此外，MDA-MB 231细胞的侵袭性通过ARHGEF 10以及Rab 8的敲低而显著降低。根据这些结果，我们提出ARHGEF 10以Rab 8依赖的方式在胞吐和肿瘤侵袭中起作用。总结：使用一种新的内部抗体的ARHGEF 10定位分析显示，ARHGEF 10定位在高尔基体出现的囊泡上。进一步的建议表明，ARHGEF 10的功能与Rab 8在这些囊泡上的定位和细胞侵袭性有关。
ABSTRACT The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)–Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner. Summary: An ARHGEF10 localization analysis using a novel in-house antibody revealed ARHGEF10 localization on vesicles emerging from the Golgi apparatus. A further proposal suggests that ARHGEF10 function is related to Rab8 localization on these vesicles and cell invasiveness.