Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity
Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity
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DOI：
10.1074/jbc.m304078200
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发表时间：
2003-10-31
影响因子：
4.8
通讯作者：
Venkateswarlu, K
中科院分区：
文献类型：
--
作者：
Venkateswarlu, K
ADP-核糖基化因子6（ARF 6）小GTP酶在刺激肌动蛋白重组和膜皱褶的途径中作为GDP/GTP调节开关发挥作用。活性ARF 6（GTP）的形成受到细胞粘连蛋白等鸟嘌呤核苷酸交换因子（GEF）的刺激，细胞粘连蛋白等鸟嘌呤核苷酸交换因子（GEF）通过普列克底物蛋白同源结构域结合脂质第二信使3，4，5-三磷酸磷脂酰肌醇，转移到激动剂刺激的细胞中的细胞膜，随后激活ARF 6。在酵母双杂交筛选中，以细胞粘连素2为诱饵，我们分离到了一个编码蛋白质的cDNA，该蛋白质被称为细胞粘连素交换因子1（IPCEF 1）的相互作用蛋白。使用酵母双杂交和谷胱甘肽S-转移酶下拉分析加上缺失突变分析，所需的特定结构域的cytohesin 2-IPCEF 1相互作用被映射到卷曲螺旋结构域的cytohesin 2和C-末端121个氨基酸的IPCEF 1。IPCEF 1还与ARF GEFs的细胞粘连蛋白家族的其他成员相互作用，表明与IPCEF 1的相互作用在ARF GEFs的细胞粘连蛋白家族中是高度保守的。通过免疫沉淀证实了哺乳动物细胞中细胞粘连素2和IPCEF 1的相互作用。免疫荧光分析显示，IPCEF 1 co-localizes与cytohesin 2在未刺激的细胞中的胞质溶胶和易位到质膜通过结合到表皮生长因子刺激的细胞中的cytohesin 2。然而，IPCEF 1的缺失突变体，缺乏细胞粘连素2结合位点未能与细胞粘连素2共同迁移到膜刺激的细胞。IPCEF 1-细胞粘连素2相互作用的功能意义通过显示IPCEF 1增加细胞粘连素2对ARF（GTP）形成的体外和体内刺激来证明。
The ADP-ribosylation factor 6 (ARF6) small GTPase functions as a GDP/GTP-regulated switch in the pathways that stimulate actin reorganization and membrane ruffling. The formation of active ARF6(GTP) is stimulated by guanine nucleotide exchange factors (GEFs) such as cytohesins, which translocate to the plasma membrane in agonist-stimulated cells by binding the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate through the pleckstrin homology domain with subsequent ARF6 activation. Using cytohesin 2 as bait in yeast two-hybrid screening, we have isolated a cDNA encoding a protein termed interaction protein for cytohesin exchange factors 1 (IPCEF1). Using yeast two-hybrid and glutathione S-transferase pull-down assays coupled with deletion mutational analysis, the specific domains required for the cytohesin 2-IPCEF1 interaction were mapped to the coiled-coil domain of cytohesin 2 and the C-terminal 121 amino acids of IPCEF1. IPCEF1 also interacts with the other members of the cytohesin family of ARF GEFs, suggesting that the interaction with IPCEF1 is highly conserved among the cytohesin family of ARF GEFs. The interaction of cytohesin 2 and IPCEF1 in mammalian cells was demonstrated by immunoprecipitation. Immunofluorescence analysis revealed that IPCEF1 co-localizes with cytohesin 2 to the cytosol in unstimulated cells and translocates to the plasma membrane via binding to cytohesin 2 in epidermal growth factor-stimulated cells. However, a deletion mutant of IPCEF1 that lacks the cytohesin 2 binding site failed to co-migrate with cytohesin 2 to the membrane in stimulated cells. The functional significance of the IPCEF1-cytohesin 2 interaction is demonstrated by showing that IPCEF1 increases the in vitro and in vivo stimulation of ARF(GTP) formation by cytohesin 2.