Identification of a novel sequence PDZ-RhoGEF that mediates interaction with the in actin cytoskeleton
Identification of a novel sequence PDZ-RhoGEF that mediates interaction with the in actin cytoskeleton
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DOI：
10.1091/mbc.e03-07-0527
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发表时间：
2004-04-01
影响因子：
3.3
通讯作者：
Wedegaertner, PB
中科院分区：
文献类型：
--
作者：
Banerjee, J;Wedegaertner, PB
Rho家族的小GTP酶是肌动蛋白细胞骨架重排的重要调节因子。Rho由Rho鸟嘌呤-核苷酸交换因子（GEF）家族的成员激活;然而，调节RhoGEF的机制尚不清楚。这份报告表明，PDZ-RhoGEF，一个亚家族的RhoGEF，含有G蛋白信号结构域的调节器的成员，是部分定位在或靠近质膜在293 T，COS-7，和Neuro 2a细胞，这种定位是一致的皮质肌动蛋白。通过使用latrunculin B破坏细胞中的皮质肌动蛋白细胞骨架防止了PDZ-RhoGEF的质膜周围定位。免疫共沉淀和F-肌动蛋白共沉淀试验表明，PDZ-RhoGEF结合肌动蛋白。广泛的缺失突变揭示了PDZ-RhoGEF中存在一个新的25个氨基酸的序列，位于氨基酸561-585，这是必要的和足够的定位到肌动蛋白细胞骨架和与肌动蛋白的相互作用。最后，与野生型PDZ-RhoGEF相比，不能与肌动蛋白细胞骨架相互作用的PDZ-RhoGEF突变体显示增强的Rho依赖性信号传导。这些结果确定与肌动蛋白细胞骨架的相互作用是PDZ-RhoGEF的一种新功能，从而暗示肌动蛋白相互作用在组织PDZ-RhoGEF信号传导中。
Small GTPases of the Rho family are crucial regulators of actin cytoskeleton rearrangements. Rho is activated by members of the Rho guanine-nucleotide exchange factor (GEF) family; however, mechanisms that regulate RhoGEFs are not well understood. This report demonstrates that PDZ-RhoGEF, a member of a subfamily of RhoGEFs that contain regulator of G protein signaling domains, is partially localized at or near the plasma membranes in 293T, COS-7, and Neuro2a cells, and this localization is coincident with cortical actin. Disruption of the cortical actin cytoskeleton in cells by using latrunculin B prevents the peri-plasma membrane localization of PDZ-RhoGEF. Coimmunoprecipitation and F-actin cosedimentation assays demonstrate that PDZ-RhoGEF binds to actin. Extensive deletion mutagenesis revealed the presence of a novel 25-amino acid sequence in PDZ-RhoGEF, located at amino acids 561-585, that is necessary and sufficient for localization to the actin cytoskeleton and interaction with actin. Last, PDZ-RhoGEF mutants that fail to interact with the actin cytoskeleton display enhanced Rho-dependent signaling compared with wild-type PDZ-RhoGEF. These results identify interaction with the actin cytoskeleton as a novel function for PDZ-RhoGEF, thus implicating actin interaction in organizing PDZ-RhoGEF signaling.