Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction
Identification of a PDZ protein, PIST, as a binding partner for Rho effector Rhotekin: biochemical and cell-biological characterization of Rhotekin-PIST interaction
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DOI：
10.1042/bj20052015
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发表时间：
2006-08-01
影响因子：
4.1
通讯作者：
Nagata, Koh-ichi
中科院分区：
文献类型：
--
作者：
Ito, Hidenori;Iwamoto, Ikuko;Nagata, Koh-ichi
在小GTPase Rho的各种效应蛋白中，Rhotekin的功能几乎是未知的。通过酵母双杂交筛选，我们已经确定了PDZ （PSD-95, disc -large and ZO-1）结构域蛋白与TC10 （Rhotekin家族的一种小GTPase）特异性相互作用]作为Rhotekin的结合伙伴。在体外以及极化和非极化MDCK （Madin-Darby犬肾）细胞中发现Rhotekin与ist相关。Rhotekin的c端SPV （Ser-Pro-Val）基序与PIST的PDZ结构域结合。在COS7细胞中，Rho的激活版本和Rac或Cdc42的激活版本明显抑制了这种结合。相比之下，TC10对结合无影响。免疫荧光分析显示，在非极化的成纤维细胞样MDCK细胞中，在高尔基体中，ist和Rhotekin共定位；在完全极化的细胞中，AJs（粘附连接）共定位。当细胞变得极化时，PIST和Rhotekin从细胞质中被招募到AJs中。在极化的MDCK细胞中，表达组成活性Rho或预防Rhotekin- ist相互作用可诱导Rhotekin的弥漫性细胞质分布。这些结果表明：(1)Rhotekin- ist相互作用的rho依赖性调节；(2)Rhotekin- ist参与AJs的募集；(3)这两种蛋白在细胞极性发育和/或维持中可能发挥作用。
Among various effector proteins for the small GTPase Rho, the function(s) of Rhotekin is (are) almost unknown. We have identified PIST [PDZ (PSD-95, Discs-large and ZO-1) domain protein interacting specifically with TC10 (a Rho-family small GTPase)] as a binding partner for Rhotekin, using yeast two-hybrid screening. Rhotekin was found to associate with PIST in vitro and in both polarized and non-polarized MDCK (Madin-Darby canine kidney) cells. The C-terminal SPV (Ser-Pro-Val) motif of Rhotekin exhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated version of Rho and partially by that of Rac or Cdc42 in COS7 cells. In contrast, TC10 had no effects on the binding. Immunofluorescence analyses revealed the co-localization of PIST and Rhotekin at the Golgi apparatus in non-polarized fibroblast-like MDCK cells and AJs (adherens junctions) in the fully polarized cells. PIST and Rhotekin are recruited from the cytosol to AJs as the cell becomes polarized. Expression of constitutively active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in polarized MDCK cells. These results suggest that there is (1) Rho-dependent regulation of Rhotekin-PIST interaction, (2) involvement of PIST in the recruitment of Rhotekin to AJs and (3) a possible role(s) for these two proteins in cell-polarity development and/or maintenance.