Quantitation of RhoA activation: differential binding to downstream effectors.
Quantitation of RhoA activation: differential binding to downstream effectors.
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RhoA 激活的定量：与下游效应器的差异结合。
DOI：
10.1080/21541248.2022.2111945
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Casanova,JamesE
中科院分区：
文献类型：
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作者：
Zhang,Yu-Wen;Torsilieri,HollyM;Casanova,JamesE
小GTPase RhoA通过激活多种下游效应通路的能力控制许多重要的细胞过程。大多数RhoA效应体含有RBD，活性RhoA和RBD之间的相互作用通常会引起效应体的构象变化，从而刺激它们的招募或活性。与GST融合的分离GTPase结合域已广泛用于所谓的下拉测定，以测量细胞裂解物中其他GTPase的激活状态。同样，含有RhoA效应Rhotekin的RBD的GST融合物已被广泛采用为测量RhoA活化的标准化工具。rbd也被用于生成荧光报告结构，以定位完整细胞中GTPase激活的位点。在本报告中，我们证明并非所有形式的活性RhoA都能够与Rhotekin RBD相互作用。一个具有组成活性的RhoA-G14V突变体，与ROCK2和mDIA1的RBD相互作用，不能结合Rhotekin RBD，这是传统的GST下拉实验和我们新建立的BRET实验所证明的。此外，不同刺激诱导的活性RhoA在细胞中也表现出对不同效应器的结合偏好。我们的数据表明，RhoA可能通过效应特异性激活来调节其下游通路的差异，并且RhoA的激活不应该仅仅由其与Rhotekin的相互作用来定义。
The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation. RBDs have also been used to generate fluorescent reporter constructs to localize sites of GTPase activation in intact cells. In this report, we demonstrate that not all forms of active RhoA are capable of interacting with the Rhotekin RBD. A constitutively active RhoA-G14V mutant, which interacted with the RBDs of ROCK2 and mDIA1, was unable to bind the Rhotekin RBD as evidenced by both conventional GST pulldown assay and our newly established BRET assay. Furthermore, active RhoA induced by different stimuli in cells also displayed binding preference for its diverse effectors. Our data demonstrate that RhoA may undergo effector-specific activation for differential regulation of its downstream pathways, and that RhoA activation should not be defined solely by its interaction with Rhotekin.