Waheed Faiza, Speight Pamela, Dan Qinghong, Garcia-Mata Rafael, Szaszi Katalin
Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital.
J Vis Exp. 2012 Mar 31(61):3932. doi: 10.3791/3932.
Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab and we have used it in kidney tubular cell lines. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.
小GTP酶Rho家族的蛋白质是细胞骨架的核心调节因子，控制着多种细胞过程，包括细胞迁移、基因表达、细胞周期进程和细胞黏附。Rho蛋白是分子开关，在结合GTP时处于活性状态，在结合GDP时处于非活性状态。它们的激活由鸟嘌呤核苷酸交换因子（GEF）蛋白家族介导。Rho-GEFs构成一个大家族，具有重叠的特异性。尽管在确定由特定信号激活的GEFs方面已经取得了很大进展，但关于这些蛋白的途径特异性调节仍有许多问题有待解决。Rho-GEFs的数量超过70种，每个细胞表达不止一种GEF蛋白。此外，这些蛋白中的许多不仅激活Rho，还激活该家族的其他成员，这进一步增加了调控网络的复杂性。重要的是，探索GEFs如何被调节需要一种方法来追踪在不同刺激激活的细胞中单个GEF的活性池。在这里，我们提供了一个逐步的方案，用于一种使用亲和沉淀测定法评估和量化可用的活性Rho特异性GEFs的方法。该测定法是几年前在伯里奇实验室开发的，我们已将其用于肾小管细胞系。该测定法利用了一种对活性GEFs具有高亲和力的“无核苷酸”突变型RhoA。该突变（G17A）使蛋白质无法结合GDP或GTP，这种状态模拟了与GEF结合的中间状态。这种突变蛋白的GST标签版本从大肠杆菌中表达和纯化，与谷胱甘肽琼脂糖珠结合，并用于从未经处理和刺激的细胞裂解物中沉淀活性GEFs。由于大多数GEFs通过翻译后修饰或从抑制性结合中释放而被激活，它们的活性状态在细胞裂解物中得以保留，并且可以通过该测定法检测到。捕获的蛋白质可以通过使用蛋白质印迹法用特异性抗体检测来探测已知的GEFs，或者通过质谱分析来鉴定由某些刺激激活的未知GEFs。
