Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS
Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS
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DOI：
10.1016/j.celrep.2020.108074
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发表时间：
2020-08-25
期刊：
影响因子：
8.8
通讯作者：
Nishida, Noritaka
中科院分区：
文献类型：
--
作者：
Zhao, Qingci;Fujimiya, Ryu;Nishida, Noritaka
小的鸟苷三磷酸酶（GTP酶）RAS在信号转导中起分子开关的作用，其突变和异常激活与肿瘤发生有关。在此，我们对非法尼基化的RAS进行实时的细胞内核磁共振（NMR）分析，以测定活的哺乳动物细胞胞质溶胶中活性GTP结合形式（fGTP）的比例随时间的变化过程。观察到的细胞内fGTP显著低于在体外对野生型RAS以及致癌突变体所测得的值，这是由于鸟苷二磷酸（GDP） - GTP交换速率（k(ex)）降低和GTP水解速率（k(hy)）增加所致。体外重建实验表明，高粘性环境促使k(ex)降低，而k(hy)的增加则受到未鉴定的胞质蛋白的刺激。本研究展示了细胞内NMR直接检测哺乳动物细胞中RAS的GTP结合水平的能力，从而揭示RAS的k(hy)和k(ex)受到多种细胞内因子的调节。
The small guanosine triphosphatase (GTPase) RAS serves as a molecular switch in signal transduction, and its mutation and aberrant activation are implicated in tumorigenesis. Here, we perform real-time, in-cellnuclear magnetic resonance (NMR) analyses of non-farnesylated RAS to measure time courses of the fraction of the active GTP-bound form (fGTP) within cytosol of live mammalian cells. The observed intracellular fGTP is significantly lower than that measured in vitro for wild-type RAS as well as oncogenic mutants, due to both decrease of the guanosine diphosphate (GDP)-GTP exchange rate (k(ex)) and increase of GTP hydrolysis rate(k(hy)). In vitro reconstitution experiments show that highly viscous environments promote a reduction of kex,whereas the increase of k(hy) is stimulated by unidentified cytosolic proteins. This study demonstrates the power of in-cell NMR to directly detect the GTP-bound levels of RAS in mammalian cells, thereby revealing that the k(hy) and kex of RAS are modulated by various intracellular factors.