Andres Douglas A, Rudolph Jennifer L, Sengoku Tomoko, Shi Geng-Xian
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky, USA.
Methods Enzymol. 2006;407:499-512. doi: 10.1016/S0076-6879(05)07040-0.
Rit (Ras-like expressed in many tissues) is the founding member of a novel subgroup within the larger Ras superfamily of small GTP-binding proteins. Although Rit shares more than 50% amino acid identity with Ras, it contains a unique effector domain in common with the closely related Rin and Drosophila Ric proteins and lacks the C-terminal lipidation motifs critical for the membrane association and biological activity of many Ras proteins. Interestingly, whereas Rit has only modest transforming ability when assayed in NIH 3T3 cells, Rit exhibits neuronal differentiation activities comparable to those of oncogenic mutants of Ras when assayed in PC12 and other neuronal cell lines. This cell-type specificity is explained in part by the ability of Rit to selectively activate the neuronal Raf isoform, B-Raf. Importantly, Rit seems to play a critical role in neurotrophin-mediated MAP kinase signaling, because Rit gene silencing significantly alters NGF-dependent MAP kinase signaling and neuronal differentiation. In this chapter, we discuss the reagents and methods used to characterize Rit-mediated signaling to MAP kinase-signaling pathways to determine the extracellular stimuli that regulate Rit activation and to characterize Rit-induced neuronal differentiation.
Rit（在多种组织中表达的类Ras蛋白）是小GTP结合蛋白Ras超家族中一个新亚组的创始成员。尽管Rit与Ras的氨基酸同一性超过50%，但它含有一个与密切相关的Rin和果蝇Ric蛋白共有的独特效应结构域，并且缺乏许多Ras蛋白的膜结合和生物学活性所必需的C末端脂化基序。有趣的是，虽然在NIH 3T3细胞中检测时Rit只有适度的转化能力，但在PC12和其他神经元细胞系中检测时，Rit表现出与Ras致癌突变体相当的神经元分化活性。这种细胞类型特异性部分是由Rit选择性激活神经元Raf亚型B-Raf的能力来解释的。重要的是，Rit似乎在神经营养因子介导的MAP激酶信号传导中起关键作用，因为Rit基因沉默会显著改变NGF依赖的MAP激酶信号传导和神经元分化。在本章中，我们讨论了用于表征Rit介导的向MAP激酶信号通路的信号传导、确定调节Rit激活的细胞外刺激以及表征Rit诱导的神经元分化的试剂和方法。
