Kupzig Sabine, Bouyoucef Dalila, Cozier Gyles E, Cullen Peter J
The Henry Wellcome Integrated Signalling Laboratories, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, United Kingdom.
Methods Enzymol. 2006;407:64-82. doi: 10.1016/S0076-6879(05)07007-2.
Two classes of proteins govern Ras activation. Guanine-nucleotide exchange factors (Ras GEFs) catalyze the activation of Ras by inducing the dissociation of GDP to allow association of the more abundant GTP, whereas GTPase-activating proteins (Ras GAPs), bind to the GTP-bound form and, by enhancing the intrinsic GTPase activity, catalyze Ras inactivation. A wide range of Ras GEFs and Ras GAPs have been identified from the various genome projects, and in a few instances, the mechanisms by which signals originating from activated receptors converge on specific GEFs and GAPs have been mapped. However, for most Ras GEFs and GAPs we have a poor understanding of their regulation. Here we focus on describing methods used to study the regulation of the GAP1 family of Ras GAPs. In particular, we emphasize how by combining biochemical, molecular, and imaging techniques, one can determine some of the complex array of mechanisms that have evolved to modulate the spatial and temporal dynamics of Ras regulation through these various Ras GAPs. By combining biochemical, molecular, and imaging techniques, we describe the visualization of the diverse and dynamic mechanisms through which stimulation of cell surface receptors leads to the regulation of these proteins. Thus, although each member of the GAP1 family performs the same basic biological function, that is, they function as Ras GAPs, each is designed to respond and decode signals from distinct second messenger pathways.
两类蛋白质调控Ras的激活。鸟嘌呤核苷酸交换因子（Ras GEFs）通过诱导GDP解离，使更丰富的GTP与之结合，从而催化Ras的激活；而GTP酶激活蛋白（Ras GAPs）则与结合GTP的形式结合，并通过增强内在的GTP酶活性来催化Ras失活。从各种基因组计划中已鉴定出多种Ras GEFs和Ras GAPs，在少数情况下，已绘制出源自活化受体的信号汇聚到特定GEFs和GAPs的机制。然而，对于大多数Ras GEFs和GAPs，我们对其调控机制了解甚少。在这里，我们重点描述用于研究Ras GAPs的GAP1家族调控的方法。特别地，我们强调如何通过结合生化、分子和成像技术，确定一些复杂的机制，这些机制已经进化出来，通过这些不同的Ras GAPs来调节Ras调控的空间和时间动态。通过结合生化、分子和成像技术，我们描述了细胞表面受体刺激导致这些蛋白质调控的多样且动态机制的可视化。因此，尽管GAP1家族的每个成员都执行相同的基本生物学功能，即它们作为Ras GAPs发挥作用，但每个成员都被设计为对来自不同第二信使途径的信号作出反应并进行解码。
