Methods for measuring RGS protein phosphorylation by G protein-regulated kinases.
Methods for measuring RGS protein phosphorylation by G protein-regulated kinases.
复制标题
通过 G 蛋白调节激酶测量 RGS 蛋白磷酸化的方法。
作者：
S. Hollinger;J. Hepler
关于G蛋白信号传导的主要参与者G蛋白信号传导的调节因子（RGS）的细胞调控知之甚少。众所周知，这些蛋白具有负调节G蛋白信号的能力，然而，它们的主要细胞功能可能超出了这种有限的作用。要全面了解RGS蛋白在细胞中的作用，需要了解它们通过短潜伏期和诱导信号（如G蛋白的激酶激活）进行调节。许多RGS蛋白在细胞中被磷酸化，对其功能和定位有不同的影响。这些研究集中在RGS14上，它包含几种G蛋白调节激酶的识别基序。本文概述了我们实验室用于研究RGS14磷酸化的程序，并描述了纯化RGS14的方法，并说明了可能遇到的并发症。本文描述了用于研究3-5环腺苷单磷酸（cAMP）依赖性蛋白激酶（PKA）、细胞外信号调节激酶（ERK）和蛋白激酶C （PKC）对RGS蛋白识别的标准方案，以及用于建立由这些激酶修饰的特定氨基酸的策略。虽然本章的重点是对RGS14的研究，但所描述的协议很容易修改为其他RGS蛋白。
Little is known about cellular regulation of the regulators of G protein signaling (RGS) proteins, principal players in G protein signaling. These proteins are known for their capacity to negatively regulate G protein signals, however, their chief cellular functions may expand beyond this limited role. Comprehensive understanding of cellular roles of RGS proteins requires knowledge of their regulation by short latency and inducible signals, such as kinase activation by G proteins. A number of RGS proteins are phosphorylated in cells, with varied effects on their function and localization. These studies focus on RGS14, which contains recognition motifs for several G protein-regulated kinases. Procedures used in our laboratory to study the phosphorylation of RGS14 are outlined, and the method used to purify RGS14 is described with notes on complications that may be encountered. Standard protocols used to investigate the recognition of RGS proteins by 3-5-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), extracellular signal-regulated kinase (ERK), and protein kinase C (PKC) are described, followed by strategies used to establish the specific amino acids modified by these kinases. Although this chapter focuses on investigations into RGS14, the protocols described are readily modified for other RGS proteins.
使用高效液相色谱和质谱法鉴定蛋白质中的磷酸化位点。
DOI：
10.1385/1-59259-059-4:87
发表时间：
2001
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
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作者：
Craig,AG
通讯作者：
Craig,AG