Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells
Genetically encoded fluorescent indicators to visualize protein phosphorylation by extracellular signal-regulated kinase in single living cells
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DOI：
10.1021/ac062171d
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发表时间：
2007-03-15
影响因子：
7.4
通讯作者：
Umezawa, Yoshio
中科院分区：
文献类型：
--
作者：
Sato, Moritoshi;Kawai, Yasutoshi;Umezawa, Yoshio
细胞外信号调节激酶（ERK）是一种丝氨酸/苏氨酸蛋白激酶，其调节多种细胞功能，例如细胞生长和分化。为了研究活细胞中ERK激活后蛋白磷酸化的时空动力学，我们开发了ERK的基因编码荧光指示剂。本指示剂基于两个绿色荧光蛋白突变体之间的荧光共振能量转移（FRET）。通过活化ERK的指示剂的磷酸化由于它们的构象改变而改变FRET效率。我们可视化的细胞质和细胞核活性的ERK使用本指标。因此，我们发现，ERK的激活持续时间是相当大的不同之间的细胞质和细胞核中的活细胞。ERK活性的亚细胞差异可能是ERK调节细胞功能的基础。目前的荧光指示剂提供了一个强大的工具来揭示蛋白磷酸化的时空动态ERK在单个活细胞。
Extracellular signal-regulated kinase (ERK) is a serine/threonine protein kinase that regulates a wide variety of cell functions, such as cell growth and differentiation. To study the spatiotemporal dynamics of protein phosphor-ylation by activated ERK in living cells, we have developed genetically encoded fluorescent indicators for ERK. The present indicators are based on fluorescence resonance energy transfer (FRET) between two green fluorescent protein mutants. Phosphorylation of the indicators by activated ERK changes the FRET efficiency due to their conformational alterations. We visualized the cytosolic and nuclear activity of ERK using the present indicators. We thus found that the activation duration of ERK is considerably different between the cytosol and nucleus in living cells. The subcellular difference in the ERK activity may be fundamental to the regulation of cell functions by ERK. The present fluorescent indicators provide a powerful tool to reveal the spatiotemporal dynamics of protein phosphorylation by ERK in single living cells.