Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells
Fluorescent indicators for Akt/protein kinase B and dynamics of Akt activity visualized in living cells
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DOI：
10.1074/jbc.m212167200
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发表时间：
2003-08-15
影响因子：
4.8
通讯作者：
Umezawa, Y
中科院分区：
文献类型：
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作者：
Sasaki, K;Sato, M;Umezawa, Y
Akt/蛋白激酶B（PKB）是调节多种细胞反应的丝氨酸/苏氨酸激酶。为了提供Akt/PKB活性的空间和时间动态信息，我们开发了Akt/PKB的遗传编码荧光指示剂。指示剂包含两个绿色荧光蛋白突变体、Akt/PKB底物结构域、柔性接头序列和磷酸化识别结构域。基于两种绿色荧光蛋白突变体之间的荧光共振能量转移，指示剂中底物结构域的磷酸化引起发射比的变化。为了使荧光指示剂表现为内源性Akt/PKB底物内皮型一氧化氮合酶和Bad，将它们分别与高尔基体靶结构域和线粒体靶结构域融合。因此，与内源性底物共定位的指示剂赋予它们对Akt/PKB磷酸化的敏感性。我们发现，高尔基体定位的指示器响应于17 β-雌二醇（E2）和胰岛素在内皮细胞中的刺激。此外，E2引起的磷酸化的血管内皮细胞中的本地化的指示剂，但没有磷酸化观察到E2或胰岛素的扩散指示剂，没有靶向结构域。结果与三个指标的差异表明，激活的Akt/PKB定位于亚细胞区室，包括高尔基体和/或线粒体，而不是在胞质溶胶中扩散，从而有效地磷酸化其底物蛋白。E2触发了Akt定位的指示剂的磷酸化，而胰岛素没有诱导这种磷酸化，这表明激活的Akt/PKB定位到线粒体是通过不同的机制在胰岛素和E2之间不同地定向的。
Akt/protein kinase B (PKB) is a serine/threonine kinase that regulates a variety of cellular responses. To provide information on the spatial and temporal dynamics of Akt/PKB activity, we have developed genetically encoded fluorescent indicators for Akt/PKB. The indicators contain two green fluorescent protein mutants, an Akt/PKB substrate domain, flexible linker sequence, and phosphorylation recognition domain. A phosphorylation of the substrate domain in the indicators caused change in the emission ratio based on fluorescent resonance energy transfer between the two green fluorescent protein mutants. To let the fluorescent indicators behave as endothelial nitric-oxide synthase and Bad, which are endogenous Akt/PKB substrates, they were fused with the Golgi target domain and mitochondria target domain, respectively. The indicators thus colocalized with the endogenous substrates conferred their susceptibilities to phosphorylation by Akt/PKB. We showed that the Golgi-localized indicator responded to the stimulation with 17beta-estradiol (E2) and insulin in endothelial cells. In addition, E2 elicited the phosphorylation of the mitochondria-localized indicator in the endothelial cells, but no phosphorylation was observed by E2 or by insulin of the diffusible indicator that has no targeting domain. The difference in the results with the three indicators suggests that the activated Akt/PKB is localized to subcellular compartments, including the Golgi apparatus and/or mitochondria, rather than diffusing in the cytosol, thereby efficiently phosphorylating its substrate proteins. E2 triggered the phosphorylation of the mitochondria-localized indicator, whereas insulin did not induce this phosphorylation, which suggests that the localization of the activated Akt/PKB to the mitochondria is directed differently between insulin and E2 via distinct mechanisms.