Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering
Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering
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DOI：
10.1073/pnas.211566798
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发表时间：
2001-12-18
影响因子：
11.1
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Zhang, J;Ma, YL;Tsien, RY
许多形式的信号转导的复杂性和特异性被广泛怀疑需要蛋白激酶和磷酸酶活性的空间微区室化，然而目前的相关成像方法，如磷酸化特异性抗体或荧光肽底物需要固定或显微注射，缺乏时间或空间分辨率。我们提出了一个基因编码的蛋白激酶A（PKA）的荧光报告组成的融合青色荧光蛋白，磷酸化氨基酸结合域（14-3-3 tau），PKA的共识底物，和黄色荧光蛋白。cAMP升高导致活细胞中黄与青色发射的比率发生25-50%的变化，这是由荧光共振能量转移中磷酸化诱导的变化引起的。报告的反应加速拴PKA全酶和本地化的细胞核减慢。我们证明，故意重新分配的底物或共定位的底物和PKA可以调节其敏感性的激酶磷酸化。PKA活性荧光报告基因的成功设计及其在研究激酶的区室化和动态调控中的应用为研究PKA与其他激酶和磷酸酶的靶向和区室化奠定了基础。
The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation of protein kinase and phosphatase activities, yet current relevant imaging methods such as phosphorylation-specific antibodies or fluorescent peptide substrates require fixation or microinjection and lack temporal or spatial resolution. We present a genetically encoded fluorescent reporter for protein kinase A (PKA) consisting of fusions of cyan fluorescent protein, a phosphoamino acid binding domain (14-3-3 tau), a consensus substrate for PKA, and yellow fluorescent protein. cAMP elevations cause 25-50% changes in the ratios of yellow to cyan emissions in live cells caused by phosphorylation-induced changes in fluorescence resonance energy transfer. The reporter response was accelerated by tethering to PKA holoenzyme and slowed by localization to the nucleus. We demonstrate that deliberate redistribution of a substrate or colocalizing a substrate and PKA can modulate its susceptibility to phosphorylation by the kinase. The successful design of a fluorescent reporter of PKA activity and its application for studying compartmentalized and dynamic modulation of kinases lays a foundation for studying targeting and compartmentation of PKA and other kinases and phosphatases.