Zhang J, Ma Y, Taylor S S, Tsien R Y
Department of Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14997-5002. doi: 10.1073/pnas.211566798.
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 (14-3-3tau), 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.
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,但目前相关的成像方法,如磷酸化特异性抗体或荧光肽底物,需要固定或显微注射,并且缺乏时间或空间分辨率。我们提出了一种用于蛋白激酶A(PKA)的基因编码荧光报告分子,它由青色荧光蛋白、一个磷酸氨基酸结合结构域(14-3-3tau)、PKA的一个共有底物和黄色荧光蛋白的融合体组成。在活细胞中,cAMP升高会导致由磷酸化诱导的荧光共振能量转移变化引起的黄色与青色发射比率发生25%-50%的变化。通过与PKA全酶拴系,报告分子的反应加速,而通过定位于细胞核则减慢。我们证明,故意重新分布底物或将底物与PKA共定位可以调节其对激酶磷酸化的敏感性。PKA活性荧光报告分子的成功设计及其在研究激酶的区室化和动态调节中的应用,为研究PKA和其他激酶及磷酸酶的靶向和区室化奠定了基础。