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Single Color Fluorescent Indicators of Protein Phosphorylation for Multicolor Imaging of Intracellular Signal Flow Dynamics

磷酸化 化学 费斯特共振能量转移 绿色荧光蛋白 荧光团 青色 蛋白质磷酸化 蛋白激酶A 双分子荧光互补 黄色荧光蛋白 融合蛋白 生物物理学 原癌基因蛋白质c-akt 钙调蛋白 荧光 生物化学 细胞生物学 生物 蛋白激酶B 重组DNA 基因 物理 艺术 视觉艺术 量子力学 酵母
作者
Yasutoshi Kawai,Moritoshi Sato,Yoshio Umezawa
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:76 (20): 6144-6149 被引量:63
标识
DOI:10.1021/ac040037s
摘要

Existing monitoring methods for protein phosphorylation involved in intracellular signal transduction in vivo are exclusively based on fluorescence resonance energy transfer, which needs the measurement of the change in fluorescence intensities at two wavelengths. Therefore, it is difficult to monitor protein phosphorylation together with other related signaling processes, such as second messengers and protein translocation. To overcome this problem, we developed novel fluorescent indicators, each containing a differently colored (cyan and green) single fluorophore. The present indicator is a tandem fusion protein containing a kinase substrate domain, a circularly permuted fluorescent protein (cpFP), and a phosphorylation recognition domain. The cpFP is obtained by dividing a green fluorescent protein mutant (GFP) at residue 144−145 and linking the carboxy and amino portions thereof with a peptide linker. The substrate domain used in this study is a peptide sequence that is phosphorylated by insulin receptor. Phosphorylation of the substrate domain induces its interaction with the phosphorylation recognition domain, which causes a conformational change in the cpFP and a change in its fluorescence. The cyan and green indicators exhibited 10% decrease and 15% increase, respectively, in their fluorescence intensities upon phosphorylation. Using this cyan indicator and GFP-tagged mitogen-activated protein kinase (MAPK), we found that insulin-induced protein phosphorylation occurred immediately upon the addition of insulin, whereas nuclear translocation of MAPK occurred 7 min later. By tailoring the substrate domains and the phosphorylation recognition domains in these cyan and green indicators, the present approach should be applicable to the in vivo analysis of a broad range of protein phosphorylation processes, together with other intracellular signaling processes.

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