Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.

Ting AY; Kain KH; Klemke RLTsien RY

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Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.

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摘要

The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20-35 changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2001年第26期|15003-15008|共6页
作者
Ting AY; Kain KH; Klemke RLTsien RY;
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作者单位

Department of Pharmacology, Howard Hughes Medical Institute, University of California at San Diego, La Jolla, CA 92093, USA.;

fizer.com;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
Genes; Reporter; Luminescent Proteins; Protein-Tyrosine Kinase; 基因; 报告; 发光蛋白质类; 蛋白质酪氨酸激酶;

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Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.

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