A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity.
A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity.
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一种绿色荧光蛋白激酶底物，可检测和定位细胞内 ERK/MAP 激酶活性。
DOI：
10.1006/abio.2001.5138
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Gocan,NC
中科院分区：
文献类型：
--
作者：
Mandell,JW;Gocan,NC
We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85–144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.
