Mandell J W, Gocan N C
Department of Pathology (Neuropathology), University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Anal Biochem. 2001 Jun 15;293(2):264-8. doi: 10.1006/abio.2001.5138.
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.
我们描述了一种多功能的细胞内ERK/丝裂原活化蛋白激酶（MAP激酶）活性报告分子：一种cDNA构建体pGFP.MBP，其编码与人髓鞘碱性蛋白85 - 144位氨基酸融合至增强型绿色荧光蛋白（GFP）C端的片段。髓鞘碱性蛋白的融合片段包含一个单一的ERK/MAP激酶磷酸化共有基序（PRTP，其中苏氨酸被磷酸化）。通过使用市售的磷酸化特异性单克隆抗体进行免疫印迹或免疫荧光可检测该特定基序的磷酸化。当通过瞬时或稳定转染在哺乳动物细胞中表达时，融合蛋白作为一种真正的激酶底物，如血清快速诱导的磷酸化被特异性MEK抑制剂阻断所证明。此外，通过GFP自发荧光可轻松观察到总底物池的定位，并且使用市售的磷酸化特异性抗体通过免疫荧光在完整固定细胞内同时检测其磷酸化程度。所述方法通常应适用于对已产生磷酸化特异性抗体的许多特定蛋白激酶底物进行细胞内分析。
