A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity

We describe a versatile intracellular reporter of ERW 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 pro tein (GFP). The fused fragment of myelin basic protein contains a single co nsensus 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 t ransient or stable transfection, the fusion protein acts as a bona fide kin ase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the t otal substrate pool is easily visualized by GFP autofluorescence and the ex tent of its phosphorylation simultaneously detected within intact fixed cel ls by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intr acellular analysis of many specific protein kinase substrates for which pho spho-specific antibodies have been produced. (C) 2001 Academic Press.