Jw. Mandell et Nc. Gocan, A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity, ANALYT BIOC, 293(2), 2001, pp. 264-268
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.