Highly efficient green fluorescent protein-based kinase substrates.
We have developed a general strategy for designing efficient protein substrates of protein kinases by attaching a phosphorylatable peptide sequence to the C-terminus of His6-tagged green fluorescent protein (GFP). We found that several C-terminal attachment sites in GFP allow for correct presentation of the phosphorylatable tail to a variety of protein kinases. Using this strategy, we have constructed highly efficient GFP-based substrates for Src, c-Abl, protein kinase A, and protein kinase C betaII protein kinases. The engineered GFP substrate for Src (GFP235IYGEFG) is 300 times more efficient than the protein most commonly used as a Src substrate-rabbit muscle enolase.
Yang
F
F
Department of Chemistry, Princeton University, Princeton, New Jersey, 08544-1009, USA.
Liu
Y
Y
Bixby
S D
SD
Friedman
J D
JD
Shokat
K M
KM
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D016391:Genes, src / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; D016315:Proto-Oncogene Proteins c-abl; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C