Highly efficient green fluorescent protein-based kinase substrates.

F Yang; Y Liu; S D Bixby; J D Friedman; K M Shokat

doi:10.1006/abio.1998.2885

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Highly efficient green fluorescent protein-based kinase substrates.

F Yang·Y Liu·S D Bixby·J D Friedman·K M Shokat

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Abstract

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.

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Amino Acid Sequence Animals Cyclic AMP-Dependent Protein Kinases Escherichia coli Genes, src Green Fluorescent Proteins Luminescent Proteins Molecular Sequence Data Phosphorylation Protein Kinase C Protein Kinases Proto-Oncogene Proteins c-abl Rabbits Sequence Homology, Amino Acid Substrate Specificity Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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F Yang, Y Liu, S D Bixby, J D Friedman, K M Shokat. 1999-01-15. Highly efficient green fluorescent protein-based kinase substrates.. https://doi.org/10.1006/abio.1998.2885

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