Method for determining protein kinase substrate specificities by the phosphorylation of peptide libraries on beads, phosphate-specific staining, automated sorting, and sequencing.
A method is described for the elucidation of the peptide substrate phosphorylation specificity of a protein kinase. Peptide libraries with two to six degenerate positions and a length of seven or nine amino acids were generated directly on Sepharose beads by solid-phase peptide synthesis according to the split-and-mix procedure. The immobilized peptides were incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA) as a model enzyme resulting in the phosphorylation of the beads that contain the recognition motif of the kinase. The beads were then stained with a new phosphate-monoester-specific fluorescent dye consisting of a complex of iron(III) with fluorescein-coupled iminodiacetic acid. A flow cytometer was used to analyze the phosphorylation efficiency and the beads with the highest phosphorylation degree were isolated by the use of a fluorescence-activated cell sorter. Pool sequencing of those beads revealed the preferred kinase motif. The results are in good agreement with data from the literature. The method lends itself to the rapid elucidation of the specificity of uncharacterized protein kinases.
Gast
R
R
Gesellschaft für Biotechnologische Forschung, AG Molecular Recognition, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Glökler
J
J
Höxter
M
M
Kiess
M
M
Frank
R
R
Tegge
W
W
D000595:Amino Acid Sequence; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000737:chemistry; D019151:Peptide Library; D010766:Phosphorylation; D020539:Sequence Analysis, Protein; D013194:Staining and Labeling; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009842:Oligopeptides; D019151:Peptide Library; D017868:Cyclic AMP-Dependent Protein Kinases