Versatile fluorescence probes of protein kinase activity.

J Am Chem Soc 2003 Nov;125(47):14248-9

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

We introduce a versatile fluorescent peptide reporter of protein kinase activity. The probe can be modified to target a desired kinase by changing the kinase recognition motif in the peptide sequence. The reporter motif contains the Sox amino acid, which generates a fluorescence signal when bound to Mg2+ present in the reaction mixture. The phosphorylated peptide exhibits a much greater affinity for Mg2+ than its unphosphorylated analogue and, thus, a greater fluorescence intensity. Product formation during phosphorylation by the kinase is easily followed by the increase in fluorescence intensity over time. These probes exhibit a 3-5-fold increase in fluorescence intensity upon phosphorylation, the magnitude of which depends on the substrate. Peptides containing the reporter functionality are phosphorylated on serine by Protein Kinase C and cAMP-dependent protein kinase and are shown to be good substrates for these enzymes. The principle of this design extends to peptides phosphorylated on threonine and tyrosine.

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Source
http://pubs.acs.org/doi/abs/10.1021/ja0380502
Publisher Site
http://dx.doi.org/10.1021/ja0380502DOI Listing
November 2003
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