Versatile fluorescence probes of protein kinase activity.
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.
Shults
Melissa D
MD
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Imperiali
Barbara
B
D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D007700:Kinetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D010455:Peptides; D011494:Protein Kinases