Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 248

A Fluorescent dye involved in metal ion chelation incorporated at the phosphorylation site

M

PKC
phosphorylation

P

Peptide substrate

B Chelation-mediated enhancement— b -Turn FocusedSox sensor

M

Src
phosphorylation

P

BTF

Peptide substrate

C Recognition-domain focused Sox biosensor

M

P

ERK
phosphorylation

Peptide substrate RDF

D Chimeric peptide/protein biosensor—The Sox-PNT ERK sensor

M

P ERK

phosphorylation

Peptide substrate

PNT Ets1

PNT Ets1

Figure 6.9 Metal-ion-induced, chelation-enhanced fluorescence. (A) Fluorescent dye
involved in metal-ion chelation incorporated at the phosphorylation site, such as the
Ca 2 þ -sensitive PKC probe. 115 (B) Chelation-mediated enhancement—b-turn-focused
(BTF) Sox sensor. 117 (C) Recognition-domain-focused (RDF) Sox biosensor. 120 (D) Chime-
ric peptide/protein biosensor—the Sox-PNT ERK sensor. 122,123

Moreover, a high-throughput approach was developed to generate Sox-
based chemosensors with the highest possible selectivity using
combinatorial libraries. 121 More recently, the RDF strategy has been
extended to engineer Sox protein biosensors that incorporate a kinase-
specific docking domain distal to the phosphorylation site, which enhances
the specificity of recognition significantly more than an extended peptide
substrate motif. For instance, a Sox-based ERK1/2 sensor (Sox-PNT)
engineered through incorporation of the PNT domain of the Ets1
transcription factor into the Sox-ERK1/2 peptide substrate sequence was
developed and readily applied to probe ERK1/2 kinase in cell lysates,
displaying high selectivity against other kinases from the JNK, p38, and
CDK families. 122 Likewise, a Sox-based p38 a -selective sensor was