Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 247

3.2.1 Ca 2 þ -chelating biosensors
Lawrence and coworkers developed a set of PKC peptide probes through
incorporation of a Ca 2 þ -sensitive fluorophore with a tetracarboxylate-
chelating site previously developed by Tsien and collaborators. 138 The fluo-
rophore was directly incorporated onto the N-terminal phosphorylation site
of a consensus PKC peptide substrate, lying within only a couple of angstroms
fromthephosphorylationsite.Theunphosphorylatedprobehaspoor affinity for
Ca 2 þ , but phosphorylation by PKC creates a divalent metal-ion-binding site,
andcoordinationofCa 2 þ by the fluorophore induces a twist that affects its spec-
tral properties, promoting a 3.6-fold increase in fluorescence 115 ( Fig. 6.9A ).
3.2.2 Mg 2 þ -chelating biosensors — Sox biosensors
Imperiali and collaborators developed a solvatochromic probe, the Sox
(sulfonamide-oxine) dye, derived from 8-hydroxyquinoline, that chelates
Mg 2 þ and undergoes fluorescent enhancement upon coordination of a
phosphate group on a phosphopeptide. 116 The Sox dye is resistant to pho-
tobleaching, is small, and causes minimal perturbation of substrate affinities
with the kinase. Based on the Sox amino acid, Imperiali and collaborators
developed a “versatile kinase kinase activity scaffold” that incorporates a
consensus phosphorylation site coupled to a b -turn sequence that
preorganizes the binding site for Mg 2 þ and a Sox dye, together with a kinase
recognition motif 117 ( Fig. 6.9B ). This b -turn-focused (BTF) design allows
the phosphorylation site to be either N- or C-terminal of the kinase recog-
nition motif. Mg 2 þ binding is weak in absence of phosphorylation
(Kd 100-300 mM) and considerably increased upon phosphorylation
(Kd 4-20 mM). Sox-based BTF biosensors were successfully developed
for PKC, PKA, and Abl substrate peptides with a phosphorylatable serine,
threonine, or tyrosine residue with consequent increases in fluorescence in-
tensity between three- and fivefold. 117 Further, this class of biosensors was
applied to develop a multiplex fluorescence-based assay of Akt, PKA, and
MK2 kinase activity in cell lysates. 118,119 To further increase the specificity
of recognition, recognition-domain-focused (RDF) chemosensors were
engineered with extended binding sequences to maximize recognition of a
variety of well-characterized Ser/Thr and Tyr kinases, including PKC,
Pim2, Akt1, MK2, PKA, Abl, Src, and IRK 120 ( Fig. 6.9C ). These
biosensors reported on phosphorylation with robust fluorescence
enhancement and high sensitivity, and were further used in 96-well plate
formats, indicating that they were amenable to HTS of kinase inhibitors.