Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 245

phosphorecognition-binding domain (for review, see Refs. 134-136 ).
Environmentally sensitive kinase biosensors have been generated through
incorporation of fluorescent probes on the phosphorylatable residue itself,
immediately adjacent to the residue or proximal to it (i.e., 2-5 residues
away). One of the first set of fluorescent peptide probes based on the
propinquity effect was generated by labeling a PKC peptide substrate at its
N-terminal serine hydroxyl moiety, thereby yielding a library of 417
compounds through incorporation of different fluorescent labels. Although
many of the dyes used did not result in a significant increase in fluorescence
of the peptide probe upon phosphorylation by PKC in vitro ,anNBD
(nitrobenzofuran)-labeled variant displayed a robust increase in fluorescence
intensity (2.5-fold) ( Fig. 6.8A ). Moreover, this peptide probe was successfully
applied to monitor PKC activity in cell lysates and further microinjected into
living cells to visualize and monitor the spatiotemporal dynamics of PKC
activity. 109 To further improve this biosensor, Lawrence and coworkers
synthesized a light-activatable variant of the NBD-PKC probe through
incorporation of a single photolytically sensitive cage on the phosphorylatable
serine group, thereby offering a precise means of controlling biosensor
availability in living cells through photoactivation. 110
Environmentally sensitive dyes will also respond to changes in their local
environment associatedwith protein-protein interactions. In the case of kinase
sensors, this has been exploited by using phosphopeptide recognition domains,
including 14-3-3, SH2, or WW domains, 83-89 that bind the phosphorylated
peptide but not the unphosphorylated species. The fluorescent probe is
consequently embedded in a hydrophobic environment, which modifies
the local polarity and consequently affects its spectral properties. This
strategy tends to enhance the fluorescence of environmentally sensitive
probes more significantly than simply proximal phosphorylation, and
further increases the selectivity of the biosensor, because the PAABD is
more likely to bind the phosphorylated substrate than any other nonspecific
protein found in cell extracts. This strategy was employed to generate a
very sensitive probe for Src tyrosine kinase based on a fluorescent peptide
labeled with dapoxyl, NBD, or Cascade Yellow two residues away from
the phosphorylation site, thereby leading to a fivefold increase in
fluorescence upon phosphorylation, thanks to interaction with an SH2
domain 111 ( Fig. 6.8B ). More recently, Gulyani et al. generated a new Src
biosensor based on a fibronectin monobody scaffold, which was employed
to quantify the dynamics of Src activity at the edge of living cells, in
correlation with protrusion and retraction activities. 112 This merobody