Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 240

osmostress on the plasma membrane, anisomycin and UV in the cytoplasm,
and etoposide in the nucleus.
AMP-activated protein kinase (AMPK) is activated when the AMP/
ATP ratio in cells is elevated as a result of energy stress. A fluorescent bio-
sensor of AMPK activity, AMPKAR, was developed to study the function
of this kinase upon cellular stress. 53 This biosensor is based on an ECFP/
cpVenus FRET pair, an FHA1 domain, and a substrate peptide. AMPKAR
exhibits enhanced FRET in response to phosphorylation, allowing probing
of the spatiotemporal dynamics of AMPK activity in living cells, thereby re-
vealing that its activation takes place in the cytosol in response to energy
stress but occurs in both the cytosol and the nucleus in response to calcium
elevation.
Focal adhesion kinase (FAK) is crucial for many cellular processes. To
visualize FAK activity and activation at different membrane microdomains,
a genetically encoded FRET biosensor was developed, which is based on an
ECFP/YPet FRET pair, an SH2 domain derived from c-Src, and a substrate
peptide derived from FAK. This biosensor was either targeted into or out-
side of detergent-resistant membrane regions at the plasma membrane. This
study revealed that FAK is activated at membrane microdomains but that its
activation mechanisms vary in response to different physiological stimuli. 61
3. FLUORESCENT PEPTIDE/PROTEIN BIOSENSORS
Concerted efforts of chemists and biologists in designing fluorescent
probes for biological applications have led to the development of a very dif-
ferent class of biosensors that do not rely on genetically encoded AFPs.
Fluorescent peptide, polypeptide, or protein biosensors constitute attractive
alternatives to genetically encoded biosensors in that they offer a high
degree of versatility, yet also a high degree of control. This class of
biosensors is engineered by exploiting peptide substrate sequences or protein
domains that bind a specific analyte or interface, which serves as platforms for
site-selective coupling of one or more fluorescent probe(s). The fluorescent
probe may be coupled by different means to the peptide backbone—most
often chemically, enzymatically, or through replacement of a fluorescent
amino acid analog (for review, see Ref. 105 ). The design allows for freedom
in the choice of the fluorescent probe, amongst a wide variety of
wavelengths and synthetic probes, 106 and for its incorporation at virtually
any position within the peptide.