Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 229

The linker between these domains is generally designed to be flexible
(i.e., rich in glycine, alanine, and proline residues), and its length has proved
to be a critical factor in affecting FRET efficiency. 90-92
Last but not least, the choice of the AFP/FRET pair is essential. Ideally,
AFPs that do not photobleach too rapidly, that have high quantum yield, and
that are red-shifted should be chosen to maximize FRET efficiency
and minimize phototoxicity. 93,94 Komatsu et al. recently proposed an
optimized backbone for genetically encoded FRET biosensors bearing
the cyan fluorescent protein (CFP) or Turquoise as donors, the yellow
fluorescent protein (YFP) or YPet as an acceptor fluorophore, and a very
long linker connecting the ligand to the sensor domain, which basically
abolishes dimerization of the FRET pair, and showed that this backbone
was ideal for both kinase and GTPase FRET biosensors reporting on
ERK, PKA, S6K, RSK, Ras, and Rac1. 92
In this section, we provide several examples to illustrate the different
strategies that have been devised in designing genetically encoded FRET
biosensors and describe how they have been employed to study the behavior
of specific kinases in living cells, rather than attempt an exhaustive review of
all the kinase biosensors that have been developed so far.
2.1. Phosphotyrosine kinase (PTK) biosensors
Ting et al. developed the first genetically encoded fluorescent reporters of
tyrosine kinases to probe the activities of Src, Abl, and EGFR (epidermal
growth factor receptor). These single-chain FRET biosensors consist of
fusions of the CFP/YFP FRET pair, an SH2 phosphotyrosine-binding
domain, and a substrate sequence, the phosphorylation of which on a critical
tyrosine promotes binding to the SH2 domain, thereby inducing a confor-
mational change that brings the two AFPs close enough to undergo FRET
between the donor and the acceptor. While the EGFR and Src indicators
were designed by combining an SH2 domain with an appropriate substrate,
separated by a linker, the Abl reporter was directly engineered by introduc-
ing a domain derived from Crk protein, which includes an SH2 domain,
two SH3 domains, and an intervening Abl phosphorylation site 46
( Fig. 6.3A and B ). The Picchu sensor was constructed in a similar fashion
to monitor specific phosphorylation by c-Abl, with a CFP/YFP FRET pair
and an SH2-2 SH3 domain derived from the CrkII adaptor protein. 47 More
recently, Mizutani et al. developed a sensitive and specific FRET biosensor,
termed Pickles, for measuring the tyrosine kinase activity of Bcr-Abl, which