Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 234

substrate sequence derived fromBad, and a PAABD domain derived from 14-
3-3n, similar to the structure of AKAR. Aktus was further fused to the Golgi-
targeting and mitochondrial-targeting domains of eNos and Bad, respectively,
so as to generate variants that would preferentially colocalize with these
endogenous substrates and thereby optimize their chances of
phosphorylation by PKB compared to the nontargeted sensor. 51 Another
PKB sensor, BKAR, was developed with the same structural design as
the CKAR reporter 69 and employed to image phosphorylation catalyzed
by PKB in real time and to investigate differences in the dynamics of this
activity in the nucleus, cytosol, and when targeted to the plasma
membrane. 52 This study revealed differences in biosensor response associated
with different subcellular compartments, inferring differences in PKB signal
propagation and termination. Another PKB reporter, AktAR, was
developed to examine dynamic Akt activity in membrane microdomains and
was based on the CFP/Venus FRET couple, an FHA1 domain, and a
FOXO1 substrate. 50 This biosensor revealed that Akt activities are
differentially regulated in different membrane microdomains and present
overall higher activity within lipid rafts.
A reporter of protein kinase D, DKAR, based on the design of BKAR
and CKAR, 52,69 was applied to investigate the dynamics of this kinase,
revealing its dependence on calcium through positive feedback regulation
of diacylglycerol production. 73
2.3. Genetically encoded ERK/MAPK biosensors
ERK1/2 (extracellular signal-regulated kinases) play central roles in growth,
cell proliferation, and differentiation. As their name suggests, these kinases
are activated following different stimuli, including mitogenic factors, hor-
mones, and cytokines, and they are themselves involved in regulating the
activity of a wide variety of substrates including transcription factors and
antiproliferative genes during G0 and G1 phases. 104 The first genetically
encoded FRET biosensor of ERK, Erkus, was designed by Sato et al. in
order to study the spatiotemporal dynamics of protein phosphorylation by
activated ERK. Erkus is a single-chain biosensor encoding the CFP and
YFP AFPs that flank an FHA2 domain from Rad53p, a short linker, and
a substrate domain derived from the EGFR T669 peptide, as well as a short
docking motif, the D-domain, derived from p90 ribosomal S6 kinase 58,80
( Fig. 6.5A ). While this biosensor allowed the visualization of the
dynamics of ERK kinase in real time, additional variants bearing either a
nuclear localization sequence (NLS) or a nuclear export sequence (NES)