Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 235

A ERK biosensor / Erkus

ERK
phosphorylation

Donor

FRET

Linker

P Acceptor

Substrate
EGFR

ERK docking
D-domain

FHA2

B ERK biosensor / EKAR

ERK
phosphorylation

FRET

Acceptor

Linker

P

Donor

Substrate
Cdc25C

WW

ERK docking
domain

Figure 6.5 Genetically encoded biosensors of ERK kinases. (A) The first genetically
encoded FRET biosensor of ERK, Erkus, is a single-chain biosensor encoding the CFP/
YFP FRET pair flanking an FHA2 domain from Rad53p, a short linker and a substrate
domain derived from EGFR T669 peptide, together with a short docking motif,
the D-domain, derived from p90 ribosomal S6 kinase. 58,80 (B) EKAR comprises a
WW domain and a substrate derived from Cdc25C, immediately flanked by an
ERK-specific docking domain, sandwiched between an mRFP/EGFP FRET pair. 57

further allowed the comparison of the cytosolic and nuclear activities of
ERK in living cells. More recently, Harvey et al. developed a different
biosensor, termed EKAR, which comprises a WW domain and a
substrate derived from Cdc25C, immediately flanked by an ERK-specific
docking domain and separated by a 72-glycine linker, sandwiched
between an mRFP and an enhanced green fluorescent protein (EGFP) 57
( Fig. 6.5B ). Expression of EKAR biosensor was observed to be essentially
nuclear; therefore, a cytoplasmic version of EKAR was developed,
EKARcyto, by appending an NES to the C-terminus of the biosensor.
Moreover, EKAR bears an ERK-specific docking site, which differs from
that of Erkus, is adjacent to the substrate, and appears to contribute to the
superior FRET signal of EKAR over Erkus. EKAR was successfully
applied to measure the spatiotemporal signaling dynamics of this kinase in
neurons from intact brain tissue by fluorescence lifetime imaging.

2.4. Cell cycle kinases: ATM, Plk, Aurora, CDK/cyclin biosensors
Several genetically encoded biosensors have been developed to study pro-
tein kinases involved in cell cycle regulation, although there have been fewer
developments in this field than in the field of protein tyrosine kinases or of