Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 231

of activation following mechanical stimuli. 76 More recently, a different var-
iant, Srcus, was engineered including a nuclear export signal, which was
employed to study Src activation by steroids in the cytosol and at the plasma
membrane. 77 Another tyrosine kinase of particular relevance to cancer is the
spleen tyrosine kinase Syk. 98 A FRET-based biosensor was developed to
image Syk tyrosine kinase activity in living cells based on an ECFP/YPet
FRET couple, a substrate derived from VAV2 and an SH2 PAABD. 78 This
biosensor allowed following and quantifying real-time activation of Syk, as-
sociated with an increase in the FRET signal, upon immunoreceptor acti-
vation and following stimulation by the platelet-derived growth factor.
In order to examine the dynamics of the ZAP-70 tyrosine kinase activity
at the immunological synapse, Randriamampita et al. developed the ROZA
biosensor ( R eporter O f Z AP-70 A ctivity). This single-chain FRET biosen-
sor bears an SH2 domain frommouse Grb2 and a peptide substrate sequence
from mouse LAT sandwiched between the CFP-YFP pair ( Fig. 6.3C ).
ROZA was applied to image ZAP-70-dependent phosphorylation in T-cell
lines and primary human lymphocytes with subcellular resolution during the
formation of an immunological synapse. 79

2.2. Biosensors for protein kinase PKA, PKB, PKC, and PKD
PKA (cAMP-dependent protein kinase) constitutes an essential enzyme
involved in major biological processes including gene expression, metabo-
lism, cell growth, and cell proliferation. 99 Genetically encoded single-chain
FRET biosensors of PKA, combining the consensus kinase substrate
sequence, with a matching PAABD sandwiched between a FRET pair of
GFP variants, were amongst the first kinase reporters developed to charac-
terize their endogenous activity in living cells in a dynamic fashion. The first
genetically encoded biosensor of PKA activity, termed ART (cAMP-
responsive tracer), was composed of BGFP/RGFP, the kinase-inducible
domain (KID) of the CREB transcription factor (cAMP-responsive
element-binding protein), which undergoes a conformational change upon
phosphorylation 64 ( Fig. 6.4A ). As opposed to most other FRET biosensors,
phosphorylation of KID by PKA disrupts the FRET between the flanking
AFPs. An entirely different biosensor called AKAR (cAMP-dependent
KAR) was later generated by Zhang et al. 65 ( Fig. 6.4B ). AKAR1 was based
on a 14-3-3 phosphorecognition domain but was later optimized and rep-
laced by an FHA domain in AKAR2, 66 so as to improve the reversibility of
the biosensor and therefore its response to cellular phosphatases, for