Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 237

checkpoint recovery. 74 This study revealed that Plk1 activation occurs sev-
eral hours before entry into mitosis, thanks to Aurora A-mediated phosphor-
ylation of Plk1, and that Bora/Aurora-A-dependent phosphorylation is a
prerequisite for Plk1 to promote mitotic entry after a checkpoint-dependent
arrest.
CDKs constitute the molecular engines that drive cell cycle progression.
In particular, CDK1/cyclin B is essential for driving cells through mitosis.
However, functional studies of this kinase have remained limited to classical
antigenic approaches following cell fixation or extraction. More recently,
Gavet and Pines developed a FRET sensor to probe CDK1/cyclin B kinase
activity in living cells based on an autophosphorylation site from cyclin B1
fused to the Polo box domain (PBD) of Plk1 and flanked by mCerulean and
YPet 56 ( Fig. 6.6B ). Thanks to this probe, imaging of CDK1/cyclin B acti-
vity was performed with high temporal precision, showing that this kinase is
inactive in G2 until just before nuclear envelope breakdown, contributing to
initiate prophase.
ATM (Ataxia telangiectasia mutated kinase) is an intracellular protein ki-
nase that coordinates the DNA damage signaling pathway upon recognition
of DNA double strand breaks. Activation of ATM leads to phosphorylation
of transducer and effector proteins that coordinate a cellular response includ-
ing DNA repair, cell cycle arrest, or apoptosis. A genetically encoded
CFP-YFP FRET reporter was developed to probe ATM kinase activity
in living cells, based on an FHA PBD and an ATM substrate sequence,
the phosphorylation of which leads to changes in FRET efficiency between
CFP and YFP associated with a conformational change of the biosensor 54
( Fig. 6.6C ). This reporter allowed monitoring the ATM activity in a specific
and quantitative fashion, providing a measurable output in response to dou-
ble strand breaks, while also providing information on the spatiotemporal
dynamics of ATM activity in living cells.
2.5. Other kinase biosensors
To visualize signal transduction based on protein phosphorylation in living
cells, Sato et al. developed genetically encoded fluorescent indicators, named
“phocuses” (fluorescent indicator for protein pho sphorylation that can be
cus tom-made) that report on phosphorylation by the insulin receptor 59
( Fig. 6.7A ). These reporters encode a CFP/YFP pair, a substrate sequence
for the insulin receptor, a flexible linker sequence, and a phosphorylation
recognition domain derived from SH2, PTB, or WW domains, or single-
chain antibodies that recognize the phosphorylated substrate sequence.