Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 239

A biosensor of the Ca 2 þ /calmodulin-bound and active form of myosin
light chain kinase (MLCK), MLCK-FIP, was developed by Chew et al. ,
based on a calmodulin-binding domain derived from MLCK and a BFP/
GFP couple that undergoes FRET in the absence of Ca 2 þ /calmodulin
and displays a decrease in FRET upon activation of MLCK through binding
of Ca 2 þ /calmodulin ( Fig. 6.7C ). This biosensor allowed imaging the spatial
and temporal pattern of MLCK activation, revealing that it is enriched at the
spindle equator during late metaphase and maximally activated just before
cleavage furrow constriction. 63
Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key
mediator of intracellular signaling in the heart. Erickson et al. developed
Camui, a FRET biosensor of CaMKII, constituted of full-length CaMKII
flanked at its N- and C-termini by CFP and YFP, respectively, which un-
dergo robust FRET in the compactly folded autoinhibited state. Activation
of the kinase results in a conformational change associated with a decrease in
fluorescence transfer between CFP and YFP. This reporter was applied to
study the activation of CamKII in living neurons and in cardiomyocytes 55
( Fig. 6.7D ).
JNK kinase is involved in signal transduction of external stimuli such as
stress and cytokines into functional responses that mediate apoptosis, prolifer-
ation, differentiation, and inflammation. Several genetically encoded fluores-
cent biosensors were engineered to detect endogenous JNK activity in living
cells, based on an EGFP/citrine FRET pair; an FHA1, FHA2, or WW
PAABD; a substrate domain derived from c-Jun or phosphoacceptor sites
from JDP2 or ATF3; and docking domains from JDP2 or JIP1. 60 The JNKAR
biosensor (for JNK activity reporter) specifically detects stress (ribotoxic and
osmotic) and cytokine (TNF- a ) induced JNK activity in living cells, in the
cytoplasm, nucleus, mitochondria, and plasma membrane, associated with a
phosphorylation-dependent increase in FRET between the two AFPs.
The stress-activated protein kinases (SAPKs) p38 and JNK are mem-
bers of the mitogen-activated protein kinase family and are important
determinants of cell fate when cells are exposed to environmental stresses
such as UV and osmostress. SAPKs are activated by SAPK kinases (SAP2Ks),
which are in turn activated by various SAP2K kinases (SAP3Ks). A geneti-
cally encoded FRET biosensor was developed to probe the dynamic behav-
ior of SAP3K activity toward the MKK6 SAP2K, which was based on a
Venus/SECFP pair, an FHA1 domain, and MKK6 substrate. 75 This biosen-
sor allowed the study of the dynamic behavior of SAP3K activation in living
cells, associated with stress stimuli such as epidermal growth factor and