Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 233

and the FHA2 domain of Rad53 sandwiched between the mCFP/mYFP
pair, which undergo FRET in the unphosphorylated state, whereas phos-
phorylation of the substrate sequence results in a conformational change that
alters the FRET ratio 69 ( Fig. 6.4C ). Targeting of this reporter to the plasma
membrane where PKC is activated revealed an oscillatory phosphorylation
in response to histamine, which is closely associated with a calcium oscilla-
tion. This original biosensor was not designed to discriminate between the
different isoforms of PKC. However, more recent developments have led to
reporters designed specifically to monitor PKCdelta activity. 70 A different
strategy was employed by Schultz et al. To develop the KCP-1 biosensor,
a PKC reporter that incorporates the GFP/EYFP FRET pair, together with
a truncated form of pleckstrin that habors a PH domain (pleckstrin homol-
ogy) and a DEP domain (Disheveled, Egl-10, pleckstrin) separated by a
14-amino acid loop kinase-specific substrate sequence bearing three phosphor-
ylation sites was designed. Upon phosphorylation of this intervening loop by
PKC, the pleckstrin moiety undergoes a conformational change, which pro-
mpts the PH and the DEP domains to interact. The major advantage of this
reporter design is that it does not rely on interactions between the substrate
and a distinct PAABD. This probe responds rapidly to PKC activation through
phorbol ester stimulation or upon activation of physiologically relevant path-
ways 71 ( Fig. 6.4D ). The KPC-1 biosensor was further engineered to KCAP-1,
which bears a PKA substrate sequence in the C-terminal loop just before the
GFP moiety. This GFP/EYFP FRET biosensor consequently responds to
PKC phosphorylation through an increase in FRET, as opposed to a decrease
in FRET induced by PKA phosphorylation, thereby allowing monitoring
PKA and PKC activities independently in living cells. 72
PKB/Akt is a serine/threonine protein kinase that plays a key role in a
wide variety of cellular processes, including glucose metabolism, apoptosis,
cell proliferation, transcription, and cell migration, and integrates many
oncogenic signals from the phosphatidylinositol 3-kinase pathway signaling
pathway, thereby promoting cellular growth, survival, and proliferation to-
gether with decreased apoptosis. Akt is constitutively phosphorylated, acti-
vated, and recruited to the plasma membrane in a large variety of solid
tumors and hematologic malignancies, and is known to be actively involved
in tumor initiation and progression. 102,103 While several reporters based on
bioluminescence have been developed, which will not be described here,
several fluorescence-based reporters have also been engineered to monitor
PKB/Akt signaling and study its dynamics in living cells. 50-52 The Aktus
reporters are based on a CFP/YFP FRET pair, a protein kinase B (PKB)