Biology Reference
In-Depth Information
A
B
PKA biosensor / ART
PKA biosensor / AKAR
PKA
phosphorylation
Donor
Linker
Acceptor
Donor
KID
CREB
1433
Substrate
FRET
Acceptor
P
PKA
phosphorylation
C
BKAR / CKAR / DKAR biosensors
P
FRET
FHA2
FRET
D
PKC biosensor / KCP-1
Substrate
FRET
PKB, PKC, PKD
phosphorylation
DEP
PH
PKC
phosphorylation
P
Substrate
Substrate
P
P
P
Figure 6.4 Genetically encoded biosensors of PKA, PKB, PKC, and PKD. (A) ART: genetically
encoded biosensor of PKA activity, composed of two AFPs joined by the kinase-
inducible domain (KID) of the CREB transcription factor (cAMP-responsive element-
binding protein).
64
Phosphorylation of KID by PKA promotes a conformational change
which promotes FRET between the flanking AFPs. (B) PKA biosensor AKAR1 based on a
1433 PAABD and a substrate peptide of PKA sandwiched between a CFP/YFP pair.
65,66
(C) BKAR, CKAR, and DKAR biosensors of PKB, PKC, and PKD kinases, respectively.
52,69,73
These reporters are constituted of a consensus phosphorylation sequence and the
FHA2 domain of Rad53 sandwiched between an mCFP/mYFP pair that undergoes
FRET in the unphosphorylated state; phosphorylation of the substrate sequence
results in a conformational change that alters the FRET ratio. (D) PKC biosensor
KCP-1 based on a GFP/EYFP FRET pair, and a domain derived from pleckstrin
constituted by a PH domain and a DEP domain flanking a loop that bears several
phosphorylation sites.
71
Upon phosphorylation of this intervening loop by PKC, the
pleckstrin moiety undergoes a conformational change, which prompts the PH and
the DEP domains to interact.
measuring the cellular dynamics of PKA activity.
67,100
More recently, a
variant SR-AKAR3 was developed to monitor PKA activity in the
sarcoplasmic reticulum in cardiomyocytes.
68
PKC kinases form a family of 15 isoforms that contribute to well-defined
signaling pathways.
101
The first genetically encoded PKC biosensor,
CKAR, was designed to incorporate a consensus phosphorylation sequence
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