Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 238

A IR reporters/phocuses

Insulin receptor
phosphorylation

Donor

FRET

Linker

P

Acceptor

PTB Substrate

B

Histone phosphorylation reporter

Histone
phosphorylation

Donor Linker

FRET

P Acceptor

Histone H3
peptide

1433

C MLCK reporter

Ca/calmodulin
binding

Donor

FRET

Ca/Cam

Ca/Cam

Acceptor Donor Acceptor

MLCK MLCK

D CamKII reporter / Camui

CamKII activation

FRET

Figure 6.7 Genetically encoded biosensors of other kinases. (A) Phocuses are fluorescent
indicators that report on phosphorylation by the insulin receptor based on CFP/YFP
FRET pair, a substrate peptide for the insulin receptor, a flexible linker sequence, and
a phosphorylation recognition domain. 59 (B) Histone phosphorylation reporter by
appending a substrate sequence from histone H3 bearing a phosphorylatable serine
residue to a phosphoserine-binding domain, derived from 14-3-3, flanked by YFP
and CFP, respectively. 62 (C) MLCK-FIP is a reporter of MLCK activity associated with
Ca 2 þ /calmodulin binding. This reporter is based on a calmodulin-binding domain de-
rived fromMLCK and a BFP/GFP couple that undergoes FRET in the absence of Ca 2 þ and
calmodulin. 63 (D) Camui, a FRET biosensor of CaMK II, constituted of full-length CaMKII
flanked by CFP and YFP, allows measurement of CaMKII activation associated with a
conformational change. 55

Upon phosphorylation, the intramolecular interaction between the substrate
and the phosphorecognition domain promotes a conformational change
which induces a consequent increase in FRET between CFP and YFP.
Lin and Ting engineered a histone phosphorylation reporter by
appending a substrate sequence from histone H3 bearing a phosphorylatable
serine residue to a phosphoserine-binding domain derived from 14-3-3
flanked by YFP and CFP, respectively 62 ( Fig. 6.7B ).