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B
Crk domain reporters of Abl, c-Abl, and Bcr-Abl
A
Src and EGFR reporter
Crk domain
CFP
CFP
Linker
YFP
YFP
SH3
SH3
Substrate
SH2
SH2
Abl / cAbl / Bcr-Abl
phosphorylation
Src / EGFR
phosphorylation
FRET
FRET
P
P
C
ROZA biosensor
CFP
Linker
Grb2
SH2
YFP
LAT
peptide
ZAP-70
phosphorylation
P
FRET
Figure 6.3 Genetically encoded protein tyrosine kinase reporters. Genetically encoded
FRET biosensors of tyrosine kinases are composed of a substrate sequence and a
matching phosphoaminoacid binding domain sandwiched between a FRET pair of
GFP variants. (A) Fluorescent reporters of Src and EGFR (epidermal growth factor recep-
tor), based on an SH2 phosphotyrosine-binding domain and a separate substrate se-
quence joined by a linker and flanked by the CFP/YFP couple.
46
(B) Structure of
fluorescent reporters of Abl,
46
c-Abl (Picchu),
47
and Bcr-Abl (pickles),
48
derived from
Crk adaptor proteins bearing an SH2 domain, two SH3 domains, and an intervening
phosphorylation site. (C) ZAP-70 biosensor ROZA constituted of the SH2 domain of
mouse Grb2 and a peptide substrate sequence derived from LAT sandwiched between
the CFP-YFP pair.
79
again incorporates one SH2 and two SH3 domains derived from CrkL, the
most characteristic substrate of Bcr-Abl, sandwiched between Venus, a var-
iant of YFP, and ECFP
48
(
Fig. 6.3B
). The high sensitivity of this biosensor
allows the assessment of Bcr-Abl activity from patient cells to monitor the
disease status and response to therapy, as well as the detection of the onset of
drug-resistant cells within a heterogeneous population.
95,96
Src is a nonreceptor tyrosine kinase that is activated by a variety of mech-
anisms and constitutes a major kinase in the signaling pathways involved in
cancer and metastasis.
97
An Src-specifc biosensor based on an ECFP/YFP
couple, an SH2 domain, and a substrate peptide derived from the c-Src sub-
strate p130cas was developed to probe this kinase and visualize its dynamics
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