From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 194

Table 5.3 Listing of validated targeting sequences for biosenseur subcellular
compartimentation
Subcellular targeted
location Targeting sequences References

Gallegos et al. 102

Cytosol LALKLAGLDI

(at C-terminal)

Gallegos et al. 102
Ananthanarayanan et al. 136

Nucleus PKKRKVEDA

(at C-terminal)

Sasaki et al. 137
Gallegos et al. 102

Golgi 33 amino residues of eNOS

(at N-terminal)

Palmer et al. 138 Endoplasmic reticulum MLLPVLLLGLLGAAAD

(at N-terminal)

þ KDEL

(at C-terminal)

MGCIKSK (at N-terminal) Violin et al. 103
Kunkel et al. 126
Gallegos et al. 102

Plasma membrane

References 102,103,126,136-138 .
Adapted from Refs. 4 .

remains the bottleneck through which every biosensor has to pass. Charac-
terization is a crucial step, as it determines the efficiency of new FRET-based
biosensors and thus requires several considerations. The results should pro-
vide evidence that the sensor generated is specific to the kinase of interest
and should assess the dynamic range of the biosensor in the presence of
the activator and/or inhibitor. This step usually relies on pharmacological
agents, the availability and specificity of which can be limited.

6.6.1 In vitro characterization
Upon expression of biosensors, in a bacterial system, for instance, lysates are
subjected to stringent purification procedures to retrieve only full-length
protein from the truncated protein produced as a result of proteolytic deg-
radation. Now, in solution (see Chapter 4) with the purified protein kinase
of interest and ATP, in a 96-well plate screening format or in a spectropho-
tometer cuvette, the properties of the biosensor are evaluated. Ratiometric
measurement methods (see Section 4.1.1 for details) are then employed un-
der different experimental conditions: before and after addition of ATP and
purified kinase, and control wells for baseline ratio determination.
In addition, phosphorylation levels of the biosensor expressed in mam-
malian cells can be estimated by immunoprecipitation and detected by