From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 188

tested in this context greatly reduced the FRET signal of the sensor. This
shows that docking sites are very important in the design of new specific
and selective KARs (see Ref. 115 for review), which was recently
highlighted in a sensor for the M-phase promoting factor. 116
6.2. Phosphoamino acid-binding domain
Upon specific phosphorylation, the PAABD recognizes and binds to the
phosphorylated substrate, resulting in a conformational change of the poly-
peptide that somehow alters in the opposite way the fluorescence emission
of the two fluorophores. The choice of a good PAABD is a key element of
KAR design. 117 Protein phosphorylation may lead to the formation of mo-
lecular signaling complexes through interactions between specific phos-
phorylated residues and binding domains. Several binding domains named
“modular domains” and “adaptors molecules” have been well characterized
and are able to activate a specific pathway. Activation of transmembrane re-
ceptors triggers phosphorylation on tyrosine residues, which results in the
recruitment and binding of adapter molecules such as Src-homology 2/3
(SH2 SH3) and phosphotyrosine binding (PTB) domains. Similarly, signal
transduction involves phosphorylation on serine/threonine residues and
thus constitutes consensus sequences recognized by other adapter molecules
including 14.3.3 proteins, forkhead-associated (FHA), domains WW
domains, and WD40 domains. The phospho-acceptor residue within the
substrate sequence then first guides the choice of the PAABD.
Several factors should be considered as they can affect the efficiency and
the reversibility of the PAABD binding to the phosphorylated substrate. For
example, in order to measure compartmentalized PKA activity in single liv-
ing cells, Zhang et al. 118 developed a genetically encoded A-kinase activity
reporter (AKAR). This first-generation PKA sensor (AKAR1) used 14-3-3
as a PAABD. Because of the high binding affinity of this PAABD toward the
phosphorylated substrate, once phosphorylated, the sensor was blocked in
closed conformation, making it weakly sensitive to cellular phosphatase ac-
tivity and thus irreversible and incompatible with dynamic measurements of
PKA activity. To circumvent this problem, 14-3-3 was replaced by an FHA
domain in subsequent versions of AKAR, 119 yielding AKAR2. When tested
in cells, it showed a better dynamic response and a reversible behavior. Sim-
ilarly, an FHA domain was also used in the first Erk biosensor Erkus. 112
Shortly afterward, the development of EKAR 42 saw the FHA domain rep-
laced by a WW domain. Although both sensors followed the same design