From FRET Imaging to Practical Methodology for Kinase Activity Sensing in Living Cells - Progress in Molecular Biology and Translational Science - page 147

histidine residues. Protein phosphorylation can alter the activity of many
proteins, causing a chain reaction leading to the phosphorylation of many
proteins involved in a particular cellular process. Conventional analytical
methods have identified and characterized various posttranslational modifi-
cations, but the major drawback is that these methods provide only a snap-
shot of the cell. In fact, in order to assess activity of protein kinases,
immunoblotting and immunocytochemistry with phospho antibodies
toward specific residues described to report on kinase activation, are global
and indirect/static approaches. They are limited to the time resolution and
the quality of the cell fractionation assay under analysis. In addition, anti-
bodies toward specific phospho residues do not really reflect activity of
the kinase of interest.
To go beyond the snapshot, tools have been developed to answer the
new challenges in today's biology quest: protein localization, interaction,
and activity, when applicable. Concerning the latter, fluorescent biosen-
sors have now become the biologist's toolbox to visualize, especially,
the spatiotemporal dynamics of kinase signaling in living systems. They
provide high sensitivity and versatility while only minimally perturbing
cell physiology.
The word “biosensor” is a rather generic term that is used to define a
wide array of systems that enable the sensing of various analytes. Typically,
a biosensor is composed of two parts having distinct functions. The first, re-
ferred to as the “bioreceptor,” recognizes the analyte and is responsible for
the selectivity and the sensitivity of the whole biosensor. The second, named
“transducer,” is in charge of conveying the signal from the recognition part
toward the adapted instrument ( Fig. 5.1 ).
This definition is, of course, applicable to molecular biosensors dedicated
to sensing biological events in living cells. Indeed, the analyte is represented
by ions such as Ca 2 þ , second messengers such as cAMP (cyclic adenosine
monophosphate), an enzymatic activity such as kinase activity, or an active
enzyme conformation. The bioreceptor or molecular recognition element
(MRE) is thus materialized by calmodulin/M13 or TroponinC/M13, Epac
(exchange protein activated by cAMP), and the substrate/phosphoamino acid-
binding domain (PAABD) respectively. The transducer element is represented
in these cases by fluorescent proteins, which, when their distance allows it, will
generate a change in their fluorescent signal or spectral properties. Thus, the
instrument dedicated to signal detection is an “optical fluorescent microscope”
or a “spectrofluorimeter.”