Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 221

1.2. Strategies for probing and studying protein kinases in vitro
and in cellulo
Despite the central role of protein kinases in physiological pathways, and
their implication as disease biomarkers in pathological disorders, there are
very fewmeans of studying their activity in real time in a noninvasive fashion
and in their natural environment. Most kinase activity studies have been tra-
ditionally performed using biochemical assays based on purified enzymes
produced as recombinant proteins from insect or mammalian cells in culture.
Commonly implemented approaches to study protein kinase activity rely
essentially on the incorporation of radioactive phosphate into artificial sub-
strates, or on antigenic approaches, which further depend on highly specific
antibodies to recognize the phosphorylated form of the kinase substrate(s).
These assays have been widely used in the laboratory and further adapted to
high-throughput drug screening, but they are irreversible endpoint assays,
which do not allow for real-time analysis of kinase activity, and further lack
the physiological cellular environment. Cell-based methods that monitor
kinase activity have been developed that rely on the incorporation of

32

P
into cells; this approach requires cell lysis, substrate protein isolation, and pu-
rification to determine its relative degree of phosphorylation by measuring
the amount of radioactivity incorporated. However, such cell-based assays
are labor intensive, show poor sensitivity, and have the disadvantage of re-
quiring high levels of radioactivity. Other cell-based assays for the study of
kinase activity involve the use of radiolabeled phosphorylation-specific an-
tibodies (i.e., antibodies that can discriminate between phosphorylated and
nonphosphorylated proteins); the phosphorylated substrate can then be
detected and quantified by immunoprecipitation, gel electrophoresis, or
Western blotting after cell lysis. Although these assays generally require
lower levels of radioactivity than 32 P-based methods, they are equally labor
intensive, time consuming, and complex to automate. Nonradioactive cell-
based methods have emerged that use an ELISA (enzyme-linked immuno-
sorbent assay) approach to measure the activation of specific kinase signaling
pathways. These kinase assays, which employ phosphorylation-specific an-
tibodies, have been demonstrated to be suitable for high-throughput drug
screening. 29 More recently, a nonradioactive method for probing kinase ac-
tivity has been developed, which is based on the use of a fluorogenic peptide
substrate (rhodamine 110, bis peptide amide) that is cleaved in its
unphosphorylated form, thereby releasing free rhodamine 110; phosphory-
lation prevents cleavage, and the compound remains as a nonfluorescent
peptide conjugate. 30 This approach has been successfully applied to