Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 255

its phosphorylation by PKC. 110 This variant offers the advantage of control-
ling both the timing and the relative concentration of biosensor released
upon illumination and was readily microinjected into HeLa cells to monitor
intracellular PKC activity. Further, a caged biosensor of PKC was micro-
injected into PTK2 cells to follow its kinase activity at mitosis, with high
temporal precision, thereby revealing that kinase activity is required for
the progression from prophase into metaphase and that robust kinase activity
of the PKCb isoform occurs just prior to nuclear envelope breakdown. This
biosensor provides “visual snapshots of intracellular kinase activity” with
high spatial and temporal control. 133 Similarly, a caged Src reporter was gen-
erated by modifying the phosphorylatable tyrosine of a self-reporting pep-
tide biosensor with a photolabile nitrobenzyl moiety and microinjected into
A549 cells expressing high levels of Src, allowing the control of the timing of
kinase sensing. 127
3.6. Peptide biosensors for cell cycle targets: CDKSENS and
CDKACT
Enzymes involved in regulation of cell cycle progression are central for pro-
cesses such as cell growth, DNA replication, and cell division, and therefore
constitute pillars of cellular physiology. Many kinases involved in coordina-
tion of cell cycle progression have been well documented to undergo mu-
tations that alter their function, aberrant expression, or hyperactivation in
human disorders, and thereby constitute attractive pharmacological targets
and biomarkers of disease. 144,145,27 However, despite the pharmacological
importance of these kinases, there are barely any means for probing these
kinases, which limits the development of diagnostic approaches to
monitor alterations in their levels or activities associated with human
cancer. The development of cell cycle reporters and biosensors to probe
some of these kinases is now paving the way for developing strategies to
detect anomalies in these kinases associated with cancer. 146
3.6.1 CDKSENS and CDKACT — Fluorescent peptide biosensors for
probing the relative abundance and activity of CDKs
Cyclin-dependent kinases (CDK/cyclins) are heterodimeric protein kinases
that coordinate cell cycle progression through phosphorylation of well-
defined enzymatic and structural targets. 147-149 CDK/cyclins are known
to contribute to sustain aberrant cell proliferation in human cancers and
constitute attractive pharmacological targets for the development of
anticancer drugs. Unfortunately, there are no direct means of studying