Fluorescent Sensors of Protein Kinases: From Basics to Biomedical Applications - Progress in Molecular Biology and Translational Science - page 256

the levels and activities of these kinases in their natural environment in living
cells. From a fundamental perspective, this limits our understanding of the
physiology of these enzymes in their natural environment. From a diagnostic
perspective, reporters that provide information on alterations in CDK/
cyclin levels and activity would provide the means of identifying cancer
cells or tumors in which these kinases constitute relevant

targets for

therapeutic intervention.
We have developed two new families of peptide biosensors that report
on the relative abundance and activity of CDK/cyclins, respectively,
through changes in fluorescence of an environmentally sensitive
probe. 113,114 CDKSENS is a biligand peptide composed of a CDK-
binding pseudosubstrate sequence and a cyclin-binding sequence that
allows docking of the biosensor onto the CDK/cyclin complex with high
affinity and specificity. This peptide bears a fluorescent probe (cyanine
dye) which undergoes significant enhancement upon recognition and
docking onto CDK/cyclin complexes. 113 CDKACT is a modular peptide
biosensor constituted of a peptide substrate, onto which a fluorescent
probe is coupled proximal to the phosphorylation site, and a PBD that
recognizes the phosphorylated peptide sequence, separated by a short
proline/glycine-rich linker. This biosensor undergoes changes in
fluorescence intensity upon phosphorylation by active CDK/cyclin
complexes and is fully reversible, thereby allowing monitoring kinase
activity in real time through changes in fluorescence intensity of an
environmentally sensitive probe. 114 Both CDKSENS and CDKACT
were validated using recombinant CDK/cyclin complexes as well as cell
extracts derived from healthy fibroblasts and cancer cell lines expressing
endogenous CDK/cyclins. The design of CDKSENS and CDKACT is
depicted in Fig. 6.14 .
CDKSENS and CDKACT constitute sensitive tools for reporting on
CDK/cyclin levels and activity in vitro and in cell extracts. Furthermore,
these peptide biosensors can be introduced into living cells, thanks to
CPPs, 107,108 thereby allowing monitoring of the CDK/cyclin levels and
activities in their natural environment through live-cell fluorescence
imaging and ratiometric quantification. Indeed, CDKSENS and
CDKACT allow the detection of subtle differences when tampering with
a single CDK or cyclin (through siRNA downregulation or through
ectopic expression) or following treatment with CDK/cyclin drugs. They
further allow the detection of the differences between different healthy
and cancer cell lines, thereby enabling identification of cells that express