Fluorescent Peptide Biosensor for Probing the Relative Abundance of Cyclin-Dependent Kinases in Living Cells 英文参考文献.docVIP

FluorescentPeptideBiosensorforProbingtheRelative AbundanceofCyclin-DependentKinasesinLivingCells LaetitiaKurzawa,MorganPellerano,J.B.Coppolani,MayC.Morris* Centre de Recherches en Biochimie Macromole′culaire, CRBM-CNRS-UMR 5237, Universite′ de Montpellier, Department of Molecular Biophysics and Therapeutics, Montpellier,France Abstract Cyclin-dependant kinases play a central role in coordinating cell growth and division, and in sustaining proliferation of cancercells,therebyconstitutingattractivepharmacologicaltargets.However,therearenodirectmeansofassessingtheir relative abundance inliving cells, currentapproaches beinglimitedtoantigenicandproteomic analysis offixed cells. In order to probe the relative abundance of these kinases directly in living cells, we have developeda fluorescent peptide biosensor with biligand affinity for CDKs and cyclins in vitro, that retains endogenous CDK/cyclin complexes from cell extracts, and that bears an environmentally-sensitive probe, whose fluorescence increases in a sensitive fashion upon recognitionofitstargets.CDKSENSwasintroducedintolivingcells,throughcomplexationwiththecell-penetratingcarrier CADY2 and applied to assess the relative abundance of CDK/Cyclins through fluorescence imaging and ratiometric quantification. This peptide biosensor technology affords direct and sensitive readout of CDK/cyclin complex levels, and reports on differences in complex formation when tampering with a single CDK or cyclin. CDKSENS further allows for detectionofdifferencesbetweendifferenthealthyandcancercelllines,therebyenablingtodistinguishcellsthatexpress high levels of these heterodimeric kinases, from cells that present decreased or defective assemblies. This fluorescent biosensor technology provides information on the overall status of CDK/Cyclin complexes which cannot be obtained through antigenic detection of individual subunits, in a non-invasive fashion which does not require cell fixation or extractionprocedures.Assuchitprovidesp