Fluorescent peptide biosensor for probing CDK6 activity in lung cancer cell extracts

International audienceCDK6 kinase regulates cell cycle progression in G1, together with CDK4, but has cell-, tissue- and developmentally-distinct functions associated with transcription, angiogenesis and metabolism. Although CDK6 constitutes an attractive cancer biomarker and target there are no means of assessing its activity in a complex environment. In this study we describe design, engineering and characterization of a fluorescent peptide biosensor derived from 6-phosphofructokinase that reports on CDK6 kinase activity through sensitive changes in fluorescence intensity. This biosensor can report on CDK6 activity in a dose-dependent fashion, thereby enabling quantification of differences in kinase activity in complex and physiologically-relevant environments. Further implementation of this biosensor in different lung and melanoma cell lines, as well as in mesothelioma cell lines derived from patients together with a CDK4 biosensor highlighted differences in kinase activity between CDK6 and CDK4 kinase. This work demonstrates the utility of these selective tools for monitoring two closely related kinases comparatively and simultaneously in the same samples, thereby offering attractive perspectives for diagnostic and therapeutic purposes

Two Protocols to Study the Interactions of Thyroid Hormone Receptors with Other Proteins and Chromatin

International audienceUnderstanding the transcriptional function of thyroid hormone receptors implies a precise analysis of their interactions with chromatin and other protein components of the cells. We present here two protocols that are routinely used in our laboratory. The first co-immunoprecipitation procedure allows addressing the capacity of proteins to form stable multiprotein complexes with TRs in cells. The chromatin affinity purification enables us to define the sites occupied by TRs on chromatin. In this case the lack of high quality antibodies is circumvented by introducing an N-terminal tag in TR, with unspecific affinity for immunoglobulins