Visualisation of the regulation of CDK/cyclin complexes by p21 (CDKN1A) in living cells using FLIM-FRET

The cell cycle is controlled in G1-phase by activities of three cyclin-dependent kinases (CDKs), CDK4/6 and CDK2, and their regulatory partners, cyclin D and cyclin E, respectively. p21 (CDKN1A), a member of the KIP/CIP family, is involved in regulation of CDK/cyclin complexes. In the present study, we analysed the impact of p21 on CDK/cyclin complex formation in living cells by fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements. Depending on the additional expression of p21, both CDK4/cyclin D3 and CDK2/cyclin E1 interactions have been favoured reflected by increased FRET efficiencies (E). Furthermore, post-translational modifications (PTM) as well as the redox status of p21 can regulate the impact on CDK/cyclin complexes. We show that phosphorylation-mimicking mutants of p21 seem to be able to affect the CDK/cyclin protein-protein interactions (PPIs), and that the reduced p21C41S variant decreased binding between CDK4 and cyclin D3. In contrast, p21C41S increased the CDK2/cyclin E1 interaction such as wild-type p21. Hence, wild-type p21 and its specific variants have an impact on the formation of distinct CDK/cyclin complexes under physiological conditions.