Phosphorylation level of the p21 PIP-box modulates interaction with PCNA in living cells

Proteins achieve their biological functions in cells by cooperation in protein complexes. In the present study, we investigated protein-protein interactions (PPIs) between the proliferating cell nuclear antigen (PCNA) and different variants of the cell cycle regulator p21 (CDKN1A) in living cells by an experimental setup comprising fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements. p21 binds with its PCNA-interacting protein-box (PIP-box) to PCNA. Depending on the localization of the post-transcriptional modification, we found that phosphorylation-mimicking mutants of p21 affected the strength of protein-protein interactions. In contrast to single phosphorylation events, the simultaneous phosphorylation of threonine 145 and serine 146, both located in p21's PIP-box, was associated with a strong reduction of the FRET efficiency (E) in protein-protein interaction measurements with PCNA. Preventing phosphorylation of T145 and S146 by mutagenesis reactions to alanine resulted in a strong PCNA/p21 interaction in living cells as evidenced by an increase in FRET efficiency (E). Using a p21ΔPIP variant containing a deleted PIP-box, the interaction with PCNA was restricted. Hence, the phosphorylation level of p21's PIP-box regulates the interaction between PCNA and distinct p21 variants under physiological conditions.