p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch.

Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition.