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

Abstract

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) p P trans PPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition.

T538 phosphorylation, Pin-ing p63-Itch stability

“…6 In their latest study, Melino and colleagues 7 elucidate aspects of p63 stability mediated by the WW domain-containing proteins Itch and Pin1, proposing a twofold regulation mechanism: whereas phosphorylation of threonine (T538) within the (T/S)PPPxY motif of p63 increases its binding affinity to the Itch WW domain, a post phosphorylation cis/trans switch catalyzed by Pin1 prolyl-isomerase regulates the final outcome of this interaction. 7 This further extends on previous observations that E3 ligases prefer the trans conformer (reviewed in 8 ) showing that the more relaxed trans conformer is indeed favorable for Itch binding. This is likely a consequence of the favorable local structural and chemical complementarity with the WW binding trench (Fig.…”

“…The twofold post phosphorylation conformational switch mechanism observed by Melino et al 7 is unlikely limited to the Itch-p63 interaction and could also be true for other WW domain interacting partners. Indeed, it has recently been shown that Pin1 directly binds to and stabilizes p63 inhibiting proteasomal degradation mediated by the E3 ligase WWP1.…”

The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System

“…The WW domains recognize the PY motif (a short proline-rich segment PPPXY) in the proline-rich domain of p63 and p73, which is located between the transactivation and DNA-binding domains [102]. Phosphorylation of threonine on this motif is crucial for WW interaction and subsequent ubiquitination by Itch/AIP4 [106]. …”

Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

“…p63-Itch binding occurs through a PPxY motif found in p63 and the WW2 domain of Itch [68]. Furthermore, this interaction is facilitated by tran/cis proline isomerization of the adjacent (T/S)P motif by the Pin1 prolyl-isomerase [183]. In accord, a p63-PPxY motif containing cyclic peptide was shown to bind Itch in vitro [64].…”