title: 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)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition.
authors.affiliation: a Dipartimento di Scienze e Tecnologie Chimiche ; University of Rome "Tor Vergata" ; Rome , Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: Melino
authors.forename: Alessia
authors.initials: A
authors.lastname: Bellomaria
authors.forename: Ridvan
authors.initials: R
authors.lastname: Nepravishta
authors.forename: Maurizio
authors.initials: M
authors.lastname: Paci
authors.forename: Gerry
authors.initials: G
authors.lastname: Melino
mesh_terms: D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D013912:Threonine / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: C081876:CKAP4 protein, human; D008565:Membrane Proteins; D013912:Threonine; D044767:Ubiquitin-Protein Ligases
keywords: CXCR4, chemokine receptor; E3 ubiquitin ligases; HECT, Homologous E6-AP Carboxyl Terminus; IPTG, isopropyl-β-D-thiogalactoside; Itch; Pin1; Ppep63, phosphorylated pep63; RHS, Rapp-Hodgkin syndrome; RP-HPLC, reverse phase high performance chromatography; TFE, 2, 2, 2-trifluoroethanol; TNF, tumor necrosis factor; TRAF6, TNF receptor-associated factor 6; cPpep63, cyclic phosphorylated pep63; p53 family; p63; pep63, p63(534–551) peptide; proline isomerization; ubiquitynation