The Ubiquitin-Protein Ligase Itch regulates p73 stability

ROSSI M, DE LAURENZI V, MUNARRIZ E, GREEN DR, LIU YC, VOUSDEN KH, CESARENI G, MELINO G.

Workshop; 12th International p53 Workshop.; 2004

p73 is a member of the p53 family of transcription factors, and like p53, has a modular structure (figure 1A). p73 shares a high degree of sequence homology with p53 and can bind to p53- responsive elements activating the transcription of p53 target genes, such as those inducing cell cycle arrest and promoting apoptosis. Unlike p53, however, p73 is expressed as different isoforms. Most of the variation generated by alternative splicing occurs at the 3' end, in a part of the sequence that does not have a counterpart in p53. At least six different p73 proteins ( ) are generated. In addition, the p73 gene exploits an alternative promoter and an extra exon (exon 3’) to generate N-terminally truncated isoforms ( Np73). These variants lack the transactivation domain and act as “dominant negatives”, blocking the function of either p53 or p73 full-length proteins. The relative levels of TA and N isoforms determine cell fate, resulting in either growth arrest and death or uncontrolled proliferation. TAp73 steady state protein levels are up-regulated in response to DNA damage in a fashion distinct from p53 while Np73 is rapidly degraded. These obervations suggest an important differential role for these isomers in carcinogenesis. Despite its importance, however, very little is known of the molecular mechanisms underlying the regulation of p73 protein steady state levels. MDM2, the E3 ubiquitin ligase that regulates the degradation of the cognate protein p53 via a proteasomal dependent pathway, binds to p73 but does not promote its degradation. In order to define the degradation pathway of p73 we searched a human cDNA library displayed on bacteriophage capsids for p73 specific binding partners. In order to identify mechanisms distinct from those of p53, as a bait we used a p73 C-terminal fragment since this region is not present in p53 and contains a PPxY (PY) sequence that has been characterized as a binding motif for a class of WW domains (figure 1A). We found that p73 binds Itch, a human ubiquitin-protein ligase (E3). The Itch mouse homologue gene is absent in the non-agouti-lethal 18H (Itchy) mice which display profound immune defects. Itch belongs to the Nedd4-like E3 family, and is characterized by a modular organization that includes: an N-terminal protein kinase C-related C2 domain; multiple WW domains; and a Cterminal HECT (homologous to the E6-associated protein carboxyl terminus) Ubiquitin (Ub)-protein ligase domain (Fig.1A). Ub-protein ligases are involved in the multistep process that leads to ubiquitination of protein substrates. In this pathway E3 catalyzes the final transfer of ubiquitin to a specific substrate, thus governing the specificity of substrate recognition. Here we show that Itch binds and ubiquitinates TAp73 and Np73, but not p53, and determines p73 proteasome-dependent degradation. We also show that Itch expression is down regulated upon DNA damage thereby allowing stabilization of the TA and N p73 proteins. Our data also show that a second as yet unidentified mechanism is responsible for selective Np73 degradation in response to DNA damage. Therefore, we have identified a relevant mechanism in the control of p73 levels both in normal as well as in stress conditions.