p73, a homolog of the tumor suppressor p53, transactivates many p53 target genes, leading to apoptosis or cellcycle arrest. p73 has recently been reported to play an important role in tumor suppression in a mouse model. Here, we show that Pirh2 physically interacted with p73 and downregulated p73 function through its E3 ligase activity. Pirh2 promoted p73 ubiquitination in vivo and in vitro. Intriguingly, Pirh2 primarily used K63-linked chains to ubiquitinate p73 in vitro, but in vivo, Pirh2 utilized K11-, K29-, K48-, and K63-linked chains to promote p73 ubiquitination. Depletion of Pirh2 by siRNA significantly reduced the ubiquitination of p73 in p53 null cells. Ectopic expression of Pirh2 repressed p73-dependent transcriptional activity, but the levels of p73 were not decreased. We consistently showed that ablation of endogenous Pirh2 restored p73-mediated transactivational activity. We found that Pirh2 repressed p73 transcriptional activity by directly inhibiting the p73 transcript, and p73 repression by Pirh2 was required for p73-dependent transcriptional activity and G 1 arrest but not for apoptosis. This study provides evidence that the ubiquitination of p73 mediated by Pirh2 represents an important pathway for controlling the suppressive function of p73. Furthermore, the data suggest a link between the transcriptional activity of p73 and its ubiquitination. Mol Cancer Res; 9(12); 1780-90. Ó2011 AACR.

Neuronal survival and death of neurons are considered a fundamental mechanism in the regulation of the nervous system during early development of the system and in adulthood. Defects in this mechanism are highly problematic and are associated with many neurodegenerative diseases. Because neuronal programmed death is apoptotic in nature, indicating that apoptosis is a key regulatory process, the p53 family members (p53, p73, p63) act as checkpoints in neurons due to their role in apoptosis. The complexity of this system is due to the existence of different naturally occurring isoforms that have different functions from the wild types (WT), varying from apoptotic to anti-apoptotic effects. In this review, we focus on the role of UBE4B (known as Ube4b or Ufd2a in mouse), an E3/E4 ligase that triggers substrate polyubiquitination, as a master regulatory ligase associated with the p53 family WT proteins and isoforms in regulating neuronal survival. UBE4B is also associated with other pathways independent of the p53 family, such as polyglutamine aggregation and Wallerian degeneration, both of which are critical in neurodegenerative diseases. Many of the hypotheses presented here are gateways to understanding the programmed death/survival of neurons regulated by UBE4B in normal physiology, and a means of introducing potential therapeutic approaches with implications in treating several neurodegenerative diseases.

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53's response to DNA damage.