Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Rami Abou Zeinab,Hong Wu,Roger P Leng,Consolaro Sergi

doi:10.1371/journal.pone.0082803

Rami Abou Zeinab, Hong Wu + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0082803

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Journal: PloS one	Publication Date: Dec 18, 2013
Citations: 34	License type: CC BY 4.0

Affiliation: Heritage Medical Research Clinic, University of Alberta

Abstract

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.

Highlights

The function of the p53 tumor suppressor gene in maintaining genomic integrity [1] through its effects on cellular processes such as DNA repair, cell cycle arrest, and programmed cell death [2,3,4] is well known
Because the self-ubiquitination process seems to be critical to Pirh2 expression and function as an E3 ligase, we designed three experiments to test the optimal conditions for Pirh2 E3 ligase activity
Because Pirh2 is an E3 ligase that regulates the p53 and p73 tumor suppressors [22,32], Pirh2 ubiquitination has always been a focus of attention among researchers investigating the p53 system

Summary

Introduction

The function of the p53 tumor suppressor gene in maintaining genomic integrity [1] through its effects on cellular processes such as DNA repair, cell cycle arrest, and programmed cell death [2,3,4] is well known. It is not surprising that 50% of human cancers are due to mutations in p53 and that the other 50% carry a wild type p53 allele but lack a functional p53 protein [5]. Tight regulation of the p53 protein is important. The precise mechanisms of p53 regulation are not fully understood, the involvement of many different proteins [6,7] and many different processes, including sumoylation [8,9,10], neddylation [11,12,13], and acetylation or other post-translational modifications [14,15,16], have been proposed. Despite the apparent involvement of numerous proteins and processes, ubiquitination has been identified as the master regulatory mechanism [17]. Poly-ubiquitination results in the addition of several ubiquitin molecules to p53, which, in turn, promotes proteosomal degradation of p53 by 26 S proteasomes [5,19]

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Results

Conclusion