Identification and Characterization of Two Novel Isoforms of Pirh2 Ubiquitin Ligase That Negatively Regulate p53 Independent of RING Finger Domains

Abstract

Pirh2 is a newly identified E3 ubiquitin ligase known to inhibit tumor suppressor p53 function via ubiquitination and proteasomal degradation. We have identified two novel Pirh2 splice variants that encode different Pirh2 isoforms and named these Pirh2B and Pirh2C. Accordingly, the full-length protein is now classified as isoform Pirh2A. The central region of Pirh2 harbors a RING finger domain that is critical for its ubiquitin ligase function. The Pirh2B isoform lacks amino acids 171-179, whereas Pirh2C is missing C-terminal amino acids 180 -261, which for each isoform results in a RING domain deletion and the abrogation of ubiquitin ligase activity. Our findings further indicate that the Pirh2B isoform but not the Pirh2C isoform is capable of binding to Pirh2A, suggesting that the C-terminal region absent in Pirh2C is critical for Pirh2-Pirh2 interactions. Similar to Pirh2A, both Pirh2B and Pirh2C interact with p53; however, interactions between p53 and Pirh2B appear stronger than those between p53 and Pirh2C. Interestingly, although both Pirh2B and Pirh2C are not able to promote in vitro p53 ubiquitination, both are capable of negatively regulating p53 protein stability and promoting the intracellular ubiquitination of p53. Furthermore, like Pirh2A, both isoforms are able to inhibit p53 transcriptional activity. We have also for the first time demonstrated that Pirh2A as well as the novel isoforms also interact directly with MDM2 within a region encompassing MDM2 acidic and zinc finger domains. It is therefore possible that Pirh2A and the novel Pirh2 isoforms identified in this study may also modulate p53 function by engaging MDM2.The tumor suppressor p53 has been implicated in a growing number of cellular processes, the most recognized of these being the initiation of signaling cascades leading to growth arrest and apoptosis (1). A large body of evidence supports the notion that p53 is capable of regulating these pathways via distinct mechanisms. It is becoming clear that p53 probably exerts its antiproliferative effects through both its well established transcription factor function as well as a transcription-independent role at the mitochondrion (1-3).Given its potent capacity to control cell fate, p53 in normal cells is held in check at multiple, often interrelated levels, including regulation of p53 protein stability, subcellular localization, and transcriptional activity. It is now well understood that p53 ubiquitination and subsequent proteasomal degradation is one of the principal mechanisms controlling these processes (4 -6). The RING domain containing ubiquitin ligase MDM2 was the first non-viral protein found to be responsible for the ubiquitin-dependent targeting of p53 for proteolysis (7,8). The importance of MDM2 in abrogating p53 function was illustrated in an embryonic lethal MDM2(Ϫ/Ϫ) mouse model in which lethality can be attributed to uncontrolled p53 activity and is rescued by p53 deletion (9, 10).Adding complexity to the once clear role of MDM2 in p53 ubiquitination is the recent discovery...

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