Ubiquitination-dependent degradation of p73 by the mitochondrial E3 ubiquitin ligase Hades

https://doi.org/10.1016/j.bbrc.2015.09.163Get rights and content

Highlights

  • Hades physically interacts with p73.

  • Hades colocalizes with p73 at the mitochondria.

  • Hades ubiquitinates p73.

  • Hades promotes the degradation of p73.

  • Exonuclear functions of p73 and p53 are negatively regulated by Hades.

Abstract

p73 is a member of the p53 family of transcription factors which plays an essential role in tumor suppression. p73 is associated with the sensitivity of cancer cells to chemotherapy and the prognosis of many cancers. In this study, we showed the ubiquitination-dependent degradation of p73 by the mitochondrial E3 ubiquitin ligase Hades. First, the binding between p73 and Hades was identified by co-immunoprecipitation experiments, and it was found that the Hades RING-finger domain mediates the interaction with p73. Immunofluorescence analysis showed that p73 moves to the mitochondria and colocalizes with Hades during etoposide-induced apoptosis. By performing in vivo and in vitro ubiquitination assays, we observed that the Hades RING-finger domain promotes ubiquitination of p73. Finally, it was shown that SiRNA-mediated depletion of Hades stabilizes p73. Taken together, our results showed that Hades mediates the ubiquitination-dependent degradation of mitochondrial p73 under apoptotic conditions. These findings suggest that Hades-mediated p73 ubiquitination is a novel regulatory mechanism for the exonuclear function of p73.

Introduction

p73 is a structural and functional homolog of the p53 tumor suppressor [1], [2], [3]. p73 contains the following domains which are analogous to p53: the transactivation domain (TAD), proline-rich domain (PR), DNA-binding domain (DBD), and tetramerization domain (TD) [4]. In addition, it has extra C-terminal domains including a sterile alpha motif (SAM) and a transcription inhibitory domain (TID) which are not present in p53. p73 is able to transactivate p53-responsive genes including p21, PUMA, and Bax [5], [6]. Like p53, p73 plays a crucial role in the regulation of cell cycle, apoptosis, and other biological functions in response to various types of stresses [1], [2], [3]. However, in contrast with p53, p73 has unique roles in development including hippocampal neurogenesis [7], metabolic control [8], and spermatogenesis [9]. Previous studies on p73-mediated apoptosis have largely focused on the transcription-dependent apoptotic mechanism of p73 within the nucleus in response to apoptotic stimuli [5]. However, several lines of evidences have shown that p73 localizes to mitochondria during apoptosis [10], [11], suggesting an exonuclear function of p73 in a manner similar to that of p53 [12], [13], [14], [15]. Sayan et al. showed that p73 and its caspase-cleaved fragments translocate to the mitochondria and augment TRAIL-induced apoptosis [10]. However, little is known about the regulatory mechanism for the exonuclear function of p73.

The steady-state protein level of p73 is primarily regulated by post-translational modifications (PTMs). In non-stressed cells, the TAp73 and DNp73 isoforms are degraded in an ubiquitination-dependent or -independent manner [16], [17]. p73 is degraded by NAD(P)H quinone oxidoreductase 1 and NADH through the ubiquitin-independent 20S proteasome [16], and undergoes ubiquitination-dependent 26S proteasomal degradation mediated by the HECT-domain E3 ubiquitin ligase Itch [17]. On the other hand, the DNp73 isoform is selectively degraded by the RING finger-domain E3 ubiquitin ligase p73-induced ring finger protein 2 (PIR2) [18] and other molecules, such and c-Jun and Yes-Associated Protein (YAP) [19], [20], [21], which leads to a greater accumulation of TAp73 than that of the DNp73 isoform, inducing TAp73-mediated apoptosis. Therefore, the apoptotic activity and thus cell fate are determined by the TA/DNp73 isoform ratio [22], [23].

E3 ubiquitin ligase Hades, also known as mitochondrial ubiquitin ligase activator of NFKB1 (MULAN), mitochondrial-anchored protein ligase (MAPL), or growth inhibition and death E3 ligase (GIDE), is anchored in the mitochondrial outer membrane via its two transmembrane domains and has a conserved C-terminal RING-finger domain (Fig. 1A) with an E3 ligase activity, facing the cytosol [24], [25]. Several proteins, including NF-κB, JNK, Akt, GABARAP, and DRP1, have recently been reported to be regulated by Hades, and these regulations are associated with various cellular functions including apoptosis [25], [26], proliferation [27], mitophagy [28], mitochondrial dynamics [29], and antiviral response [30]. Mitochondrial E3 ubiquitin ligase Hades was previously shown to negatively regulate the exonuclear function of p53. Jung et al. found that Hades reduces the stability of mitochondrial p53 via a mechanism that requires the RING-finger domain with its E3 ubiquitin ligase activity [26]. They showed that Hades polyubiquitinates p53 independent of MDM2 by targeting lysine24 in the p53 TAD and inhibits a p53-dependent mitochondrial cell death pathway by blocking the interaction between p53 and Bcl-2.

In this study, we showed that Hades acts as a novel E3 ubiquitin ligase for mitochondrial p73. First, mitochondrial E3 ubiquitin ligase Hades was identified as a new binding partner of p73 by co-immunoprecipitation. By performing fluorescence imaging analysis, we observed that, in response to apoptotic stress, p73 translocates to the mitochondria and colocalizes with Hades. The in vivo and in vitro ubiquitination assays showed that p73 is polyubiquitinated by Hades, and the Hades RING-finger domain mediates the ubiquitination of p73. In addition, the SiRNA knockdown of Hades showed that Hades mediates p73 degradation through ubiquitination. Taken together, this study showed that Hades negatively regulates mitochondrial p73 by ubiquitination-dependent degradation which could be a novel regulatory mechanism for the exonuclear function of p73.

Section snippets

Plasmids and recombinant proteins

The full-length p73 and Hades were purchased from Source BioScience. The Hades RING-finger domain was generated by PCR amplification. The ubiquitin gene and destination vectors (pDEST-GFP-N, pDEST-mCherry-N, pDEST-HA-N, and pDEST-FLAG-N, pDEST-His) were kindly provided by Dr. J. Park (Korea University, Seoul, Korea). Using Gateway cloning, p73, full-length Hades, Hades RING-finger domain, and ubiquitin genes were subcloned into the Gateway cloning system entry vector (pDONR207) and subsequent

Hades physically interacts with p73

A previous study showed that Hades is an E3 ubiquitin ligase for p53 and its RING-finger domain mediates the ubiquitination of p53 [26]. To investigate whether p73, one of the p53 family members, is a novel substrate of Hades, co-immunoprecipitation experiments were first performed with full-length Hades and the Hades RING-finger domain (Fig. 1A). To examine whether full-length Hades interacts with p73 under apoptotic conditions, H1299 cells were co-transfected with GFP-full length Hades and

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (No. 2010-0028631 and NRF-2012M3C1A3671508), by the Bio-Synergy Research Project (NRF-2012M3A9C4048759) of the Ministry of Science, ICT and Future Planning through the National Research Foundation and by the KAIST Future Systems Healthcare Project from the Ministry of Education, Science and Technology. We are grateful to J. Park for the pDEST-GFP-N, pDEST-mCherry-N,

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