SAG/RBX2 is a novel substrate of NEDD4-1 E3 ubiquitin ligase and mediates NEDD4-1 induced chemosensitization.

Weihua Zhou,Jie Xu,Yi Sun,Yongchao Zhao

doi:10.18632/oncotarget.2246

Weihua Zhou, Jie Xu + Show 2 more

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https://doi.org/10.18632/oncotarget.2246

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Abstract

Sensitive to apoptosis gene (SAG), also known as RBX2, ROC2, or RNF7, is a RING component of SCF E3 ubiquitin ligases, which regulates cellular functions through ubiquitylation and degradation of many protein substrates. Although our previous studies showed that SAG is transcriptionally induced by redox, mitogen and hypoxia via AP-1 and HIF-1, it is completely unknown whether and how SAG is ubiquitylated and degraded. Here we report that NEDD4-1, a HECT domain-containing E3 ubiquitin ligase, binds via its HECT domain directly with SAG's C-terminal RING domain and ubiquitylates SAG for proteasome-mediated degradation. Consistently, SAG protein half-life is shortened or extended by NEDD4-1 overexpression or silencing, respectively. We also found that SAG bridges NEDD4-1 via its C-terminus and CUL-5 via its N-terminus to form a NEDD4-1/SAG/CUL-5 tri-complex. Biologically, NEDD4-1 overexpression sensitizes cancer cells to etoposide-induced apoptosis by reducing SAG levels through targeted degradation. Thus, SAG is added to a growing list of NEDD4-1 substrates and mediates its biological function.

Highlights

Protein ubiquitylation is carried out by a three-step enzymatic cascade, involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase [1]
The protein levels of Sensitive to apoptosis gene (SAG) and NEDD4-1 are inversely correlated and SAG binds to NEDD4-1 via its RING domain
Our results indicate that SAG-NEDD4-1 binding is mediated by the HECT domain on NEDD4-1 and the RING domain on SAG

Summary

Introduction

Protein ubiquitylation is carried out by a three-step enzymatic cascade, involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase [1]. The multi-unit Cullin RING ligase (CRL) is the largest family of E3 ubiquitin ligases with its RING component being either RBX1 or RBX2 [5, 6]. Both family members contain an evolutionarily conserved RING finger domain at the C-terminus, which is required for the ubiquitin ligase activity of CRLs [7, 8]. Our most recent results showed that SAG is an onco-cooperating gene, required for lung tumorigenesis induced by a mutant Kras [13]. Our previous studies showed that SAG is subjected to the regulation of AP-1 [14] and HIF-1 [15] at the transcriptional level, it is unknown how SAG is regulated at the post-translational level and by which E3 ligase

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