SAG/RBX2 E3 ligase complexes with UBCH10 and UBE2S E2s to ubiquitylate β-TrCP1 via K11-linkage for degradation.

Peng Kuang,Mingjia Tan,Qiang Zhang,Weihua Zhou,Yi Sun

doi:10.1038/srep37441

Peng Kuang, Mingjia Tan + Show 3 more

Open Access

https://doi.org/10.1038/srep37441

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Abstract

SAG/RBX2 and RBX1 are two family members of RING components of Cullin-RING ligases (CRLs), required for their enzymatic activity. Previous studies showed that SAG prefers to bind with CUL5, as well as CUL1, whereas RBX1 binds exclusively to CULs1–4. Detailed biochemical difference between SAG and RBX1, and whether SAG mediates cross-talk between CRL5 and CRL1 are previously unknown. Here we report that the levels of SAG and β-TrCP1 are inversely correlated, and SAG-CUL5-βTrCP1 forms a complex under physiological condition. SAG-CUL5, but not RBX1-CUL1, negatively modulates β-TrCP1 levels by shortening its protein half-life through promoting its ubiquitylation via atypical K11-linkage. Consistently, chemical inducers of SAG reduced β-TrCP1 level. Furthermore, SAG mainly binds to E2s UBCH10 and UBE2S known to mediate K11 linkage of ubiquitin, whereas RBX1 exclusively binds to E2s CDC34 and UBCH5C, known to mediate K48 linkage of ubiquitin. Finally, silencing of either UBCH10 or UBE2S, but not UBCH5C, caused accumulation of endogenous β-TrCP1, suggesting that β-TrCP1 is a physiological substrate of SAG-UBCH10C/UBE2S. Our study, for the first time, differentiates SAG and RBX1 biochemically via their respective binding to different E2s; and shows a negative cross-talk between CRL5 and CRL1 through SAG mediated ubiquitylation of β-TrCP1.

Highlights

Protein ubiquitylation is a post-translational modification, that via modulating protein stability, activity, or localization[1] regulates many cellular pathways including proinflammatory signaling, DNA damage response, and apoptosis[2,3]
It has been established that CDC34 or UBCH5C E2s couples with CRL1, known as SCF (SKP1-Cullin1-F box protein) E3, to assemble the ubiquitin chain via the K48 linkage[11], whereas UBCH10/UBE2C and UBE2S couples with APC/C (Anaphase Promoting Complex/cyclosome) E3 to assemble the ubiquitin chain via the K11
We found that SAG-CUL5-mediated ubiquitylation of β-TrCP1 is via the K11 linkage, achieved by SAG binding to K11 linkage E2s, UBCH10/UBE2C and UBE2S, and silencing of either UBCH10 or UBE2S caused β-TrCP1 accumulation

Summary

Introduction

Protein ubiquitylation is a post-translational modification, that via modulating protein stability, activity, or localization[1] regulates many cellular pathways including proinflammatory signaling, DNA damage response, and apoptosis[2,3]. Protein ubiquitylation is catalyzed by an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase, that is responsible for substrate recognition[4], and catalyzes the transfer of ubiquitin directly from the E2 to the substrate[5] Multiple run of this cascade reaction results in polyubiquitylation of a substrate[6]. It appears that their E3 ligase activity is biochemically interchangeable in carrying out an in vitro polyubiquitylation reactions when RBX1 or SAG, used as the source of E3, was purified from transfected cells through immunoprecipitation[18,19] It is, totally unknown whether SAG and RBX1 bind to different E2s to assemble different linkage of ubiquitin chains. Our study revealed, for the first time, that there is a negative cross-talk between CRL1 and CRL5 through SAG-mediated ubiquitylation and degradation of β-TrCP1, and that CRL5 E3 can mediate K11-linked ubiquitin chain through SAG binding to specific E2s

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