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Cereblon Promotes the Ubiquitination and Proteasomal Degradation of Interleukin Enhancer-Binding Factor 2

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Abstract

Interleukin enhancer-binding factor 2 (ILF2) forms a heterodimer with interleukin enhancer-binding factor 3 (ILF3) via double-stranded RNA-binding motif and zinc finger associated domain and thus regulates gene expression and cancer cell growth. However, how ILF2 is degraded in cells remains elusive. In this work, using stable isotope labeling by amino acids in cell culture (SILAC) quantitative proteomics, we find that ILF2 is downregulated in cells expressing cereblon (CRBN). Using affinity purification and immunoblotting analysis, we demonstrate that CRBN interacts with ILF2 and functions as a substrate receptor of the cullin-4 RING E3 ligase complex. Biochemical experiments disclose that CRBN expression reduces ILF2 protein level and this reduction is diminished when the proteasome is inhibited. Upon protein synthesis inhibition, the degradation of ILF2 is enhanced by CRBN. Moreover, CRBN promotes the ubiquitination of ILF2 and thus results in the ubiquitin-mediated proteasomal degradation. Analyses of previously identified post-translational modification sites and the crystal structure of ILF2 discover the potential ubiquitination sites on ILF2. Through mutagenesis and biochemical experiments, we further reveal that the K45R mutation completely abolishes the effect of CRBN on ILF2, suggesting that this is the key residue responsible for its ubiquitination. Taken together, we identify an E3 ligase that regulates ILF2 and uncover a molecular pathway for its degradation. This work might be helpful to elucidate the molecular mechanism by which CRBN regulates diverse cellular functions.

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Acknowledgements

This work was supported by the National Key R&D Program of China (2019YFA0802400), National Natural Science Foundation of China (31670833), Talent Program in Six Major Disciplines in Jiangsu Province (SWYY-080), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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  1. Qihui Lian and Yuan Gao contributed equally.

Authors and Affiliations

  1. Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, Soochow University, 199 Ren’ai Road, Suzhou, 215123, Jiangsu, China

    Qihui Lian, Yuan Gao, Qian Li, Xian He, Xiaogang Jiang & Guoqiang Xu

  2. Department of Oncology, Haian Hospital of Traditional Chinese Medicine, Haian, 226600, Jiangsu, China

    Zhongjian Pu

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  1. Qihui Lian
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Contributions

GX and ZP conceived the concept; QL, YG, XH, QL, and XJ performed the experiments and analyzed the data. GX and ZP wrote the manuscript and all authors revised the manuscript.

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Correspondence to Zhongjian Pu or Guoqiang Xu.

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Additional information

In commemoration of Prof. Harold A. Scheraga This work was written to memorialize Harold A. Scheraga of the Baker Laboratory of Chemistry and Chemical Biology, Cornell University for his long-term dedication to scientific research and for fostering hundreds of students and postdocs in experimental and computational biophysics. The last author of this paper, Guoqiang Xu, was trained as a postdoctoral associate in the Scheraga laboratory between 2003 and 2005. It was during his tenure in Prof. Scheraga′s laboratory that he initiated his scientific career in experimental biological sciences. Prof. Scheraga was enthusiastic and persistent about the research in biochemistry and biophysics. He was a critical thinker and always demonstrated to others the perfectness and dedication required to conduct research. These aspects influenced Guoqiang’s scientific career in the long run. Prof. Scheraga’s personality also shaped Guoqiang and sculpted his career development. We will always cherish the memory and exciting moments we had with Prof. Scheraga.

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Lian, Q., Gao, Y., Li, Q. et al. Cereblon Promotes the Ubiquitination and Proteasomal Degradation of Interleukin Enhancer-Binding Factor 2. Protein J 39, 411–421 (2020). https://doi.org/10.1007/s10930-020-09918-9

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