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

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.

• Authors: Qihui Lian, Yuan Gao, Qian Li, Xian He, Xiaogang Jiang, Zhongjian Pu, Guoqiang Xu
• Journal: The protein journal (Protein J) Vol. 39 Issue 5 Pg. 411-421 (10 2020) ISSN: 1875-8355 [Electronic] Netherlands
• PMID: 33009960 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Adaptor Proteins, Signal Transducing
• CRBN protein, human
• ILF2 protein, human
• Nuclear Factor 45 Protein
• Ubiquitin-Protein Ligases
• Proteasome Endopeptidase Complex

Topics

• Adaptor Proteins, Signal Transducing (genetics, metabolism)
• HEK293 Cells
• Humans
• Nuclear Factor 45 Protein (genetics, metabolism)
• Proteasome Endopeptidase Complex (genetics, metabolism)
• Proteolysis
• Ubiquitin-Protein Ligases (genetics, metabolism)
• Ubiquitination