Ubiquitin-dependent Degradation of Interferon Regulatory Factor-8 Mediated by Cbl Down-regulates Interleukin-12 Expression

Jie Zhao,Huabao Xiong,Yibang Chen,Jay C Unkeless,Hua Gu,Lloyd Mayer,Sidong Xiong,Hongxing Li,Keiko Ozato,Bo Huang,Hee Jeong Kong

doi:10.1074/jbc.m414296200

Abstract

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation. IRF-8 induced in peritoneal macrophages by interferon-gamma and lipopolysaccharide was degraded rapidly, and degradation of IRF-8 was blocked by MG132, the proteasome inhibitor, but inhibitors of calpain and lysosomal enzymes had no effect. The ubiquitination of IRF-8 was shown by co-immunoprecipitation from RAW264.7 macrophages retrovirally transduced with IRF-8 and hemagglutinin-ubiquitin. The dominant negative ubiquitin mutants K48R and K29R inhibited IRF-8 degradation in 293T cells, confirming the relationship between ubiquitination of IRF-8 and its degradation. IRF-8 carboxyl-terminal truncation mutants were not ubiquitinated and were consequently stable, indicating that the carboxyl-terminal domain of IRF-8 controls ubiquitination. The ubiquitin-protein isopeptide ligase (E3) that ubiquitinated IRF-8 was likely to be Cbl, which formed a complex with IRF-8, demonstrable by both immunoprecipitation and gel filtration. Furthermore, IRF-8 stability was increased by dominant negative Cbl, and IRF-8 ubiquitination was significantly attenuated in Cbl-/- cells. Reflecting increased stability and expression, the IRF-8 carboxyl-terminal deletion mutant induced interleukin (IL)-12 p40 promoter activity much more strongly than IRF-8 did. Furthermore, IRF-8-induced IL-12 p40 synthesis in RAW264.7 cells was enhanced by dominant negative Cbl, and peritoneal macrophages from Cbl-/- mice showed increased IL-12 p40 protein production. Taken together, these results suggest that the proteasomal degradation of IRF-8 mediated by the ubiquitin E3 ligase Cbl down-regulates IL-12 expression.

Highlights

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation
IRF-8 in Peritoneal Macrophages Is Degraded by the Proteasome—We have shown previously that IFN-␥ and LPS synergistically induce IRF-8 protein expression in macrophages (11), and we examine the stability of IRF-8
Thioglycollateelicited macrophages from C3H/OUJ mice activated for 6 h with IFN-␥ (10 ng/ml) and LPS (1 ␮g/ml) were incubated with fresh medium in the presence or absence of the proteasome inhibitor MG132 (10 ␮M), and at various time intervals cell lysates were assayed by immunoblotting for IRF-8

Summary

Introduction

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation. IRF-8-induced IL-12 p40 synthesis in RAW264.7 cells was enhanced by dominant negative Cbl, and peritoneal macrophages from Cbl؊/؊ mice showed increased IL-12 p40 protein production. Taken together, these results suggest that the proteasomal degradation of IRF-8 mediated by the ubiquitin E3 ligase Cbl down-regulates IL-12 expression. IRF-8 and IRF-1 synergistically activate the expression of IL-12 (18), which is a pivotal cytokine driving the immune system toward a T help type (Th) 1 response (19) Both IRF-8- and IRF-1-deficient mice are highly susceptible to several pathogens, including Listeria monocytogenes, Yersinia enterocolitica, and Leishmania major (20 –22), due to the inability of these mice to generate a Th1 cell-mediated response directed against intracellular pathogens. The regulated expression of IRF-8 and IRF-1 controls the character of immune responses, and the expression

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Conclusion