Ubiquitin-dependent degradation of interferon regulatory factor-8 mediated by Cbl down-regulates interleukin-12 expression.
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.
Xiong
Huabao
H
Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, USA. xionghbl@yahoo.com
Li
Hongxing
H
Kong
Hee Jeong
HJ
Chen
Yibang
Y
Zhao
Jie
J
Xiong
Sidong
S
Huang
Bo
B
Gu
Hua
H
Mayer
Lloyd
L
Ozato
Keiko
K
Unkeless
Jay C
JC
D000818:Animals; D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D050835:Interferon Regulatory Factors; D018664:Interleukin-12 / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D012097:Repressor Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D000099185:Interferon Regulatory Factor-8
D050835:Interferon Regulatory Factors; D018664:Interleukin-12; D011518:Proto-Oncogene Proteins; D050721:Proto-Oncogene Proteins c-cbl; D012097:Repressor Proteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099185:Interferon Regulatory Factor-8; C493849:Cbl protein, mouse