Identification of Distinct Signaling Pathways Leading to the Phosphorylation of Interferon Regulatory Factor 3

Marc J Servant,Benjamin Ten Oever,Cecile Lepage,Lucia Conti,Sandra Gessani,Ilkka Julkunen,Rongtuan Lin,John Hiscott

doi:10.1074/jbc.m007790200

Abstract

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.

Highlights

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state
Multiple Forms of interferon regulatory factor-3 (IRF-3) Phosphoprotein—C-terminal phosphorylation of interferon regulatory factors (IRF)-3 following paramyxovirus infection is a prerequisite for its nuclear translocation, association with CBP/p300 co-activators, and transcriptional activation [13, 16, 17, 28]
virus activated kinase (VAK) activity is relatively easy to detect in extracts from virus-infected cells, since phosphorylated IRF-3 migrates slower in SDS-PAGE than nonphosphorylated IRF-3 [13, 16, 17], a phenomenon observed with many phosphoproteins

Summary

Introduction

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. Numerous studies suggest that the IKK␤ catalytic subunit is required for IKK and NF-␬B kidney; CIP, calf intestine alkaline phosphatase; TNF, tumor necrosis factor; MeV, measle virus; NDV, Newcastle disease virus; VSV, vesicular stomatitis virus; JNK, c-Jun N-terminal kinase; PAGE, polyacrylamide gel electrophoresis; aa, amino acid(s); PMSF, phenylmethylsulfonyl fluoride; HAU, hemagglutinating units; WCE, whole cell extracts; RANTES, regulated on activation normal T cell expressed; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to a conformational change, dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, stimulation of DNA binding and transcriptional activities [3, 13,14,15,16,17]. As with NF-␬B activation, the rate-limiting step in this process is C-terminal phosphorylation of IRF-3 by an uncharacterized virus activated kinase (VAK) activity

Methods

Results

Conclusion