Protein Kinase Cα Is Involved in Interferon Regulatory Factor 3 Activation and Type I Interferon-β Synthesis

Jolyn Johnson,Valentina Albarani,Muriel Nguyen,Michel Goldman,Fabienne Willems,Ezra Aksoy

doi:10.1074/jbc.m700421200

Abstract

Protein kinase C (PKC) isoforms are critically involved in the regulation of innate immune responses. Herein, we investigated the role of conventional PKCalpha in the regulation of IFN-beta gene expression mediated by the Toll-like receptor 3 (TLR3) signaling pathway. Inhibition of conventional PKC (cPKC) activity in monocyte-derived dendritic cells or TLR3-expressing cells by an isoform-specific inhibitor, Gö6976, selectively inhibited IFN-beta synthesis induced by double-stranded RNA polyinosine-polycytidylic acid. Furthermore, reporter gene assays confirmed that PKCalpha regulates IFN-beta promoter activity, since overexpression of dominant negative PKCalpha but not PKCbeta(I) repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-kappaB-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKCalpha inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN-beta and Tank-binding kinase-1. Additional biochemical analysis demonstrated that Gö6976-treated dendritic cells exhibited IRF-3 phosphorylation, dimerization, nuclear translocation, and DNA binding activity analogous to their control counterparts in response to polyinosine-polycytidylic acid. In contrast, co-immunoprecipitation experiments revealed that TLR3-induced cPKC activity is essential for mediating the interaction of IRF-3 but not p65/RelA with the co-activator CREB-binding protein. Furthermore, PKCalpha knock-down with specific small interfering RNA inhibited IFN-beta expression and down-regulated IRF-3-dependent promoter activity, establishing PKCalpha as a component of TLR3 signaling that regulates IFN-beta gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN-beta synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN-beta induced via both TLR-dependent and -independent pathways.

Highlights

Type I interferons (IFNs),4 comprising IFN-␤ and the IFN-␣ family are central to the innate immunity of mammals and the development of effective adaptive immune responses against viruses and tumors [1, 2]
Protein kinase C (PKC)␣ Is Involved in Toll-like receptor 3 (TLR3)-mediated Induction of interferon regulatory factor 3 (IRF-3)-dependent Promoter Activities—In the set of experiments, we investigated the effects of inhibiting poly(I:C)-induced conventional PKC activity on human IFN-␤ promoter activity in TLR3-expressing 293 cells
The poly(I:C)-induced transcriptional activity of Inhibition of Conventional PKCs Does Not Interfere with Gal4-IRF-3 was inhibited with increasing concentrations of ATF-2 and c-Jun Activation—We evaluated the role of PKC␣-siRNA, whereas the control GFP-siRNA had no effect conventional PKC (cPKC) in TLR3-mediated activation of ATF-2/c-Jun, which is on its activity (Fig. 8B)

Summary

Introduction

Type I interferons (IFNs),4 comprising IFN-␤ and the IFN-␣ family are central to the innate immunity of mammals and the development of effective adaptive immune responses against viruses and tumors [1, 2]. PKC␣ Is Involved in TLR3-mediated Induction of IRF-3-dependent Promoter Activities—In the set of experiments, we investigated the effects of inhibiting poly(I:C)-induced conventional PKC activity on human IFN-␤ promoter activity in TLR3-expressing 293 cells.

Results

Conclusion