Protein kinase Cα is involved in interferon regulatory factor 3 activation and type I interferon-β synthesis

Abstract

Protein kinase C (PKC) isoforms are critically involved in the regulation of innate immune responses. Herein, we investigated the role of conventional PKCα in the regulation of IFN-β 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-β synthesis induced by double-stranded RNA polyinosinepolycytidylic acid. Furthermore, reporter gene assays confirmed that PKCα regulates IFN-β promoter activity, since overexpression of dominant negative PKCα but not PKCαI repressed interferon regulatory factor 3 (IRF-3)-dependent but not NF-κB-mediated promoter activity upon TLR3 engagement in HEK 293 cells. Dominant negative PKCα inhibited IRF-3 transcriptional activity mediated by overexpression of TIR domain-containing adapter inducing IFN-β 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 coactivator CREB-binding protein. Furthermore, PKCα knockdown with specific small interfering RNA inhibited IFN-β expression and down-regulated IRF-3-dependent promoter activity, establishing PKCα as a component of TLR3 signaling that regulates IFN-β gene expression by targeting IRF-3-CREB-binding protein interaction. Finally, we analyzed the involvement of cPKCs in other signaling pathways leading to IFN-β synthesis. These experiments revealed that cPKCs play a role in the synthesis of IFN-β induced via both TLR-dependent and -independent pathways. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.