Phosphoproteomics Combined with Quantitative 14-3-3-affinity Capture Identifies SIRT1 and RAI as Novel Regulators of Cytosolic Double-stranded RNA Recognition Pathway

• Published in: Molecular & cellular proteomics Vol. 13; no. 10; pp. 2604 - 2617
• Main Authors: Öhman, Tiina, Söderholm, Sandra, Hintsanen, Petteri, Välimäki, Elina, Lietzén, Niina, MacKintosh, Carol, Aittokallio, Tero, Matikainen, Sampsa, Nyman, Tuula A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2014; The American Society for Biochemistry and Molecular Biology
• Subjects: 14-3-3 Proteins - metabolism; Cardiovirus Infections - immunology; Cardiovirus Infections - metabolism; Cell Line; Cytosol - metabolism; Encephalomyocarditis virus - genetics; Encephalomyocarditis virus - immunology; Humans; Immunity, Innate; Intracellular Signaling Peptides and Proteins - metabolism; Keratinocytes - cytology; Keratinocytes - immunology; Keratinocytes - metabolism; Keratinocytes - virology; Phosphorylation; Proteomics - methods; Repressor Proteins - metabolism; RNA, Double-Stranded - immunology; RNA, Double-Stranded - metabolism; RNA, Viral - immunology; RNA, Viral - metabolism; Signal Transduction; Sirtuin 1 - metabolism
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M114.038968

Viral double-stranded RNA (dsRNA) is the most important viral structure recognized by cytosolic pattern-recognition receptors of the innate immune system, and its recognition results in the activation of signaling cascades that stimulate the production of antiviral cytokines and apoptosis of infected cells. 14-3-3 proteins are ubiquitously expressed regulatory molecules that participate in a variety of cellular processes, and 14-3-3 protein-mediated signaling pathways are activated by cytoplasmic dsRNA in human keratinocytes. However, the functional role of 14-3-3 protein-mediated interactions during viral dsRNA stimulation has remained uncharacterized. Here, we used functional proteomics to identify proteins whose phosphorylation and interaction with 14-3-3 is modulated by dsRNA and to characterize the signaling pathways activated during cytosolic dsRNA-induced innate immune response in human HaCaT keratinocytes. Phosphoproteome analysis showed that several MAPK- and immune-response-related signaling pathways were activated after dsRNA stimulation. Interactome analysis identified RelA-associated inhibitor, high-mobility group proteins, and several proteins associated with host responses to viral infection as novel 14-3-3 target proteins. Functional studies showed that RelA-associated inhibitor regulated dsRNA-induced apoptosis and TNF production. Integrated network analyses of proteomic data revealed that sirtuin1 was a central molecule regulated by 14-3-3s during dsRNA stimulation. Further experiments showed that sirtuin 1 negatively regulated dsRNA-induced NFκB transcriptional activity, suppressed expression of antiviral cytokines, and protected cells from apoptosis in dsRNA-stimulated and encephalomyocarditis-virus-infected keratinocytes. In conclusion, our data highlight the importance of 14-3-3 proteins in antiviral responses and identify RelA-associated inhibitor and sirtuin 1 as novel regulators of antiviral innate immune responses.