Development of a Phosphoproteomic Screen of Innate Immune Signalling: Identification and Characterisation of a Novel Phosphorylation of NFkB1/p105

Abstract

Toll-like receptors (TLRs) expressed by antigen-presenting cells of the innate immune system, such as macrophages, detect microbial products and activate signalling cascades that initiate specific gene expression programmes that define the subsequent adaptive immune response. However, it is poorly understood how TLR-specific responses arise, as many of the signalling components are common to multiple TLRs, and it is likely that as yet undiscovered phosphorylations of signalling proteins contribute to specificity of TLR pathways. TLR4, the receptor for lipopolysaccharide (LPS), is used as a model system for TLR signalling, as it activates many of the signalling mechanisms utilised by other TLRs, and I attempted to discover novel regulatory phosphorylations in LPS-activated RAW 264.7 macrophages. Because it is not yet possible to accurately predict post-translational modifications from genomic data, the exact sites of phosphorylation have to be identified experimentally, and the method of choice for this is mass spectrometry-based phosphoproteomics. Using an optimised phosphoproteomics workflow and stringent filtering criteria, I identified 445 phosphorylation sites in unstimulated and LPS-treated RAW macrophages, several of which were potential LPS-induced regulatory phosphorylations, including a previously uncharacterised phosphorylation of the NF-κB protein p105, a key regulator of TLR and other inflammatory signalling pathways. Following validation of a phospho-specific p105 antibody, I demonstrated that the novel phosphorylation of p105 was induced by LPS, other TLR ligands, tumour necrosis factor (TNF)-α, and prostaglandin E2 in RAW macrophages and primary human macrophages, and by interleukin (IL)-1β in 4 primary human skin fibroblasts. Moreover, the LPS-induced phosphorylation of p105 was blocked by the protein kinase A (PKA) inhibitor H89. These results indicate that the novel phosphorylation of p105 may be important for the regulation of TLR and other inflammatory signalling pathways in both hemopoietic and non-hemopoietic cells.