Molecular & Cellular Proteomics : Mcp
Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria which induces strong pro-inflammatory reactions of mammals. These processes are triggered upon sequential binding of LPS to CD14, a GPI-linked plasma membrane raft protein, and to the TLR4/MD2 receptor complex. We have found earlier that upon LPS binding CD14 triggers generation of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a lipid controlling subsequent pro-inflammatory cytokine production. Here we show that stimulation of RAW264 macrophage-like cells with LPS induces global changes of the level of fatty-acylated, most likely palmitoylated, proteins. Among the acylated proteins which were upregulated in those conditions were several enzymes of the phosphatidylinositol cycle. Global profiling of acylated proteins was performed by metabolic labeling of RAW264 cells with 17ODYA, an analogue of palmitic acid functionalized with an alkyne group, followed by detection and enrichment of labeled proteins using biotin-azide/streptavidin and their identification with mass spectrometry. This proteomic approach revealed that 154 fatty-acylated proteins were upregulated, 186 downregulated and 306 not affected in cells stimulated with 100 ng/ml LPS for 60 min. The acylated proteins affected by LPS were involved in diverse biological functions, as found by Ingenuity Pathway Analysis. Detailed studies of 17ODYA-labeled and immunoprecipitated proteins revealed that LPS induces S-palmitoylation, hence activation, of type II phosphatidylinositol 4-kinase (PI4KII) , which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate, a PI(4,5)P2 precursor. Silencing of PI4KII and PI4KII inhibited LPS-induced expression and production of pro-inflammatory cytokines, especially in the TRIF-dependent signaling pathway of TLR4. Reciprocally, this LPS-induced signaling pathway was significantly enhanced after overexpression of PI4KII or PI4KII; this was dependent on palmitoylation of the kinases. However, the S-palmitoylation of PI4KII, hence its activity, was constitutive in RAW264 cells. Taken together the data indicate that LPS triggers S-palmitoylation and activation of PI4KII, which generates PI(4)P involved in signaling pathways controlling production of pro-inflammatory cytokines.