Phosphoproteome profiling of toll-like receptor response to different ligand stimulation in macrophages identifies differentially activated pathways and phosphorylation kinetics

Tuesday, September 23, 2014 — Poster Session III
12:00 p.m. – 2:00 p.m.	FAES Academic Center	NIAID	IMMUNO-16

* FARE Award Winner

Authors

• V Sjoelund
• M Smelkinson
• A Nita-Lazar

Abstract

Macrophages that encounter a pathogen are usually stimulated by a combination of TLR engaged by distinct ligands on the microbe. As a first step to understanding the integrated signaling under such complex conditions, we have investigated the differences in the phosphoprotein signaling cascades triggered by individual TLR4, 2 and 7 ligands. We performed a global quantitative and early post-stimulation kinetic analysis of the phosphoproteome of mouse macrophages. For each ligand-TLR pair, we found in particular marked elevation of phosphorylation of components of the cytoskeleton. Phosphorylation of elements involved in phagocytosis was only seen in response to TLR2 and 4 ligands. Changes in proteins involved in endocytosis-related signaling pathways were delayed in response to TLR2 versus TLR4 ligands. In particular, using a PKC inhibitor, we investigated the role of MARCKS, a cytoskeletal phosphoprotein that is phosphorylated in response to the TLR ligands, in macrophage migration and in TLR4 endosome trafficking. These findings reveal that the phosphoproteomic response to engagement of distinct TLRs varies both in the major targets within the cell that undergo modification and the kinetics of such changes. Our results have important implications for understanding how macrophages sense and respond to pathogens that confront the immune system.

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