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Temporal phosphorylation dynamics analysis of TLR stimulation: role of MARCKS in TLR signaling

Friday, November 08, 2013 — Poster Session IV

2:00 p.m. – 4:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • A Nita-Lazar
  • I Fraser


The Toll-like receptors (TLRs) are a family of pathogen recognition receptors that alert the host to the presence of pathogens. Cytokines are among the most important genes to be regulated by TLR signaling, and given their role in the orchestration of the inflammatory response, mechanisms of modulating their production have garnered a lot of interest. We investigated the changes in the phosphoproteome post activation of TLR4, TLR2/1 and TLR7 in C57 macrophages at 5 different time points using triple SILAC. On average, we identified 500 reproducible phosphosites per receptor. We found changes in the kinetics of response in regards to endocytosis. Functional bioinformatics analysis identified networks involved in actin cytoskeleton signaling, RhoGTPase signaling and phospholipase C signaling, amongst others that were upregulated post stimulation of the macrophages. We found 24 phosphosites that were statistically differentially phosphorylated at a given time point between 2 different TLRs. A third of these proteins are involved in the cytoskeleton, including MARCKS. The phosphorylation state of MARCKS was found to play a role in the ability of the macrophages to migrate post-LPS stimulation. We also found that MARCKS was transiently colocalized with the early endosome post LPS stimulation.

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