Phosphoproteomic Analysis of Differentially Ligated Human Neutrophils Elucidates Downstream Tyrosine Phosphorylation Events

This study is a first step towards profiling the tyrosine phosphoproteome of human neutrophils upon binding to a pathogen‐associated molecular pattern (PAMP). β‐glucans, a class of long‐chain polymers of glucose, are a major structural component of the fungal cell wall that are recognized by human neutrophils in the absence of opsonins. The neutrophil response to fungi occurs in infected tissues such that extravasated cells coincidentally receive signals from the extracellular matrix and PAMPs, such as β‐glucan. We hypothesize that differential ligation of neutrophils by fibronectin ± β‐glucan will alter cellular function and these functions will result in identifiable changes in pTyr signaling. Indeed, neutrophils adhered to fibronectin + IMPRIME‐PGG‐β‐glucan formed distinctive clusters that were not present when they adhered to fibronectin alone. The clusters were dependent on β 2 integrin CR3 and β 1 integrin VLA‐3 activity. We used a global tyrosine phosphoproteomics approach to identify phosphopeptides that were significantly and reproducibly dependent on ligand. Upon fibronectin + β‐glucan treatment, 68 phosphopeptides were upregulated and 13 were downregulated as compared to fibronectin alone. A novel role for ERK resulted from this work and extended our understanding of the mechanism underlying the finely regulated response of neutrophils to fungal recognition molecules. NIH GM066194