Regulation of IL-1 signaling through control of focal adhesion assembly

Abstract

IL-1 signaling is adhesion-restricted in many cell types, but the mechanismthat drives it is not defined. We screened for proteins recruited to nascent adhesions in IL-1-treated human fibroblasts with tandem mass tag-mass spectrometry.We used fibronectin bead preparations to enrich 10 actin-associated proteins. There was a 1.2 times log 2-fold enrichment of actin capping protein (ACP) at 30min after IL-1 stimulation.Knockdown (KD) of ACP by siRNA reduced IL-1-induced ERK activation(by 56%, matrix metalloproteinase-3 (MMP-3) expression by 48%, and MMP-9 expression by 62% (in all reductions, P < 0.01). Confocal or structured illumination microscopy showed that ACP was diffused throughout the cytosol but strongly accumulated at the ruffled border of spreading cells. ACP colocalizedwith nascent paxillin- and vinculin-containing adhesions at the ruffled border, but not with mature adhesions in the center. ACP KD promoted the formation of large, stable adhesions. Immunoprecipitation and proximity ligation analysis showed thatACPwas associatedwith the IL-1 signal transduction proteinsmyeloid differentiation factor 88 (MyD88) and IL-1 receptor-associated kinase (IRAK) at the ruffled border of the leading edge. IL-1-induced phospho-ERK andMyD88 or IRAK colocalized at the leading edge. We concluded that ACP is required for recruitment and function of IL-1 signaling complexes in nascent adhesions at the leading edge of the cell.