The recruitment of the interleukin-1 (IL-1) receptor-associated kinase (IRAK) into focal adhesion complexes is required for IL-1β-induced ERK activation

The interleukin-1 (IL-1) receptor colocalizes with focal adhesion complexes (FACs), actin enriched structures involved in cell adhesion and signaling in fibroblasts and chondrocytes, The colocalization of FACs and IL-1 receptors has been implicated in the restriction of IL-1 signaling transduction to ERK; however, the mechanism of this restriction and the requirement of IL-1 receptor-associated proteins have not been characterized. We determined if the association kinetics of the interleukin-1 receptor-associated kinase (IRAK) colocalizes with FACs and the requirement for IRAK in IL-1-dependent ERK activation. Human gingival fibroblasts were incubated with collagen-coated beads to induce the assembly of FACs at sites of cell-bead contact. Immunoblot analysis of bead-isolated FACs showed a time-dependent assembly of the focal adhesion proteins beta-actin, vinculin, and talin, which was blocked by the actin monomer sequestering toxin latrunculin B, Although no IRAK was isolated with FACs from unstimulated cells, phosphorylated IRAK was transiently associated with FACs isolated from IL-1 beta-stimulated fibroblasts, Fibroblasts plated on tissue culture plastic (which permitted the formation of focal adhesions) showed phosphorylation of ERK, JNK, and p38, Cells plated on poly-L-lysine (to prevent the formation of focal adhesions) showed activation only of JNK and p38. ERR activation was partially restored by incubating cells plated on poly-L-lysine with collagen-coated beads before IL-1 stimulation, Cells treated with latrunculin B or swinholide A, which caused a progressive depolymerization of actin filaments, showed a reduction or elimination of IL-1-induced ERK activation, respectively. Fibroblasts electroinjected with a mouse monoclonal anti-IRAK antibody to block the recruitment of IRAK into FACs failed to activate ERK after IL-1 treatment, indicating that FAG-associated IRAK is required for the activation of ERK. These data indicate that the integrity of actin filament arrays and the recruitment of IRAK into focal adhesions are involved in the restriction of IL-1 signaling to ERK.