The Department of Biomedicine

BBB seminar: Christopher A. McCulloch

Use of tandem mass tag spectrometry for identifying novel molecules in IL-1 signal transduction

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Christopher A. McCulloch
Matrix Dynamics Group, University of Toronto, ON, Canada

Interleukin-1 (IL-1) is an important inflammatory mediator and modulates fibroblast metabolism to promote matrix degradation. Previous studies showed that IL-1-induced calcium flux and mitogen-activated protein kinase signaling are dependent on focal adhesion formation, suggesting a matrix-dependent restriction system for IL-1 signaling. However, the mechanism by which focal adhesions restrict IL-1 signaling to mitogen-activated protein kinases such as ERK is not well-defined. In human gingival fibroblasts we screened for IL-1-stimulated, focal adhesion-associated proteins, which were isolated with fibronectin-coated beads and detected by tandem mass tag (TMT) mass spectrometry. Notably, we found that the abundance of F-actin capping protein in focal adhesion-associated proteins was enhanced after treatment with IL-1 in time-course experiments. Cells treated with siRNA to knock down capping protein showed marked reductions of IL-1-induced ERK activation and MMP-3/MMP-9 expression. By immunostaining and imaging with structured illumination microscopy, capping protein was distributed throughout the cytosol and accumulated at the leading edge. Capping protein co-localized with nascent paxillin and vinculin-immunostained adhesions at the leading edge, but not with mature focal adhesions. Knock down of capping protein perturbed focal adhesion turnover as indicated by the formation of large adhesions. After IL-1 stimulation, capping protein spatially associated with the IL-1 signaling transduction proteins MyD88 and IRAK-1, and phosphorylated ERK, especially at the leading cell edge. These data indicate that tandem mass spectrometry can help to identify unexpected regulatory proteins involved in inflammatory signaling. In particular, capping protein may play an important role in recruiting IL-1 signaling complexes to nascent focal adhesions and there restrict IL-1 signal transduction at the leading edge of the cell.

Chairperson: Donald Gullberg, Department of Biomedicine