SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover |
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Authors: | Michael SkalskiNamit Sharma Karla WilliamsAndrew Kruspe Marc G Coppolino |
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Institution: | Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 |
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Abstract: | Integrin signaling is central to cell growth and differentiation, and critical for the processes of apoptosis, cell migration and wound repair. Previous research has demonstrated a requirement for SNARE-dependent membrane traffic in integrin trafficking, as well as cell adhesion and migration. The goal of the present research was to ascertain whether SNARE-dependent membrane trafficking is required specifically for integrin-mediated signaling. Membrane traffic was inhibited in Chinese hamster ovary cells by expression of dominant-negative (E329Q) N-ethylmaleimide-sensitive fusion protein (NSF) or a truncated form of the SNARE SNAP23. Integrin signaling was monitored as cells were plated on fibronectin under serum-free conditions. E329Q-NSF expression inhibited phosphorylation of focal adhesion kinase (FAK) on Tyr397 at early time points of adhesion. Phosphorylation of FAK on Tyr576, Tyr861 and Tyr925 was also impaired by expression of E329Q-NSF or truncated SNAP23, as was trafficking, localization and activation of Src and its interaction with FAK. Decreased FAK-Src interaction coincided with reduced Rac activation, decreased focal adhesion turnover, reduced Akt phosphorylation and lower phosphatidylinositol 3,4,5-trisphosphate levels in the cell periphery. Over-expression of plasma membrane-targeted Src or phosphatidylinositol 3-kinase (PI3K) rescued cell spreading and focal adhesion turnover. The results suggest that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3K-dependent pathway. |
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Keywords: | CHO Chinese hamster ovary ECM extracellular matrix FN fibronectin FA focal adhesions FAK focal adhesion kinase NSF N-ethylmaleimide-sensitive fusion protein PI3K phosphatidylinositol 3-kinase PIP3 phosphatidylinositol 3 4 5-trisphosphate SNARE soluble NSF attachment protein receptor |
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