S100A6 is a calcium binding protein expressed mainly in fibroblasts and epithelial cells. Interestingly, S100A6 is also present in extracellular fluids. Recently we have shown that S100A6 is secreted by WJMS cells and binds to integrin β1 (Jurewicz et al., 2014). In this work we describe for the first time the mechanism of S100A6 secretion and signaling pathways activated by the S100A6-integrin β1 complex. We show that colchicine suppressed the release of S100A6 into the cell medium, which indicates that the protein might be secreted via a tubulin–dependent pathway. By applying double immunogold labeling and immunofluorescence staining we have shown that S100A6 associates with microtubules in WJMS cells. Furthermore, results obtained from immunoprecipitation and proximity ligation assay (PLA), and from in vitro assays, reveal that S100A6 is able to form complexes with α and β tubulin in these cells, and that the S100A6-tubulin interaction is direct. We have also found that the S100A6 protein, due to binding to integrin β1, activates integrin-linked kinase (ILK), focal adhesion kinase (FAK) and p21-activated kinase (PAK). Our results suggest that binding of S100A6 to integrin β1 affects cell adhesion/proliferation due to activation of ILK and FAK signaling pathways.

S100A6是主要在成纤维细胞和上皮细胞中表达的钙结合蛋白。有趣的是，S100A6也存在于细胞外液中。最近，我们发现S100A6由WJMS细胞分泌，并与整联蛋白β1结合（Jurewicz等，2014）。在这项工作中，我们首次描述了S100A6整合素β1复合物激活S100A6分泌和信号传导途径的机制。我们显示秋水仙碱抑制了S100A6向细胞培养基的释放，这表明该蛋白可能是通过微管蛋白依赖性途径分泌的。通过应用双重免疫金标记和免疫荧光染色，我们已经显示S100A6与WJMS细胞中的微管结合。此外，从免疫沉淀和邻近结扎测定（PLA）以及从体外试验显示，S100A6能够与这些细胞中的α和β微管蛋白形成复合物，并且S100A6-微管蛋白之间的相互作用是直接的。我们还发现，由于与整合素β1结合，S100A6蛋白激活了整合素连接的激酶（ILK），粘着斑激酶（FAK）和p21活化的激酶（PAK）。我们的结果表明，由于ILK和FAK信号通路的激活，S100A6与整联蛋白β1的结合会影响细胞黏附/增殖。