DBNLS232 regulates formation of filopodia and promotes cell migration in A549 cells

Abstract

Objective To investigate the role of site-specific phosphorylation of adaptor protein Drebrin-like (DBNL) in regulating the formation of filopodia and the migration and movement of lung cancer A549 cells. Methods DBNL was stably knocked down by shRNA in A549 cells, and then its effects on migration and movement of lung cancer cells were analyzed. Online proteome database, site-directed mutagenesis and indirect immunofluorescence assay were used integratively to analyze the effect of wild type DBNL and 3 mutant types (S146E and T165E, S232E and S232A) on F-actin arrangement, formation of filopodia and migration and movement in A549 cells. Co-immunoprecipitation assay was applied to confirm phosphorylation of DBNL S232. Results DBNL knockdown significantly inhibited the migration and movement of A549 cells (P 0.05), however, the phosphorylation of DBNL S146, T165 and S232 were predominantly increased in lung cancer tissue when compared to adjacent tissue (P < 0.001). In addition, indirect-immunofluorescence assay indicated that DBNL S232E which was enriched around the membrane that mimic phosphorylation improved the formation of filopodia and the movement of cells (P < 0.001), but not DBNL wild type and other mutations (T165E, S232E). Furthermore, unphosphorylated mutation DBNL S232A obviously suppressed the formation of filopodia (P < 0.01). Finally, prediction for phosphorylation on online found that p38 and CDK5 kinases could potentially phosphorylate DBNL S232. Conclusion Phosphorylation of DBNL S232 in lung cancer A549 cells promotes the formation of filopodia and enhances the movement of lung cancer cells.

Keywords

• lung cancer
• dbnl
• phosphorylation
• cell movement
• filopodia