Figure 1
From: hnRNPK S379 phosphorylation participates in migration regulation of triple negative MDA-MB-231 cells
Aurora-A phosphorylates hnRNPK at Serine 379 and its kinase activity is correlated with breast cancer cell migration. (a) Detection of in vitro hnRNPK S379 phosphorylation mediated by Aurora-A kinase. Recombinant GST-hnRNPK proteins were incubated with a commercial Aurora-A kinase in the presence or absence of ATP at 37 °C for 16 h. Levels of hnRNPK S379 phosphorylation were determined by Western blot analysis using S379 phosphorylation-specific polyclonal antibodies. (b) Detection of in vivo hnRNPK S379 phosphorylation in HEK293 cells upon amplification of Aurora-A. HEK293 cells were transfected with Flag-hnRNPK and synchronized at G2/M phase using nocodazole to amplify Aurora-A. Subsequently, hnRNPK was immunoprecipitated using Flag antibody and the protein analyzed to measure its S379 phosphorylation level. (c) Aurora-A kinase activity is correlated with the migration of MDA-MB-231 cells. A total of 3 × 104 MDA-MB-231 cells were seeded into Transwells for the migration assay in the presence of DMSO only, 1 μM, 5 μM and 10 μM of Aurora-A inhibitor (AAI). After 20 h, the migrated cells were fixed using 10% formaldehyde and stained with crystal violet for imaging analysis. (d) Quantification of suppression of migration of MDA-MB-231 cells upon inhibition of Aurora-A kinase. Counts of migrated cells per field in triplicate experiments were quantified in bar graphs with standard deviation. Data is shown as mean ± SD, and * indicates significant difference compared to wild type (*p < 0.05, n = 4). The full blot images are shown in Supplementary Fig. S5.
