Fig. 4

AURKA phosphorylates NKX3.1 via direct phosphorylation at S28, 101, and 209 in vitro and in C4-2 and 22Rv1 cells. A AURKA phosphorylates NKX3.1 at S28, S101 and S209. Kinase assays were conducted as indicated in Materials and Methods. B S28, S101 and S209 are the only AURKA sites on NKX3.1, as the phospho-resistant triple mutant (3A-NKX3.1) is not phosphorylated by AURKA. The top panel is the autoradiograph. The bottom panel shows the corresponding Coomassie blue-stained gel. C AURKA phosphorylates NKX3.1 at S28, S101 and S209 in C4-2 cells. Ectopically expressed HA-tagged NKX3.1 was pulled down using HA antibody and phospho-serine levels of NKX3.1 were measured in NKX3.1-C4-2 along with 3A-NKX3.1-C4-2 cells in response to inhibition of AURKA (1 μM MLN8237, 12 h). D Quantification of p-Ser levels (relative to total NKX3.1 levels) obtained from three independent experiments. All values were normalized against wild-type NKX3.1 overexpressing cells without AURKA inhibition (**P < 0.01). E S28, S101 and S209 are the only three sites of AURKA-mediated phosphorylation of NKX3.1 in 22Rv1 cells. NKX3.1-22Rv1 and 3A-NKX3.1-22Rv1 cells were either treated with DMSO control or 1 μM AURKA inhibitor (MLN8237) for 12 h, followed by NKX3.1 immunoprecipitation using HA antibody after which phospho-Ser and NKX3.1 levels were probed using Western blot analysis. F Quantification of pSer levels of NKX3.1 in 22Rv1 cells in response to AURKA inhibition. The bar graph is representative of data obtained from three independent experiments, normalized to NKX3.1-22Rv1 cells without AURKA inhibition, (***P < 0.001)