Figure 1.

Aurora Kinase-A (AURKA) associates and directly phosphorylates Y-box binding protein-1 (YBX1) at two sites and regulates its localization in C4-2 and 22Rv1 cells. (A) YBX1 is a substrate of AURKA in vitro. AURKA-TPX2 was mixed with either [³²P]ATP (lane 1), or 6x-His–YBX1 and [³²P]ATP (lane 2) in a kinase assay for 30 min. Lane 3 shows YBX1 mixed with [³²P]ATP. (B) YBX1 shows comparable phosphorylation levels as ALDH1A1, a known AURKA substrate. YBX1 and ALDH1A1 were subjected to kinase assay with AURKA and [³²P]ATP (lanes 2 and 3, respectively). Lanes 1 and 4 are controls with YBX1 and ALDH1A1, respectively, with [³²P]ATP. Lane 5 shows AURKA with [³²P]ATP. (C) AURKA phosphorylates YBX1 at T62 and S102. Phospho-resistant YBX1 single mutants were exposed to an in vitro kinase assay using AURKA-TPX2 and [³²P] ATP (lanes 3 and 4, respectively). WT YBX1 with [³²P] ATP was used as a positive control (lane 2). Lanes 5, 6 and 7 show WT, T62 and S102 mutants exposed to [³²P] ATP, respectively in the absence of AURKA. (D) Histogram shows % change in phosphorylation of WT and phospho-dead single mutants of YBX1 from three independent experiments. (E) T62 and S102 are the only AURKA sites on YBX1, as 2A-YBX1 mutant is not phosphorylated by AURKA (lane 3). Lane 2 shows WT YBX1 with AURKA and [³²P] ATP. (F) YBX1 associates with AURKA in cells. YBX1 IP was conducted in C4-2 cells, and AURKA levels analyzed (lane 3). IgG (lane 1) and AURKA (lane 2) were used as negative and positive controls, respectively. (G) AURKA binds YBX1 in C4-2 cells. AURKA IP was conducted in C4-2 cells, and YBX1 levels analyzed (lane 2). IgG (lane 1) and YBX1 (lane 3) were used as negative and positive controls, respectively. (H) AURKA inhibition using MLN8237 inhibits nuclear translocation of YBX1 in C4-2 cells. C4-2 cells were treated with 1 μM MLN8237 for 12 h, and AURKA subcellular localization analyzed using AURKA-specific antibody (green). DAPI is shown in blue. (I) AURKA depletion inhibits nuclear translocation of YBX1. C4-2 cells were exposed to control or AURKA shRNA for 30h, fixed and stained with YBX1 antibody (green) or DAPI (blue). >100 cells were analyzed from multiple random frames. (J) Subcellular fractionation of scrambled shRNA-treated and AURKA shRNA-treated C4-2 cells confirms reduced nuclear translocation of YBX1 upon AURKA depletion. C4-2 cells were exposed to control or AURKA shRNA for 30 h prior to fractionation. (K) AURKA inhibition using MLN8237 prevents nuclear translocation of YBX1 in 22Rv1 cells. 22Rv1 cells were treated with 1 μM MLN8237 for 12 h, and AURKA subcellular localization analyzed using AURKA-specific antibody (green). (L) AURKA depletion inhibits nuclear translocation of YBX1 in 22Rv1 cells. 22Rv1 cells were exposed to control or AURKA shRNA for 30 h, fixed and stained with YBX1 antibody (green) or DAPI (blue). (M) Subcellular fractionation confirms reduced nuclear translocation of YBX1 upon AURKA depletion in 22Rv1 cells. 22Rv1 cells were exposed to control or AURKA shRNA for 30 h prior to fractionation.