Fig. 5
AURKA-mediated phosphorylation of ALDH1A1 at T267, T442, and T493 regulates its protein stability and subcellular localization. a AURKA phosphorylates ALDH1A1 at T267, T442, and T493. Phospho-dead single mutants of ALDH1A1 were subjected to an in vitro kinase assay using AURKA/TPX2 and [32P]ATP. b T267, T442, and T493 are the only AURKA sites on ALDH1A1, as the 3A-ALDH1A1 mutant is not phosphorylated by AURKA. c AURKA regulates the subcellular localization of ALDH1A1 via phosphorylation. HA-tagged wild-type and 3A-ALDH1A1-expressing stable cells were fixed and immunostained with DAPI (blue) and antibody against HA (green). More than 100 cells were analyzed from multiple random frames. Representative data are shown. d Double staining of total ALDH1A1 and ectopically expressed ALDH1A1 in wild-type and 3A-expressing BxPC3 cells. e AURKA regulates the subcellular localization of ALDH1A1 via phosphorylation of all three sites. More than 100 cells were analyzed from multiple random frames. Representative data are shown. f AURKA promotes ALDH1A1 levels by phosphorylation. BxPC3 cells were infected with HA-tagged wild-type ALDH1A1 or 3A-ALDH1A1 retrovirus. After 30 h, protein levels were analyzed using AURKA, HA, and actin antibodies. g AURKA inhibits ALDH1A1 ubiquitylation by phosphorylating T267, T442, and T493. h AURKA increases ALDH1A1 levels by phosphorylation at T267, T442, and T493. i Graphical representation of ALDH1A1 levels in BxPC3 cells expressing either wild-type or mutant ALDH1A1. Average values of wild-type and mutant ALDH1A1 levels from three independent experiments are depicted in the graph. **p < 0.01, compared to control analyzed by two-way analysis of variance. j AURKA inhibits ALDH1A1 ubiquitylation by phosphorylating T267, T442, and T493 sites. BxPC3 cells expressing either wild-type ALDH1A1, T267A-ALDH1A1, T442A-ALDH1A1, T493A-ALDH1A1, or 3A-ALDH1A1 were co-infected with AURKA shRNA along with 6x-His-ubiquitin. Ubiquitylated proteins were isolated and analyzed as described in Methods. Each experiment was done at least three independent times. Representative data are shown