Fig. 3

AURKA stabilizes ALDH1A1 protein levels and increases its enzymatic activity. a AURKA prevents ALDH1A1 degradation. AURKA-BxPC3 and BxPC3 cells were treated with cycloheximide for 2 and 4 h, and AURKA and ALDH1A1 levels analyzed. b Graphical representation of AURKA degradation rate. The results of densitometric scanning are shown graphically with AURKA signal normalized to actin signal. AURKA half-life is ~2 h. The significance of the difference between means was determined by Duncan’s multiple range test, *p < 0.05. c Graphical representation of ALDH1A1 degradation rate. ALDH1A1 half-life is ~2 h, *p < 0.05. d AURKA stabilizes ALDH1A1 by inhibiting its ubiquitylation. Each experiment was done at least three independent times. Representative data are shown. e, f AURKA increases ALDH1A1 enzymatic activity. Comparative spectrophotometric analysis of ALDH1A1 activity upon phosphorylation by AURKA. ALDH1A1 activity with and without ATP and AURKA were used as controls. The increase in ALDH1A1 activity observed in the presence of ATP can be overcome by stringent purification of ALDH1A1 (compare e and f). g Stoichiometry of ALDH1A1 phosphorylation by AURKA after 1.5 and 6 h. h, i Coupling the change in ALDH1A1 activity with stoichiometry of ALDH1A1 phosphorylation. Two identical reaction mixtures were treated with cold ATP to measure ALDH1A1 activity (h, i), or with cold ATP spiked with 0.01 μCi of [³²P]ATP (g). After 1.5 and 6 h kinase reactions, ³²P incorporation (g) and ALDH1A1 activity (h, i) were measured. j Dephosphorylation of ALDH1A1 by calf-intestinal alkaline phosphatase (CIP) decreases its dehydrogenase activity. Each experiment was done at least three independent times. Representative data are shown