Fig. 6

AURKA-mediated phosphorylation of ALDH1A1 modulates its oligomeric state and dehydrogenase activity. a Monomeric ALDH1A1 and three AURKA phosphorylation sites (4WB9). T267 is within the NAD⁺ binding pocket, T442 is within the catalytic site, and T493 is within the oligomerization domain. b ALDH1A1-phosphorylation-resistant mutants have diminished enzymatic activity. c Phosphorylation of ALDH1A1 by AURKA triggers ALDH1A1 oligomers to dissociate to the monomeric form. The same membrane is shown as a short (top) and long (bottom) exposure for clarity. d Average relative changes in tetramer and monomer abundance derived from three independent experiments. The intensities of tetramer and monomer at each time were normalized independently against time 0. Quantification of tetramer and monomer intensities was derived from the short exposure (**p < 0.01 monomer at 0 and 30 min) from three independent experiments. e Average ratio of normalized monomer to tetramer as a function of time. To account for the large difference in tetramer and monomer abundance, the intensity of each band was normalized against the tetramer intensity at time zero and the ratio of monomer to tetramer was plotted as a function of time. After 30 min, a significant increase in the monomer to tetramer was observed (***p < 0.01 at 0 and 30 min). f Activity staining of phosphorylation-induced ALDH1A1 monomer harbors high dehydrogenase activity. Purple color is used to denote it as activity-based staining. g Coomassie G-250 stain of panel f. h Quantification of oligomer-specific ALDH1A1 activities from three independent experiments (***p < 0.001 at 0 and 30 min) analyzed by two-way analysis of variance. Specifically, monomer and tetramer intensities of activity and Coomassie stain at 30 min of phosphorylation were normalized against the unphosphorylated tetramer control (basal activity), then the normalized activity of each oligomer was divided by its respective normalized Coomassie signal to generate the plot shown. i Phos-tag staining of phosphorylated ALDH1A1. j Coomassie G-250 stain of panel i. k Quantification of normalized Phos-tag intensities with respect to total protein levels from three independent experiments. Protein quantification was carried out in the same way as described for the activity assay (***p < 0.001 for monomer at 0 and 10 min and 0 and 30 min analyzed by two-way analysis of variance). The red color denotes it as a phosphorylation-specific signal. Data used to generate the summary statistics shown in panels d, e, h, and k are reported in Additional file 5