Fig. 3.

Cdk5 directly phosphorylates ALDH1A1 at S75 and S274. a The Cdk5-p25 complex (Cdk5/p25) phosphorylates ALDH1A1 at S75 and S274. Recombinant 6× His-tagged wild-type and ALDH1A1 mutants were subjected to a kinase assay with Cdk5-p25. b S75 and S274 are the only Cdk5 sites on ALDH1A1, as the 2A-ALDH1A1 mutant is not phosphorylated by Cdk5. c Glutamate stimulates nuclear translocation of Cdk5 and ALDH1A1 in ALDH1A1-HT22 cells. ALDH1A1-HT22 cells were treated with glutamate for 0–12 h with or without roscovitine, followed by immunostaining with anti-HA and Cdk5 antibody. Representative pictures are shown. d Quantification of the subcellular localization of ALDH1A1 in the cell nucleus versus the cytoplasm. The graphs show the mean ± SEM of the relative fluorescence intensity with respect to control cells. *p < 0.05 versus nucleus fraction control analyzed by two-way analysis of variance. e Subcellular fractionation of ALDH1A1 in glutamate-treated ALDH1A1-HT22 cells in the absence or presence of roscovitine. Alpha-tubulin is the cytoplasmic marker and lamin A is the nuclear marker. N nuclear fraction, C cytoplasmic fraction. f Cdk5 knockdown inhibits nuclear translocation of ALDH1A1. Cdk5-shRNA-infected ALDH1A1-HT22 cells were treated with glutamate. Representative pictures are shown. g Quantification of the subcellular localization of ALDH1A1 in the cell nucleus versus the cytoplasm. The graphs show the mean ± SEM of the relative fluorescence intensity with respect to control cells