Fig. 6: Cdk5 phosphorylates SIRT2 and thereby mediates the nuclear translocation of SIRT2.

a Liquid chromatography-mass spectrometry after a kinase assay in vitro demonstrated that the Ser331 and b Ser335 sites of SIRT2 were phosphorylated by Cdk5. c Phosphorylation levels of SIRT2 were detected in forebrain cortical tissue obtained from forebrain neuron-specific Cdk5 knockout mice and WT mice. Phosphorylation of SIRT2 was detected in primary culture neurons using immunoprecipation and western blotting with anti-phosS/TP and anti-SIRT2 antibodies. d SDS-PAGE used for the Cdk5/p35 kinase assay in vitro. Purified GST-SIRT2 WT, S331A, S335A, and S331AS335A (AA) fusion proteins were mixed with active Cdk5/p35 and ATP, and the reaction mixture was analyzed using western blotting with anti-phosS/TP and anti-GST antibodies. e HEK293 cells were transiently transfected with Flag-SIRT2 WT or Flag-SIRT2 mutation DD (double mutations at S331D and S335D) plasmids for 24 h, and subsequently, the cytoplasmic and nuclear distribution of SIRT2 (green) was detected using immunofluorescence staining. The scale bar represents 10 μm. f Immunoblotting assays for SIRT2 was performed using an anti-Flag antibody in HEK293 cells transfected with Flag-SIRT2 WT or Flag-SIRT2-DD. g Quantification of SIRT2 protein levels shown in f (n = 3). All data are presented as the means ± SD. Statistical analyses were conducted using two-way ANOVA followed by Tukey’s post hoc test in g. *P < 0.05, ***P < 0.001.