GSK3α phosphorylates the S848 residue of Dyn2. (A and B) Phosphorylation of Dyn2 in response to GSK3α overexpression. HA-Dyn2^WT or HA-Dyn2^S848A were coexpressed with WT, CA, or KI forms of GSK3α in Hela cells. After precipitation with anti-HA antibody, the phosphorylated Dyn2 was detected with anti-phospho-serine antibody, normalized with HA intensity, and shown in B (n = 3 experimental replicates). (C and D) Isoform specific activity of GSK3 on Dyn2. HA-Dyn2^WT or HA-Dyn2^S848A were co-expressed with CA GSK3α or GSK3β in Hela cells. After precipitation with anti-HA antibody, the phosphorylated Dyn2 was detected with anti-phospho-serine antibody, normalized with HA intensity, and shown in D (n = 3 experimental replicates). (E and F) In vitro kinase assay. 0.8 μg purified Dyn2^WT or Dyn2^S848A were incubated with ATP in the presence or absence of 10 ng purified GSK3α. After 30-min or 1-h incubation, the phosphorylated Dyn2 was quantified by Western blotting with anti–p-Dyn2^S848 antibody. The intensity of p-Dyn2^S848 was quantified with ImageJ, normalized with Dyn2 signal and shown in F (n = 3 experimental replicates). Data are shown as average ± SD and analyzed with one-way ANOVA. *P < 0.05; **P < 0.01. Molecular weight is in kD. Source data are available for this figure: SourceData F6.