GSK3α phosphorylates the S848 residue of Dyn2. (A) Analysis of potential kinases for Dyn2^S848. Screenshot of the prediction results for Dyn2^S848 kinases using Scansite motif scans (https://scansite4.mit.edu/#scanProtein). Lower score indicates better match of protein sequence to the optimal binding pattern. (B) Analysis of Dyn2 phosphorylation under GSK3α overexpression. HA-Dyn2^WT or HA-Dyn2^S848A were coexpressed with GSK3α CA or KI in Hela cells. After precipitated with anti-HA antibody, the phosphorylated Dyn2 was detected with Dyn2 phospho-S848-specific (p-S848) antibodies. (C) MS analysis of Dyn2 phosphorylation by GSK3α. Recombinant Dyn2 was incubated with GSK3α and ATP in kinase assay buffer for 1 h. The reaction was then analyzed for phosphorylation with LC-MS/MS. The 838-IPPGIPPGVPpSR phosphopeptide containing phosphorylated Ser848 was identified. The MS/MS spectrum of this phosphopeptide is shown. The m/z of (3+) charged phosphopeptide is 422.89 with mass accuracy of <5 ppm. The mass difference between the y1 and y2 ions is consistent with phosphorylation at S848. Of note, the MS/MS spectrum is interfered by another peptide from Dyn2 (258-FFLSHPAYRH) with identical mass to our targeted phosphopeptide containing pS848. (D) The expression of HA-tagged GSKα or -β in lentiviral-infected L6 myoblasts was examined by Western blotting using anti-HA antibody. Molecular weight is in kD. Source data are available for this figure: SourceData FS4.