Fig 1: CAMKIIδ phosphorylates and destabilizes FOXO3A. A) Identification of FOXO3A S7 and S12 phosphorylation by CAMKIIδ from proteomic analysis. The mass shift of phosphorylation is 79.9663. Determination of S7: The theoretical molecular weight of SPA's three amino acid residues is 255.12. The calculation (b9−b6 = 1135.61−800.4249 = 355.185) matches the theoretical shift for SPA plus phosphorylation, calculated as (255.12+79.9663 = 355.086). Thus, it can be determined that the serine residue in SPA underwent phosphorylation modification. Determination of S12: The theoretical molecular weight of the peptide plus the charge is 2909.4. The actual mass shift across the entire peptide was calculated as (m/z × charge = 1023.118 × 3 = 3069.354), suggesting there are two phosphorylation sites on this peptide (2909.4+79.9663 × 2 = 3069.33). Furthermore, the y2−y1 fragment analysis indicates that S26 did not undergo phosphorylation modification. Since S7 was confirmed to be phosphorylated, it can be inferred that S12 is another phosphorylation site. B) HEK293 cells transfected with 3xFLAG‐FOXO3A, and CAMK2D‐HA or CAMK2G‐HA plasmids were co‐immunoprecipitated with HA‐beads; protein levels of 3xFLAG‐FOXO3A were analyzed by western blot. C) (left) In vitro kinase assay of CAMKIIδ on FOXO3A wild‐type and site mutants. HEK293 cells were transfected with 3xFLAG‐FOXO3A wild‐type, 3xFLAG‐FOXO3A S7A, S12A, and S7/12A plasmids. Proteins were then pulled down by FLAG beads, followed by an in vitro kinase assay supplied with Ca2+, calmodulin, and ATP. Total phosphorylated serine and threonine levels were detected by western blots. (right) In vitro kinase assay of CAMKIIδ and CAMKIIγ on FOXO3A protein. D) Radioisotope‐based in vitro kinase assay was performed on purified FOXO3A protein using 4 nM CAMKIIγ and 0.1 nM CAMKIIδ. E) Mutu CAMK2D wild‐type and knockout cells were treated with cycloheximide (CHX) for the indicated times. FOXO3A protein levels were then analyzed by western blot with Hsp90 as the loading control (left panel). Protein levels were measured with densitometric intensity. FOXO3A levels were quantified relative to Hsp90 levels and graphed as the percentage of remaining FOXO3A protein after treatment (right panel). F) Wild‐type BCL cells were treated with MG132, chloroquine, or DMSO control for indicated times, and FOXO3A expression was determined by western blot. GAPDH was used as a loading control. G) Venn diagram showing identified potential ubiquitylation regulator for FOXO3A by overlapping the MS results and online prediction. H) HEK293 cells transfected with vector control, WT or S7&12 mutant 3xFLAG‐FOXO3A were co‐immunoprecipitated with FLAG‐beads; protein levels of USP7 were analyzed by western blot. I) HEK293 cells overexpressing FLAG‐FOXO3A were transfected with HA‐ubiquitin and treated with 0.5µM USP7 inhibitor for 24 h. FOXO3A immunoprecipitated with FLAG beads, and HA‐ubiquitin was examined by western blot. All the results shown here, except panel A and G, are representative of three independent experiments. T‐test was used for analysis, unless otherwise indicated.