CASK is modified by SUMO1 at residue K679. (A) A schematic diagram of CASK, CASK deletion mutants, and SUMO1-CASK fusions. Some of the constructs had two versions: one was Myc tagged at the N-terminal end, and the other had no extra tag. (B) COS1 cells were cotransfected with plasmids expressing 1 μg Myc-tagged SUMO1, 2, or 3 and 1 μg CASK. After 24-h incubation, total cell extracts were harvested and immunoprecipitated with CASK antibody. Immunoblotting analysis was then performed sequentially using CASK antibody to monitor CASK proteins and Myc tag antibody to detect Myc-tagged SUMOs. (C) 1.2 μg CASK deletion mutants was cotransfected with 0.4 μg SUMO1 into COS1 cells to evaluate the SUMO1-modified region of CASK by immunoprecipitation-immunoblotting analysis as described in B except that Myc tag antibody was used first for immunoblotting. The arrowhead points to the position of endogenous full-length CASK in COS1 cells. Arrows indicate the positions of SUMO1-CASK deletion mutants. (D) Two putative SUMO1-modified consensus motifs, guanylate kinase⁶⁴⁸LE and TK⁶⁷⁹QE, were predicted in the sequence of rat CASK, and site-directed mutagenesis was performed to change lysine 648 and 679 to arginine. COS1 cells were cotransfected with 0.5 μg SUMO1 and 0.5 μg of wild-type CASK, K648R, or K679R mutant and subjected to immunoprecipitation-immunoblotting analysis. (B and D) Arrows indicate the positions of SUMOylated CASK; arrowheads point to the positions of unmodified full-length CASK.