Identification and characterization of SUMO target lysine residues in WASp. (A) GPS-SUMO tool predicted SUMO-acceptor K-residues and SUMO-interaction motif (SIM) in different WASp domains (WH1, CRIB, Basic, GBD, PPP, VCA). K-residues circled in red were experimentally mutated (K>R), whereas K476 circled in green is a natural pathogenic mutation (K476E). These 6 K-residue mutants were included in SUMOylation assays. (B) Comparison of amino acid sequences of putative SUMO motifs (S*motif-1; S*motif-2) and SIM motif of WASp from yeast to human. Hydrophobic cluster SUMO motif (HCSM) preceding S*motif-1 is boxed in red. (C) Disease-causing mutations (n = ∼61 patients reported thus far) occurring in the SUMO, HCSM, and SIM motifs of WASp are shown (see supplemental Figure 3D for additional details). V75M (n = ∼22 patients) occurring in S*motif-1/HCSM that initially presents as XLT and in some patients progresses to serious disease (clinical severity grade 5 with autoimmunity and/or malignancy)⁷²  is highlighted with red lettering. (D) In vitro SUMOylation assays were performed as described in legend to Figure 2A with full-length, wild-type WASp (FL-wt), K>R single mutants, penta-SUMO-site mutant (PSM), and mock (untransfected) as substrates immunoprecipitated with anti-FLAG Ab from HeLa cells transfected with the indicated constructs. Western blot with anti-FLAG or -WASp Ab is shown. The data are representative of 2 biologic replicates shown as Gel-1 and Gel-2 to highlight subtle interassay variations in the SUMOylation patterns (see supplemental Figure 3 for WASp*SUMO construct sequences and expression by flow cytometry). (E) The indicated FLAG-tagged mutants (PSM, K>R, and disease-causing) and FL-wt were stably transfected (or mock transfected) in the normal human T_H-cell line, and the expression of the mutants on day 7 of T_H1-skewing/TCR-activation was monitored by anti-WASp and -FLAG antibodies western blot. β-Actin served as loading control. (F) Transfected mutants immunoprecipitated with anti-FLAG antibody from nuclear (nu) and cytoplasmic (cy) fractions of NEM-treated, normal T_H1-skewed/TCR-activated cells were used as substrate for the in vivo SUMOylation assays as described in the legend to Figure 2C. The same blot was sequentially reprobed with the indicated antibodies. Multiple SUMO*WASp conjugates are indicated as *S1/S2/S3 bands (molecular weight ≥ 85 kDa). The purity of nuclear and cytoplasmic fractions was monitored with Histone H3 and LAMP-1 immunoblotting, respectively. (G-H) Two-step sequential immunoprecipitation (1stIP: FLAG, 2nd IP: Myc) performed on the (G) whole cell extracts or (H) nuclear extract generated from T_H1-skewed Jurkat cells stably transfected with the indicated mutants and immunoblotted with the indicated antibodies. Molecular mass of indicated bands is in kilodaltons. NEM^+/−, treated/nontreated with NEM.