Mutations in LCP1 and WNK1 increase cell fitness and favor cell growth and survival. (A) Schema of the nonsense mutation in LCP1 found in CLL005. (B) LCP1-mutant cells have altered DNA response upon gamma-irradiation. HEK293 cells stably expressing wild-type, mutant, or control constructs were treated with 10 Gy. Levels of gamma-H2AX and phosphorylated forms of ATM, ATR, DNA-PK, and GAPDH were assessed using immunoblot. (C) Both wild-type and mutant LCP1 physically interact with ATM protein. HEK293 cells stably expressing wild-type, mutant, or control constructs were transiently transfected with an ATM-expressing construct (with N-terminal His tag). Forty-eight hours after transfection, cells were either treated or not treated with irradiation. Immunoprecipitations were performed on protein lysates using anti-His beads or anti-GFP as well as isotype control antibodies followed by immunoblot against GFP or ATM protein. Shown are immunoprecipitates from untreated cells. Irradiation does not change the physical association between these proteins (Supplemental Fig. S8). (D) Cells with mutant LCP1 have greater overall survival after gamma-irradiation compared with cells with wild-type LCP1 with or without ATM inhibitor treatment. The survival rate was calculated by normalization to each group of nonirradiated cells using colony assays performed on six-well plates. Mean ± SD; n = 3. (E) Crystal structure of WNK1 kinase domain (PDB entry 4Q2A). Val403 is located underneath the activation loop of WNK1. Mutation of Val403 to Phe potentially disrupts activation loop folding and affects kinase activity. (F) Cells with mutant WNK1 demonstrate a faster progression from G1 to S phase. HEK293 cells were induced to express either wild-type or mutant WNK1 for 48 h. After synchronization by serum deprivation for 24 h, cell cycle was assessed by EdU assay 24 h after return to complete media. The mean percentage of cells (±SD; n = 3) in different phases of the cell cycle is shown.
