Mutant JAK3s confer cytokine independence in BaF3 cells. (A) BaF3 cells transduced with JAK3s were counted daily after IL-3 withdrawal. WT JAK3 transduced and untransduced BaF3s did not survive unless supplemented with IL-3. Those transduced with mutant JAK3s became cytokine independent by 7 days. (B) BaF3 cells expressing mutant JAK3s showed constitutive autophosphorylation and phosphorylation of STAT5 (Y694) and AKT (S473) without IL-3. All Western blot analyses were performed using whole-cell lysates except pSTAT3 and pJAK3 which were blotted from immunoprecipitates. (C) Flow cytometry histograms show Vybrant dyecycle violet staining of stable BaF3 lines. Proportion of cells in cell-cycle stages G1 or S/G2 are expressed as percentage of total cells above the peaks. Mutant JAK3-transduced BaF3 cells were maintained without IL-3. A572V, L156P, and E183G mutants showed comparable proportions of cells in S/G2 phase. R172Q is more closely comparable with WT JAK3 expressing BaF3 cells. Data shown are representative of 2 independent experiments. (D) Semi-log graph shows JAK3 protein in BaF3 cells expressing WT or mutant JAK3 after treatment with 100 μg/mL cycloheximide for 0, 0.5, 1, 2, and 3 hours. The respective half-lives were calculated from linear regression of 5 total experiments (R2 > 0.90). A572V, L156P, E183G, and R172Q are all significantly more stable than WT JAK3.
