Mutation of 2 intracellular proline residues in CD123 prevents IL-3 signaling. (A) Site-directed mutagenesis of CD123 was used to generate proline to alanine substitutions (336 PRIP 339 → ARIA). (B) Cells expressing huβc and CD123-high-WT or CD123-high-ARIA were starved of cytokine for 6 hours and then stimulated in a titration of human IL-3 for 48 hours. Proliferation was measured by the addition of 0.5 µCi of 3H-thymidine/well for the final 5 hours of stimulation. Data are represented as percentage of maximum proliferation in 100 ng/mL mouse SCF ± standard error of the mean (SEM), n = 3. (C) Cells were starved in media containing 0.5% serum for 6 hours and then stimulated ± 100 ng/mL huIL-3 for 10 minutes at 37°C. Western blotting was as previously described in Figure 1. (D) Site-directed mutagenesis of murine CD123 was used to generate proline to alanine substitutions (365 PPIP 368 → APIA). (E) WT FL cells expressing murine CD123-high-WT or CD123-high-APIA were starved of cytokine and then stimulated with a titration of murine IL-3. Proliferation was measured as previously. Data are represented as percentage of maximum proliferation of the EV line in 1000 ng/mL IL-3 ± SEM, n = 3. (F) Cells were starved and stimulated with a titration of murine IL-3 for 10 minutes at 37°C. Western blotting was as described previously. Statistical analysis: #nonsignificant difference, *P < .05, **P < .01, ***P < .001. Solid blue lines/bars, EV; hashed or solid red lines/bars, CD123-high-WT; dotted or solid green lines/horizontal bars, CD123-high-APIA.
