FIGURE 2.

Activation of JAK2-V617F by IL27Ra requires functional JAK2-V617F kinase and FERM domains. A, an empty expression vector (lanes 1–3) and an expression vector for IL27Ra (lanes 4–9) were transfected into 293T cells along with expression vectors for JAK2-V617F (VF) (lanes 1 and 5), JAK2-R683G (RG) (lanes 2 and 8), JAK2-K539L (KL) (lanes 3 and 9), wild type JAK2 (WT) (lane 4), a FERM domain mutant JAK2-V617F,Y114A (VF-YA) (lane 6), and a kinase domain mutant JAK2-V617F,K882E (VF-KE) (lane 7). Two days after transfection, cells were lysed and an equal amount of lysate protein was analyzed by immunoblotting using antibodies to detect pJAK2 (Tyr¹⁰⁰⁷/Tyr¹⁰⁰⁸), JAK2, pSTAT5 (Tyr⁶⁹⁴), STAT5, and IL27Ra, as indicated. B, BaF3 cells co-expressing either control vector (top graph) or IL27Ra (center graph), with wild type JAK2 (closed circles), JAK2-V617F (closed squares), JAK2-V617F,K882E (open circles), or JAK2-V617F,Y114A (open triangles) were washed and plated in the absence of cytokine on day 0. Total viable cells were determined by trypan blue exclusion over time. In the same experiment, BaF3 cells co-expressing a control vector with JAK2-R683G (closed circles) or IL27Ra with JAK2-R683G (closed squares) were washed of cytokine and viability was assessed over time (bottom graph). The broken lines indicate the viable cell count dropped below the limit of detection of the hemocytometer. C, cytokine-dependent BaF3 cells co-expressing either an empty vector (lanes 1, 2, and 8) or IL27Ra (lanes 3–6 and 9) with wild type JAK2 (WT) (lanes 1 and 3), JAK2-V617F (VF) (lanes 2 and 4), JAK2-V617F,K882E (lane 5), JAK2-V617F,Y114A (lane 6), or JAK2-R683G (lanes 8 and 9) were starved of cytokine for 3 h. Cells were then lysed and an equal amount of lysate protein was analyzed by immunoblotting for pJAK2 (Tyr¹⁰⁰⁷/Tyr¹⁰⁰⁸), JAK2, pSTAT5 (Tyr⁶⁹⁴), STAT5, pERK (Thr²⁰²/Tyr²⁰⁴), ERK, and IL27Ra, as indicated.