Constitutive signaling of TpoR induced by CALR mutants is JAK2-dependent. JAK2 requirement for STAT5 transcriptional activation was tested by luciferase assay in JAK2-deficient cell, γ2A, transfected with CALR wild-type or mutants, STAT5, and TpoR. (A) CALR mutants were unable to induce STAT5 activation downstream of TpoR in the absence of JAK2. (B) This observation was confirmed by luciferase assay with the TpoR box 1 dead mutant deficient in JAK2 binding. (C) Schematic representation of Gaussia princeps luciferase complementation assay used to test JAK2 dimerization in HEK293-derived BOSC cells. (D) Close proximity between the C-terminal kinase domains of JAK2 activation is induced by CALR del52 and ins5 only in the presence of TpoR. Values shown represent the average of 3 pooled independent experiments, each performed with 3 biological replicates ± standard error of the mean (SEM). Statistical analysis (jmp pro11) was performed by the nonparametric multiple comparisons Steel test with a control group; *P < .05, **P < .01, ***P < .001. (E) Immunoprecipitation using anti-HA antibody from lysates of HEK293 cells transiently transfected with respective CALR (wild-type or del52) and HA-TpoR constructs, followed by western blotting with the indicated antibodies. Phosphorylated form of JAK2 interacted with TpoR only in the presence of CALR del52 but not wild-type. (F) JAK2 (Y1007/1008), STAT3 (Y705), STAT5 (Y694), TpoR (Y626), ERK1/2, (T202/Y204) and Akt (T308) activation by CALR del52 in Ba/F3 cells in the absence of Tpo. The condition +Tpo for CALR wild-type probed with pY-TpoR, pY-ERK1/2, pT-AKT, and β-actin was run in the same gel, but before CALR del52 minus Tpo. It had been displaced for symmetry.