Tyrosine 201 is required for constitutive activation of JAK2V617F and its downstream signaling. (A-C) Ba/F3-EpoR cells expressing JAK2WT, JAK2V617F, and JAK2V617F/Y201F were deprived of cytokine and serum for 6 hours. Tyrosyl phosphorylation of JAK2 (A), Stat5 (B), and Shp2 (C) was detected by immunoprecipitation with specific antibodies against JAK2, Stat5, and Shp2, followed by immunoblotting with phosphotyrosine antibody (4G10). Membranes were reprobed with total antibodies. Note that constitutive tyrosine phosphorylation of JAK2, Stat5, and Shp2 induced by JAK2V617F was significantly inhibited by the Y201F mutation. (D) Ba/F3-EpoR cells expressing JAK2WT, JAK2V617F, and JAK2V617F/Y201F were cytokine and serum-starved followed by stimulation with Epo (3 U/mL) for the indicated times. Cell lysates were prepared and directly immunoblotted with phospho-specific antibodies or total antibodies as indicated. (E) Histograms demonstrate the fold changes in phosphorylation of JAK2, Stat5, Shp2, p70S6K, Akt, and Erk2 compared with the phosphorylation levels in cells expressing JAK2WT. All the data are normalized for the JAK2WT value at 15 minutes, which is set to 1. Data from 3 independent experiments are shown as mean ± SEM. *P < .05 (1-way ANOVA). **P < .005 (1-way ANOVA). Notably, Y201F mutation markedly inhibited the activation of JAK2V617F and downstream signaling pathways, including Stat5, Shp2, p70S6 kinase, Akt, and Erk.