Tyrosine 201 promotes interaction of Stat5 and Shp2 with JAK2V617F. (A-B) JAK2WT, JAK2V617F, or JAK2V617F/Y201F was coexpressed with Stat5A (A) or Shp2 (B) into 293T cells. Stat5A or Shp2 was immunoprecipitated from cell lysates using antibodies against Stat5A (A) or Shp2 (B). Coprecipitated JAK2, Stat5A, and Shp2 were determined by immunoblotting using specific antibodies as indicated. (C-D) Ba/F3-EpoR cells expressing JAK2WT, JAK2V617F, or JAK2V617F/Y201F were cytokine and serum deprived before harvesting. Cell lysates were immunoprecipitated by an anti-Stat5 (C) or anti-Shp2 antibody (D) and subjected to immunoblotting with the indicated antibodies. Note that the Y201F mutation significantly inhibited the interaction between JAK2V617F and Stat5 as well as the interaction between JAK2V617F and Shp2. (E) Y201 is not required for dimerization of JAK2WT or JAK2V617F. The 293T cells were cotransfected with Myc- and HA-tagged JAK2WT or different JAK2 mutants as indicated. Myc-tagged JAK2WT or mutants were efficiently coimmunoprecipitated with HA-tagged JAK2WT or JAK2 mutants. Notably, Y201F mutation did not affect the self-association or dimerization of JAK2WT or JAK2V617F.