JAK2^ex13InDel and JAK2^V617F use similar mechanisms of constitutive kinase activation. (A) Residues L583-A586 are located in the N-lobe of the pseudokinase domain of JAK2 (JH2) that faces the catalytically active kinase domain (JH1) (top left). L583-A586 forms the loop between the N-terminal JH2 αC and the β3 strand in JAK2^WT (top right, light blue). This loop is structurally close to V617 and the JH2 αC, which is altered in the presence of the V617F mutation. Compared with the loose conformation of JH2 αC in JAK2^WT (bottom right), the JH2 αC of JAK2^V617F exhibits a rigid helix (bottom right). A strikingly similar conformational change in JH2 αC is observed in the JAK2^ex13InDel mutant (bottom left). (B) To demonstrate that E596 is also critical for JAK2^ex13InDel activity, we cultured Ba/F3 cells expressing JAK2^E596R, JAK2^E596R/V617F, and JAK2^{ex13InDel/E596R} with or without IL-3. JAK2^{ex13InDel/E596R} did not demonstrate IL-3–independent growth (n = 3).