The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains

• Kinetic analysis of recombinant JAK2 proteins revealed a random Bi–Bi mechanism.
• The JH2 domain reduced Vmax 5–20 fold in JH1JH2WT and JH1JH2V617F.
• The JH2 domain increased Ka (ATP) 4–10 fold for the 10 substrate peptides tested.
• The V617F mutation increased Vmax 4–5 fold.
• The SH2–JH2 linker increased inhibition by JH2 and reduced the affinity for ATP.

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi–Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2–JH2 linker region participates in controlling activity by reducing the affinity for ATP.