Abstract
Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the Km for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or Km for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.
Highlights
Most cytokine receptors lack intrinsic kinase activity and rely on Janus kinases (JAKs) for signaling [1,2]
Protein purification and substrate identification HIS-tagged JH1-JH2WT, JH1-JH2V617F and JH1 domains from Janus kinase 2 (JAK2) (Figure 1A) were produced in Sf9 cells using a Bac-toBac expression system and were purified using a two-step purification protocol
After the discovery of the JAK2 V617F mutation in myeloproliferative neoplasms (MPN) patients, several other mutations in the JH2 domain of JAKs have been identified in MPN, and in lymphoid and myeloid leukemia and multiple myeloma patients [14,39]
Summary
Most cytokine receptors lack intrinsic kinase activity and rely on Janus kinases (JAKs) for signaling [1,2]. The N-terminal segment (JH7-JH3 domains) contains a FERM (band 4.1 ezrin, radixin and moesin) domain as well as an atypical SH2 (Srchomology-2 domain)-like domain which have been shown to mediate association with the membrane-proximal region of cytokine receptors [1]. The JH2 domain, located between the SH2-like and the JH1 domains, has been predicted to be catalytically inactive due to the lack of essential amino-acids in the catalytic consensus motifs of kinases and has been classified as a pseudokinase domain [3]. The JH2 domain has been shown to have an important regulatory role in JAK activation [4,5]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call