Analysis of jak2 catalytic function by peptide microarrays: The role of the JH2 domain and V617F mutation

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 Kmfor 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 Kmfor 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

Analysis of Janus Tyrosine Kinase Phosphorylation and Activation

Activation of Janus kinases (Jaks) occurs through autophosphorylation of key tyrosine residues located primarily within their catalytic domain. Phosphorylation of these tyrosine residues facilitates access of substrates to the active site and serves as an intrinsic indicator of Jak activation. Here, we describe the methods and strategies used for analyzing Jak phosphorylation and activation. Tyrosine-phosphorylated (active) Jaks are primarily detected from cell extracts using anti-phosphotyrosine-directed Western blot analysis of Jak-specific immunoprecipitates. Additionally, receptor pull-down and in vitro kinase assays can also be utilized to measure cellular Jak catalytic activity. In addition to tyrosine phosphorylation, recent evidence indicates Jaks can be serine phosphorylated upon cytokine stimulation, however the lack of commercially available antibodies to detect these sites has hindered their analysis by Western blot. However, phosphoamino acid analysis (PAA) has been employed to monitor Jak serine and threonine phosphorylation. Over the past decade, remarkable advances have been made in our understanding of Jak function and dysfunction, however much remains to be learned about their complex regulatory mechanisms

The pseudokinase domain of JAK2 is a dual-specificity protein kinase that negatively regulates cytokine signaling

Human JAK2 tyrosine kinase mediates signaling through numerous cytokine receptors. The JAK2 JH2 domain functions as a negative regulator and is presumed to be a catalytically inactive pseudokinase, but the mechanism(s) for its inhibition of JAK2 remains unknown. Mutations in JH2 lead to increased JAK2 activity, contributing to myeloproliferative neoplasms (MPNs). Here we show that JH2 is a dual-specificity protein kinase that phosphorylates two negative regulatory sites in JAK2: Ser523 and Tyr570. Inactivation of JH2 catalytic activity increased JAK2 basal activity and downstream signaling. Notably, different MPN mutations abrogated JH2 activity in cells, and in MPN (V617F) patient cells phosphorylation of Tyr570 was reduced, suggesting that loss of JH2 activity contributes to the pathogenesis of MPNs. These results identify the catalytic activity of JH2 as a previously unrecognized mechanism to control basal activity and signaling of JAK2