Analysis of Jak2 Catalytic Function by Peptide Microarrays: The Role of the JH2 Domain and V617F Mutation

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.

Structure of the pseudokinase–kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition

“…5). The wild-type pseudokinasekinase construct was ∼100-fold less active than the kinase domain alone, recapitulating a similar effect reported for JAK2 (14) and indicating that the pseudokinase has a direct role in inhibiting TYK2 enzymatic activity. Comparison of the four TYK2 analogs that mimic JAK-associated mutations showed that all of the mutants were more active than wild type pseudokinasekinase, with the R744G and R901S mutants approaching levels of activity seen for the isolated kinase domain (Fig.…”

“…Because the TYK2 kinase activation loop is found in an active conformation in our structure, activation loop phosphorylation likely does not have a role in regulating pseudokinase-mediated inhibition. Given that isolated JAK kinase domains are significantly active (14,32,37) and pseudokinase-kinase interface mutations increase kinase activity, we speculate that, in the context of receptor-induced JAK dimerization, a displacement event, direct or perhaps allosteric, relieves the inhibitory influence of the pseudokinase domain, allowing the kinase domain to autophosphorylate and become the active phospho-transfer catalyst required for downstream signaling.…”

“…However, these JAK2 phosphorylation sites are not universally conserved in the other JAK family members (13), suggesting this may be a JAK2-specific regulatory pathway. Moreover, the in vitro activity of the isolated JAK2 pseudokinase-kinase protein is reduced and the K m value for ATP increased compared with the kinase domain alone (14), suggesting there may also be a more direct mechanism by which the pseudokinase regulates enzymatic activity.…”

“…Functional screening for constitutively active JAK2 alleles has also identified several point mutations in the pseudokinase N-lobe (18). Importantly, JAK2 V617F is a kinase-activating mutation, increasing JAK2 activity in both cell-based assays (17) and in vitro in the context of the isolated JAK2 pseudokinase-kinase module (14). Analogous mutations made in both JAK1 (L653F) and TYK2 (V678F) also activate their respective kinase domains (19).…”

JAK kinase targeting in hematologic malignancies: a sinuous pathway from identification of genetic alterations towards clinical indications

“…24 Using the recently solved crystal structure of JH1-JH2 from TYK2, this intramolecular inhibitory interface has been localized near the JH1 catalytic site and is predominantly mediated by the N-lobes of each domain. 25 The JH1-JH2 crystal structure of TYK2 supports an in cis mode of inhibition with the pseudokinase domain inhibiting the directly adjacent kinase domain within the same protein.…”