ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation

Abstract

Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wildtype JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches.T he Janus kinases (JAK1-3, TYK2) are a family of nonreceptor tyrosine kinases with essential functions in the regulation of hematopoiesis, the immune system, and cellular metabolism. JAKs interact specifically with various cytokine receptors and couple cytokine binding to cytoplasmic signaling cascades, including the signal transducers and activators of transcription (STAT) pathway. JAKs consist of an N-terminal FERM domain, an SH2-like (Src homology 2) domain, a pseudokinase domain (JAK homology 2, JH2), and the C-terminal tyrosine kinase domain (JH1). JH2 mediates critical regulatory functions in JAKs and primarily serves to inhibit basal JH1 activity. Experimental deletion of JH2 increases JH1 activity in full-length JAK in the absence of stimulation (1-3), and in recombinant systems addition of JH2 suppresses JH1 activity (4-6). JH2 is, however, also required for ligand-induced activation of full-length JAKs in cell (1-3, 6, 7). The regulatory functions of JH2 are corroborated by the multitude of human disease mutations identified in the domain. The most common JAK2 mutation, V617F, leads to cytokine-independent signaling through the exclusively JAK2-dependent homotypic receptors for erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), and thrombopoietin (8). The V617F mutation is found in ∼95% of patients with polycythemia vera (PV) (9...

Glycolytic Stimulation Is Not a Requirement for M2 Macrophage Differentiation

“…JAK kinases are well known as ATP-dependent kinases and can be blocked by ATP-competitive inhibitors (Hammarén et al, 2015). Indeed, STAT6 phosphorylation can be blocked by an ATP competitive inhibitor of JAK (Figure 6a).…”

Structural models of full-length JAK2 kinase

“…The backside of a protein kinase domain is known to be dynamically coupled with the ATP-binding P-loop (i.e., the β1-β2 loop). 45 This model suggests that ATP-binding by JH2 helps to maintain the high constituent activity of V617F 26 by modulating the conformation of the backside.…”

“…21 It has been proposed that the dimerization releases the JH1 kinase domains from the autoinhibitory monomer structure, allowing trans-phosphorylation of the two JH1 domains at the Y1007 and Y1008 residues in the activation loop. 22 It has also been suggested that the JH2 domain is likely to be crucial to the dimerization, and that JAK2 mutations such as F595A, 23,24 F739R, 25 and others that abrogate ATP binding to JH2 26 weaken the constitutively high kinase activity of the V617F mutant, possibly by undermining the structural integrity of the JH2 domain and thus hindering JAK2 dimerization.…”

Molecular insights into regulation of JAK2 in myeloproliferative neoplasms

“…A detailed understanding of the pathogenic activation of JAK2 may afford an opportunity to develop novel small-molecule therapeutics for MPNs, which would act to inhibit the basal hyperactivity of V617F, but allow normal cytokine stimulation of wild-type JAK2. Although a difficult challenge, several regions of JAK2 JH2, including the ATP-binding pocket 41 and aC, 33,[37][38][39] are potential targets for such compounds.…”