Modulation of Activation-Loop Phosphorylation by JAK Inhibitors Is Binding Mode Dependent

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...

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“…JAK2 V617F hyperphosphorylation (32). Our characterization of the nucleotide binding and thermal stability of JH2 ATP binding site mutants (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Hammarén

Ungureanu

Grisouard

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

123

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“…Indeed, structural studies showed that the isolated JH1 domains of the four Jaks complexed with ATP-competitive inhibitors adopt an active conformation with an exposed loop (42)(43)(44). With regard to intact Jaks, specific ATP competitive inhibitors that block catalytic function also promote activation loop phosphorylation in cells (45). Moreover, some JH2 mutants of Tyk2 and Jak3 have been described that are catalytically dead but constitutively phosphorylated on the activation loop.…”

Section: Discussionmentioning

confidence: 99%

Two Rare Disease-Associated Tyk2 Variants Are Catalytically Impaired but Signaling Competent

Gakovic

Ragimbeau

et al. 2013

The Journal of Immunology

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Tyk2 belongs to the Janus protein tyrosine kinase family and is involved in signaling of immunoregulatory cytokines (type I and III IFNs, IL-6, IL-10, and IL-12 families) via its interaction with shared receptor subunits. Depending on the receptor complex, Tyk2 is coactivated with either Jak1 or Jak2, but a detailed molecular characterization of the interplay between the two enzymes is missing. In human populations, the Tyk2 gene presents high levels of genetic diversity with >100 nonsynonymous variants being detected. In this study, we characterized two rare Tyk2 variants, I684S and P1104A, which have been associated with susceptibility to autoimmune disease. Specifically, we measured their in vitro catalytic activity and their ability to mediate Stat activation in fibroblasts and genotyped B cell lines. Both variants were found to be catalytically impaired but rescued signaling in response to IFN-α/β, IL-6, and IL-10. These data, coupled with functional study of an engineered Jak1 P1084A, support a model of nonhierarchical activation of Janus kinases in which one catalytically competent Jak is sufficient for signaling provided that its partner behaves as proper scaffold, even if inactive. Through the analysis of IFN-α and IFN-γ signaling in cells with different Jak1 P1084A levels, we also illustrate a context in which a hypomorphic Jak can hamper signaling in a cytokine-specific manner. Given the multitude of Tyk2-activating cytokines, the cell context–dependent requirement for Tyk2 and the catalytic defect of the two disease-associated variants studied in this paper, we predict that these alleles are functionally significant in complex immune disorders.

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“…79 Conventional (type 1) adenosine triphosphate-competitive JAK inhibitors bind to and stabilize the active conformation of JAKs, whereas type 2 inhibitors bind to and stabilize the inactive conformation, preventing the kinase from cycling to the active conformation. 81 The JAK2 inhibitor persistence is a phenomenon in which JAK-STAT signaling is reactivated despite the presence of a type 1 JAK2 inhibitor via heterodimerization between activated JAK2 and other JAKs, such as JAK1 or TYK2.…”

Section: Other Jak Inhibitors In Development For Mfmentioning

confidence: 99%

JAK2 inhibitors for myeloproliferative neoplasms: what is next?

Bose

Verstovšek

2017

Blood

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Since its approval in 2011, the Janus kinase 1/2 (JAK1/2) inhibitor ruxolitinib has evolved to become the centerpiece of therapy for myelofibrosis (MF), and its use in patients with hydroxyurea resistant or intolerant polycythemia vera (PV) is steadily increasing. Several other JAK2 inhibitors have entered clinical testing, but none have been approved and many have been discontinued. Importantly, the activity of these agents is not restricted to patients with JAK2 V617F or exon 12 mutations. Although JAK2 inhibitors provide substantial clinical benefit, their disease-modifying activity is limited, and rational combinations with other targeted agents are needed, particularly in MF, in which survival is short. Many such combinations are being explored, as are other novel agents, some of which could successfully be combined with JAK2 inhibitors in the future. In addition, new JAK2 inhibitors with the potential for less myelosuppression continue to be investigated. Given the proven safety and efficacy of ruxolitinib, it is likely that ruxolitinib-based combinations will be a major way forward in drug development for MF. If approved, less myelosuppressive JAK2 inhibitors such as pacritinib or NS-018 could prove to be very useful additions to the therapeutic armamentarium in MF. In PV, inhibitors of histone deacetylases and human double minute 2 have activity, but their role, if any, in the future treatment algorithm is uncertain, given the availability of ruxolitinib and renewed interest in interferons. Ruxolitinib is in late-phase clinical trials in essential thrombocythemia, in which it could fill an important void for patients with troublesome symptoms.

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Modulation of Activation-Loop Phosphorylation by JAK Inhibitors Is Binding Mode Dependent

Cited by 113 publications

References 47 publications

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

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

Two Rare Disease-Associated Tyk2 Variants Are Catalytically Impaired but Signaling Competent

JAK2 inhibitors for myeloproliferative neoplasms: what is next?

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