Crystal structures of the JAK2 pseudokinase domain and the pathogenic mutant V617F

Bandaranayake, R.M.; Ungureanu, Daniela; Shan, Yibing; Shaw, David E.; Silvennoinen, Olli; Hubbard, Stevan R.

doi:10.1038/nsmb.2348

Cited by 208 publications

(257 citation statements)

References 48 publications

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“…Secondary structure analysis of αC showed that the time αC residues (587-602) were in an α-helical conformation increased upon ATP binding from 41% to 65% in wild type and from 73% to 84% in V617F (Fig. S6D) (16). Taken together, these data imply a stabilizing effect for ATP binding on JH2 and a difference in the αC region of ATP-bound JAK2 JH2 V617F compared with wild type.…”

Section: Molecular Dynamic Simulations Imply a Stabilizing Effect Forsupporting

confidence: 62%

“…The crystal structure of JAK2 JH2 V617F is highly similar to the wild-type structure (16), with small changes in αC and the β3-αC linker and a slight change in the topography of the ATP binding pocket due to alignment of Phe617, Phe595, and Phe594 (the latter two in αC) in V617F (Fig. S1).…”

Section: Resultsmentioning

confidence: 83%

“…The effects of nucleotide binding to the structure of JH2 are subtle: mainly rigidification of αC in the ATP-bound form (16). Why is it then that the loss of nucleotide binding has a pronounced effect in V617F but not in wild type?…”

Section: Discussionmentioning

confidence: 99%

“…Molecular dynamic simulations were performed to explore the structural consequences of JH2 ATP binding. JAK2 JH2 wild type and V617F were simulated for 3.5 μs in the presence of ATP, and the results were compared with simulations of the domains without ATP (16). An overall reduction in flexibility of JH2 was observed upon ATP binding, with reductions in root-mean-square fluctuation and deviation (Fig.…”

Section: Molecular Dynamic Simulations Imply a Stabilizing Effect Formentioning

confidence: 99%

“…Additionally, JAK2 JH2 was found to possess weak kinase activity in vitro and autophosphorylate two regulatory sites: Ser523 in the SH2-JH2 linker and Tyr570 in JH2 itself (17). The structure of JAK2 JH2 V617F is highly similar to wild-type JH2 but shows a rigidified C helix (αC) in the kinase N lobe and a slightly altered ATP binding cleft (16). These structural differences, however, do not provide an obvious explanation for the mechanism of pathogenic activation.…”

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confidence: 99%

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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.

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123

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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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Section: Molecular Dynamic Simulations Imply a Stabilizing Effect Forsupporting

confidence: 62%

Section: Resultsmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Molecular Dynamic Simulations Imply a Stabilizing Effect Formentioning

confidence: 99%

mentioning

confidence: 99%

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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.

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123

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JAK Family Inhibitors for Autoimmune Diseases

Borzilleri

Hart

Moslin

et al. 2021

Burger's Medicinal Chemistry and Drug Discovery

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The JAK family of non‐receptor tyrosine kinases mediates the signaling of proinflammatory cytokines that contribute to the pathogenesis of numerous autoimmune diseases. While cytokine‐directed therapies are available to treat immune‐driven diseases, oral small‐molecule inhibitors of the JAK family (Jakinibs) have been approved as viable alternatives for the treatment of RA, PsA, and IBD. These first‐generation pan‐JAK inhibitors target the highly conserved catalytically active kinase domains in a reversible, ATP‐competitive manner, albeit in a nonselective fashion. Novel approaches to target alternative binding modes through, for example irreversible or allosteric kinase inhibition, have led to the identification of the next generation of agents, which demonstrate improved JAK family selectivity. This improved selectivity offers the potential for greater and perhaps broader efficacy by allowing for higher dosing, while avoiding undesired side effects. This article provides an overview of the JAK‐STAT signaling pathway and outlines the challenges associated with the discovery of selective JAK inhibitors while highlighting a unique allosteric TYK2 inhibitor. For each of the agents discussed, a synopsis of the medicinal chemistry efforts leading to a particular molecule is provided along with a brief summary of available preclinical and clinical efficacy and safety data.

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A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

Gorantla,

Mueller,

Albers‐Leischner

et al. 2023

Molecular Oncology

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Tyrosine‐protein kinase (janus kinase; JAK)–signal transducer and activator of transcription (STAT) signaling plays a pivotal role in the development of myeloproliferative neoplasms (MPNs). Treatment with the potent JAK1/JAK2‐specific inhibitor, ruxolitinib, significantly reduces tumor burden; however, ruxolitinib treatment does not fully eradicate the malignant clone. As the molecular basis for the disease persistence is not well understood, we set out to gain new insights by generating ruxolitinib‐resistant cell lines. Surprisingly, these cells harbor a 45 kDa JAK2 variant (FERM‐JAK2) consisting of the N‐terminal FERM domain directly fused to the C‐terminal kinase domain in 80% of sublines resistant to ruxolitinib. At the molecular level, FERM‐JAK2 is able to directly bind and activate STAT5 in the absence of cytokine receptors. Furthermore, phosphorylation of activation‐loop tyrosines is dispensable for FERM‐JAK2‐mediated STAT5 activation and cellular transformation, in contrast to JAK2‐V617F. As a result, FERM‐JAK2 is highly resistant to several ATP‐competitive JAK2 inhibitors, whereas it is particularly sensitive to HSP90 inhibition. A murine model of FERM‐JAK2 leukemogenesis showed an accelerated MPN phenotype with pronounced splenomegaly. Notably, most current protocols for the monitoring of emerging JAK variants are unable to detect FERM‐JAK2, highlighting the urgent need for implementing next‐generation sequencing approaches in MPN patients receiving ruxolitinib.

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Crystal structures of the JAK2 pseudokinase domain and the pathogenic mutant V617F

Cited by 208 publications

References 48 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

JAK Family Inhibitors for Autoimmune Diseases

A newly identified 45‐kDa JAK2 variant with an altered kinase domain structure represents a novel mode of JAK2 kinase inhibitor resistance

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