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

Ungureanu, Daniela; Wu, Jiangxue; Pekkala, Tuija; Niranjan, Yashavanthi; Young, Clifford; Jensen, Ole N.; Xu, Chong-Feng; Neubert, Thomas A.; Skoda, Radek C.; Hubbard, Stevan R.; Silvennoinen, Olli

doi:10.1038/nsmb.2099

Cited by 239 publications

(213 citation statements)

References 40 publications

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“…Recombinant proteins were expressed in Sf9 cells as detailed earlier (17). After cell collection and Ni-NTA purification, protein was either used as such [JAK2(536-812-6xHis) for TSA] or subjected to anion exchange purification as described earlier (17) [JAK2(513-827-6xHis) and JAK1(553-856-6xHis) for MANT-ATP binding assays].…”

Section: Methodsmentioning

confidence: 99%

“…The crystal structure of JAK2 JH2 (16) revealed a prototypical kinase-domain fold that binds ATP (17), but with a noncanonical binding mode. 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).…”

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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: Methodsmentioning

confidence: 99%

mentioning

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.

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123

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“…Additionally, the JAK2 pseudokinase and kinase domains have been reported to coimmunoprecipitate, and coexpression of the JAK2 pseudokinase domain inhibits activity of the isolated kinase domain (10). Recent work on the JAK2 pseudokinase domain indicates that it has weak catalytic activity and autophosphorylates two inhibitory sites within the SH2-pseudokinase linker and pseudokinase N-lobe (11,12). 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.…”

Section: Jak1 | Jak3mentioning

confidence: 99%

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

Lupardus¹,

Ultsch²,

Wallweber³

et al. 2014

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

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Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state. JAK1 | JAK3H elical bundle cytokines of the interleukin and IFN families regulate a wide variety of immune and cellular growth responses (1). These signaling pathways are initiated by cytokinemediated receptor dimerization and subsequent activation of nonreceptor tyrosine kinases called Janus kinases (JAKs) that are associated with the receptor intracellular domains (2). JAK activation leads to phosphorylation of the receptor and recruitment of STAT family transcription factors, which are then phosphorylated by the JAK and translocated to the nucleus where they induce gene transcription. The JAK family consists of four members (JAK1, JAK2, JAK3, and tyrosine kinase 2 or TYK2), each of which binds a distinct set of cytokine receptor subtypes and hence has a unique gene knockout phenotype, ranging from embryonic lethality due to neuronal and erythropoietic defects (JAK1 and JAK2, respectively) (3-5), to profound immune system deficiencies (JAK3 and TYK2) (6).The JAK kinases are large (∼1,150-aa) multidomain proteins whose primary sequences were initially organized into seven JAK-homology (JH) domains that reflected distinct regions of high sequence homology (7). Subsequent structure predictions indicated that JAKs contain four distinct domains: N-terminal 4.1, Ezrin, Radixin, Moesin (FERM) and Src homology 2 (SH2) domains, followed by C-terminal pseudokinase and kinase domains (7,8) (Fig. 1A). The FERM and SH2 domains constitute the receptor-binding module (2), whereas the pseudokinase, which has a canonical kinase fold but lacks key catalytic residues, is thought to regulate kinase activity. Deletion of the pseudokinase in JAK2 and JAK3 increases basal kinase activity and deregulates signaling through cognate receptors (9, 10). Additionally, the JAK2 pseudokinase and kinase domains have been reported to coimmunoprecipitate, and coexpression of the JAK2 pseudokinase domain inhibits activity of the isolated kinase domain (10). Recent work on the JAK2 pseudokinase domain indicates that it has weak catalytic activity and autophosphorylates two inhibitory sites within the SH2-pseudokinase linker and pseudokinase...

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“…All the mice developed an aggressive lethal mixed T-cell lympho-and myeloproliferative disease after a latency of 5-10 weeks. 8 Similarly, transgenic mice expressing the ETV6-JAK2 (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) variant from an SRα promoter/enhancer element developed a fatal CD8 + T-cell acute leukemia. 11 Interestingly, breeding of the SRα-ETV6-JAK2 transgenics into a Cde-/-background with a block in T-cell development resulted in the development of B-cell lymphoma/leukemia.…”

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confidence: 99%

“…Interestingly, particular breakpoints were found resulting in variant fusion proteins that were associated with different disease phenotypes. Peeters and colleagues cloned a t(9;12)(p24;p13) from a patient with early pre-B cell acute lymphoblastic leukemia (ALL) leading to a fusion of exon 4 of ETV6 to exon 17 of JAK2 (ETV6-JAK2, [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. In addition, they identified a t(9;15;12)(p24;q15;p13) in a patient with atypical CML in a transformation that fused exon 5 of ETV6 to exon 12 of JAK2 (ETV6-JAK2, 5-12).…”

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confidence: 99%

Modeling ETV6-JAK2-induced leukemia: insights from the zebrafish

Schwaller

2012

Haematologica

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C loning and functional characterization of a large number of chromosomal translocations revealed that aberrant activation of protein kinases is a key event for expansion of the malignant clone in chronic myeloproliferative disorders, as well as in acute leukemia. The best known example is t(9;22)(q34;q11), leading to the expression of the BCR-ABL1 (BCR-ABL) tyrosine kinase fusion associated with chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). In 1997, two studies reported that the JAK2 gene on the short arm of chromosome 9p24 encoding for just another cytoplasmic protein tyrosine kinase is involved in rare chromosomal translocations resulting in fusions to the ETS-family member ETV6 (also known as TEL). Interestingly, particular breakpoints were found resulting in variant fusion proteins that were associated with different disease phenotypes. Peeters and colleagues cloned a t(9;12)(p24;p13) from a patient with early pre-B cell acute lymphoblastic leukemia (ALL) leading to a fusion of exon 4 of ETV6 to exon 17 of JAK2 (ETV6-JAK2, 4-17). In addition, they identified a t(9;15;12)(p24;q15;p13) in a patient with atypical CML in a transformation that fused exon 5 of ETV6 to exon 12 of JAK2 (ETV6-JAK2, 5-12).1 In parallel, Lacronique and colleagues cloned a t(9;12)(p24;p13) from blasts from a childhood T-cell ALL patient leading to a fusion of ETV6 exon 5 to JAK2 exon 19 containing only the JH1 tyrosine kinase domain (ETV6-JAK2, 5-19).2 Since their original description, very few additional cases of hematologic malignancies with ETV6-JAK2 have been reported. 3 Later, additional translocations involving the JAK2 gene were identified, such as t(9;22)(q34;q11.2) leading to a BCR-JAK2 fusion, t(8;9)(p22;p24) leading to a fusion to the pericentrilar material 1 (PCM1) gene, or t(4;9)(q21;p24) fusing JAK2 to the SEC31A gene. [4][5][6] Interestingly, in all of the PCM1-JAK2 positive cases reported so far, large parts of JAK2 (exon 9) were included and associated with a wide disease phenotype spectrum, including atypical CML, myelodysplasia/-proliferation with erythroid hyperplasia, or T-cell lymphoma. To address the biological activity of an ETV6-JAK2 fusion, several groups over-expressed the three fusion variants in IL-3 dependent murine Ba/F3 cells. Expression of all ETV6-JAK2 variants rendered Ba/F3 cells IL-3-independent with similar efficacy. Transformation of the cells by ETV6-JAK2 expression was associated with constitutive activation of the signal transducers and activators of transcription (STATs), predominantly of STAT5, but also STAT1 and STAT3, and expression of several putative STAT targets such as oncostatin M, CIS or PIM1. 8-10To model ETV6-JAK2 disease in vivo, we transplanted bone marrow cells with retroviral expression of the 5-19 fusion variant in lethally irradiated mice. All the mice developed an aggressive lethal mixed T-cell lympho-and myeloproliferative disease after a latency of 5-10 weeks. 8 Similarly, transgenic mice expressing the ETV6-JAK2 (5-19) variant from an SRα ...

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

Cited by 239 publications

References 40 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

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

Modeling ETV6-JAK2-induced leukemia: insights from the zebrafish

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