Amino acid residue E543 in JAK2 C618R is a potential therapeutic target for myeloproliferative disorders caused by JAK2 C618R mutation

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Abstract

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays a key role in the hematopoietic and immune responses. The acquired JAK2 C618R somatic mutation is detected in a subset of myeloproliferative disorders (MPDs) patients and presumed to be a biomarker for MPDs. However, how JAK2 C618R mutation causes MPDs is still unclear. Our results indicate that the amino acid residue E543 in JAK2 C618R is indispensable for its constitutive activation. When the glutamic acid at this position was mutated to alanine (E543A) in the JAK2 C618R, its activity significantly decreased. However when the glutamic acid was mutated to the acidic amino acid, aspartic acid, JAK2 C618R activity changed little. These results suggest that there is an interaction between the amino acid residue R618 and E543, and that this interaction is crucial to sustain the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effects on the function of wild type JAK2 (WT JAK2). This study suggests that the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by the JAK2 C618R mutation.

Highlights

JAK2 C618R mutation is presumed to be a biomarker for MPDs. ► The E543 and R618 interaction was indispensable for JAK2 C618R activity and structural stability. ► Mutations disrupted this interactions reduced the proliferation of JAK2 C618R expressing Ba/F3 cells. ► The residue E543 single mutations did not affect the function of WT JAK2. ► The residue E543 might be a potential target for specific inhibitors to treat MPDs.

Introduction

Janus kinases (JAKs)1 are key effectors in controlling immune responses and maintaining hematopoiesis [1], [2]. These non-receptor tyrosine kinases play key roles in several important intracellular signaling pathways, including JAK/STAT pathway which is the key mediator of cytokine signaling [3], [4]. Due to the pivotal roles of JAKs in cytokine signaling, specific JAKs inhibitors are therapeutically deployed in diseases caused by deregulated cytokine activity [1], [2], [3], [4].

Janus kinase 2 (JAK2) belongs to the JAKs family and is an essential component of erythropoietin receptor signaling pathway. JAK2, like other JAKs family members, is a multidomain protein possessing seven conserved JAK homology (JH) domains 1–7 [5]. The C-terminal JH1 domain is a highly conserved kinase domain and responsible for ATP binding and substrate phosphorylation. The JH2 domain which was presumed to be a pseudokinase is proven to be a kinase now [6]. This JH2 domain phosphorylates amino acid residues Ser523 and Tyr570 in JAK2 and plays vital roles in keeping JAK2 in low activity [7]. Previous study suggests that deletion of JH2 domain in JAK2 leads to remarkable increase in kinase activity, while fusion of JH2 domain to the JH2 domain deleted JAK2 decreases its activity [8]. Thus the JH2 domain is essential for JAK2 auto-inhibition. Furthermore homology models of JAK2 indicate that the interactions between JH1 and JH2 domains hold JH1 domain in a closed, inactive conformation [9]. Therefore, mutations altering the JH1/JH2 domains interactions might lead to JAK2 constitutive activation [2], [10].

Deregulation of JAK2 kinase activity is a common event in various types of cancer especially in hematological neoplasias such as the myeloproliferative disorders (MPDs). MPDs was first described by William Dameshek in 1951 as a class of stem cell derived hematological disorders that included essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). MPDs are clinically characterized by the presence of increased JAK2 activity in peripheral red blood cells, platelets or neutrophils along with bone marrow fibrosis, respectively [11]. Several research groups have reported that the JAK2 V617F mutation was responsible for the MPDs phenotype in a large percentage of afflicted individuals by constitutive activation of JAK2 and the subsequent cytokine independent signaling pathogenesis [8], [9], [10], [12]. The identification of JAK2 somatic activating mutations in most MPDs patients led to the clinical development of JAK2 kinase inhibitors [9], [10], [11], [12], [13]. Although JAK2 inhibitors therapies improve MPD associated splenomegaly and systemic symptoms, these inhibitors do not discriminate the WT and mutant JAK2s and lead to side effects [14]. Therefore to look for potential mutant JAK2 specific inhibitor targets for MPD is of great significance [14]. Recently, the acquired JAK2 C618R somatic mutation, another biomarker for MPDs, is reported [9], [13]. This mutation has been identified in a considerable portion of MPDs patients, including 10–15% PV patients and 18–25% ET and PMF patients [9], [13]. However, the molecular mechanism of how JAK2 C618R mutation results in its gain-of-function phenotype remains unclear. Although the structure of JAK2 has never been crystallized, the mimic structure of JAK2 was obtained by homology modeling [9], [15], [16]. The homology models suggest that the amino acid residue C618 is located at the joint of the β-4 and β-5 sheets of JH2 connected by hydrogen bonds and anchored by surrounding residues. The hydrogen bond between R564 and D544 plays vital roles in maintaining JH1 and JH2 domains interactions and keeping JH1 domain in a closed, inactive conformation. According to homology models of JAK2 C618R mutation in this study, the hydrogen bond between R564 and D544 is disrupted by the obtained interaction between the amino acid residues R618 and E543 which might lead to JAK2 C618R constitutive activation (Fig. 1). However, these questions remain: does the C618R mutation leads to JAK2 hyperactivation and causes constitutive activation of downstream signal transduction, as the homology models have suggested? Does the amino acid residue E543 play an important role in maintaining JAK2 C618R constitutive activation?

In this study, homology structures of WT JAK2 and JAK2 C618R suggest that the interaction between E543 and R618 plays a key role in maintaining JAK2 C618R constitutive activation. Moreover, results from enzyme kinetics and luciferase assay experiments imply that the residue E543 is indispensable for the constitutive activation of JAK2 C618R, so mutations that abolished the interaction between E543 and R618 obviously decreased the activity of JAK2 C618R. Furthermore, the mutation (E543D) that repaired the E543 and R618 interaction rescued the constitutive activation of JAK2 C618R. More importantly, the E543 single mutation had no effect on the function of WT JAK2. Thus, the amino acid residue E543 might be a potential target for specific inhibitors to treat MPDs caused by JAK2 C618R mutation.

Section snippets

Modeling the structure of WT and mutant JAK2s

The full-length homology model of JAK2 that was kindly provided by Dr. Romano Kroemer [17] was used for subsequent molecular dynamics simulations. The simulations were performed using the NAMD analysis package developed by the Theoretical and Computational Biophysics Group at the University of Illinois at Urbana-Champaign [18]. High hydrophilic cavities inside the protein were filled with water molecules using the program DOWSER [19], and the protein was enclosed in a water box with 10 Å

Structural analysis of mutant JAK2s

In order to investigate the effects of mutations on the structure of JAK2, the structures of WT and mutant JAK2s were predicted by homology modeling. The homology models suggest that the hydrogen bond between R564 and D544 in the JH2 domain is vital in maintaining JH1 and JH2 domains interactions and keeping JH1 domain in a closed, inactive conformation. In the JAK2 C618R mutant, R618 interacted with E543, and disrupted the hydrogen bond between R564 and D544 and the interactions between JH2

Acknowledgments

The present investigation was supported by Grants from the Natural Science Foundation of China (30971281, 81200375, 81000210, 30901753 and 81100349), Natural Science Foundation of Jiangsu Province (BK2012140) and the China Postdoctoral Science Foundation funded project (2012M511324).

References (39)

  • H.E. Broxmeyer

    Blood

    (2011)
  • X. Lu et al.

    J. Biol. Chem.

    (2008)
  • T. Kisseleva et al.

    Gene

    (2002)
  • J. Boudeau et al.

    Trends Cell Biol.

    (2006)
  • R.L. Levine et al.

    Blood

    (2008)
  • J.H. Yoo et al.

    Cancer Genet. Cytogenet.

    (2009)
  • I.S. Lucet et al.

    Blood

    (2006)
  • J.B. Rouget et al.

    Biophys. J.

    (2010)
  • A. Tefferi

    Blood

    (2012)
  • N. Seubert et al.

    Mol. Cell

    (2003)
  • S.M. Kelly et al.

    Biochim. Biophys. Acta

    (2005)
  • S.M. Kelly et al.

    Biochim. Biophys. Acta

    (1997)
  • G. Wernig et al.

    Cancer Cell

    (2008)
  • A. Quintas-Cardama et al.

    Blood

    (2010)
  • J.S. Rawlings et al.

    J. Cell Sci.

    (2004)
  • A. Tefferi

    N. Engl. J. Med.

    (2012)
  • D. Ungureanu et al.

    Nat. Struct. Mol. Biol.

    (2011)
  • P. Saharinen et al.

    Mol. Biol. Cell

    (2003)
  • T.S. Lee et al.

    BMC Struct. Biol.

    (2009)
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