Janus kinase-2 (JAK2) mediates signaling by various cytokines, including erythropoietin and growth hormone. JAK2 possesses tandem pseudokinase and tyrosine-kinase domains. Mutations in the pseudokinase domain are causally linked to myeloproliferative neoplasms (MPNs) in humans. The structure of the JAK2 tandem kinase domains is unknown, and therefore the molecular bases for pseudokinase-mediated autoinhibition and pathogenic activation remain obscure. Using molecular dynamics simulations of protein-protein docking, we produced a structural model for the autoinhibitory interaction between the JAK2 pseudokinase and kinase domains. A striking feature of our model, which is supported by mutagenesis experiments, is that nearly all of the disease mutations map to the domain interface. The simulations indicate that the kinase domain is stabilized in an inactive state by the pseudokinase domain, and they offer a molecular rationale for the hyperactivity of V617F, the predominant JAK2 MPN mutation.
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This work was supported in part by the US National Institutes of Health (grant R21 AI095808; S.R.H.) and the Medical Research Council of Academy of Finland, Sigrid Juselius Foundation, Medical Research Fund of Tampere University Hospital, Finnish Cancer Foundation and Tampere Tuberculosis Foundation (O.S.). The APRE-Rluc and pRG-TK plasmids were gifts from D. Levy and J. Belasco (both at New York University School of Medicine), respectively. We thank R. Bandaranayake for crystallographic support, A. Philippsen for animation support and W.T. Miller, M. Mohammadi and M.P. Eastwood for critical reading of the manuscript.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Analysis of the initial 14 JAK2 JH2-JH1 simulations, and contact surfaces in the final model.
(a) Analysis of 14 JAK2 JH2-JH1 simulations. From each of the 14 3.0-µs simulations, starting from an arbitrary JH2-JH1 non-contacting pose, 300 snapshots (10-ns interval) were evaluated using both EMPIRE and OSCAR scoring functions15,16. The score of each snapshot from each simulation was plotted in the two-dimensional energy space with a unique color. The dots corresponding to the simulation that generated pose 2, the JH2-JH1 interaction that was pursued further (see Fig. 1), are colored red. (b) Time evolution of the orientation of JH1 and its center of mass for each of the 14 initial simulations (see Fig. 1). For each time snapshot in the simulations, the JH1 orientation is represented by the angles of the JH1 principal axes of inertia relative to the principal axes of JH1 in the final model. Also shown for each snapshot is the distance of the JH1 center-of-mass from the JH1 center-of-mass in the final model. JH2 in each snapshot is superimposed on JH2 in the final model prior to the angle and distance calculations. As shown, pose 2 (end of simulation 2) bears resemblance to, but is non-trivially different from the pose in the final model. Only after addition of the JH2-JH1 linker and further simulation (2 Extended) does this pose converge towards the final model. Other than simulation 2, simulation 12 also reached a JH2-JH1 pose that bears resemblance to the pose in the final model. (c) Surface representations of JH2 and JH1 (from final model) in “open book” view, in which JH2 has been rotated clockwise by 90° (vertical axis) and JH1 counterclockwise by 90°, with respect to the orientation in Fig. 2a, to reveal the interaction surface. Left, in cyan outline are residues in JH2 (residues 537–810) that are within 4.0 Å of an atom in JH1, and in green outline are residues in JH2 that are within 4.0 Å of an atom in the SH2-JH2 linker (residues 520–536). Right, same as left, except for JH1 (residues 840–1132) relative to JH2 (orange outline) and the SH2-JH2 linker (green outline). Activating mutations in JH1 and JH2 are colored pink and labeled. Other residues of interest are also labeled, as are the N and C lobes of the kinase domains.
(a) Luciferase activities of wild-type and mutant JAK2 measured using an APRE-luc reporter to assess endogenous STAT3-dependent transcription in COS7 cells. The firefly luciferase activity of each sample was normalized to that of renilla luciferase (luciferase ratio) and plotted as fold-change relative to the wild-type JAK2 (WT) luciferase ratio (set to 1.0). Average values and standard deviations were derived from triplicate samples. (b) Analysis of JAK2 mutants D873R and D873N in JH1 alone. Representative western blot of JAK2 JH1 (HA-tagged) immunoprecipitated from transfected HEK 293T cells and probed for JAK2 Tyr1007-Tyr1008 phosphorylation (pJAK2) (top) or protein levels (HA) (bottom). A reference molecular-weight marker (in kDa) is indicated on the left. (c) Epo-dependent activation of JAK2 mutants. JAK2-deficient γ2A cells transfected with the indicated JAK2 and STAT5 (HA-tagged) plasmids were either left untreated (−) or stimulated with Epo (+), and the whole cell lysates were probed for JAK2 Tyr1007-Tyr1008 phosphorylation (pJAK2, top), STAT5 phosphorylation (pSTAT5, middle), or protein levels (HA, bottom). Reference molecular-weight markers (in kDa) are indicated on the left.
(a) Analysis of R683E, D873N and R683E D873N. The distance between the Cα atoms of Glu592 (JH2) and Arg947 (JH1), interacting residues in the JH2-JH1 model (see Fig. 2e), is plotted as a function of simulation time. In the activating mutations R683E and D873N, the two residues (Glu592, Arg947) separated, whereas for wild type (WT) and the double mutation, the distance remained relatively stable. (b) Salt-bridge analysis for JAK2 Glu592-Arg947. WT, E592R and E592R R947E were simulated for 7.5 µs each. Plotted are the distances between select residues as a function of simulation time. Shown in solid lines are the distance trajectories between native residues, and shown in dashed lines are distance trajectories in which at least one of the residues involved has been introduced by mutation. To simplify the salt-bridge presentation, the actual distances displayed are between Cζ of Arg588, Arg592 or Arg947 (to account for Nɛ, Nη1, and Nη2 of arginine) and either Cδ of Glu592 or Glu947 (to account for Oɛ1 and Oɛ2 of glutamic acid) or P of pSer523 (to account for O1P, O2P and O3P of phosphoserine). Thus, the representative distance for a salt bridge is ∼3.8 Å rather than ∼2.7 Å (typical nitrogen-oxygen distance). Gray regions indicate the approximate distance range for salt bridges.
Conformational changes in JH1 and analysis of V617F. (a) Radius of gyration (Rg, an overall measure of the size) of JH1 as a function of simulation time (same simulations as in Fig. 4a). (b) Root-mean-square deviation (RMSD) for Cα atoms in JH1 as a function of simulation time, after aligning Cα atoms in JH2 for each time frame with the Cα atoms in their initial positions. Both simulations were initiated from an identical JH2-JH1 pose with Ser523 and Tyr570 phosphorylated. In one simulation, the JH1 activation loop was phosphorylated at Tyr1007 and Tyr1008 (orange), and in the other simulation (green), the activation loop was unphosphorylated. A high RMSD is indicative of a high degree of structural deviation from the JH2-JH1 configuration (the autoinhibitory pose) shown in Fig. 2a. (c–e) Analyses of JAK2 MPN mutation V617F. In previous studies, Ungureanu et al.11 showed that Ser523 and Tyr570 are poorly phosphorylated in activated JAK2 mutants such as V617F. Thus, to simulate V617F JH2-JH1 properly, we left Ser523 (and Tyr570) unphosphorylated and, for comparison purposes, we also simulated unphosphorylated wild type (WT). F595A, in αC of JH2 and proximal to Val617, was shown to suppress V617F14,28, and therefore we also simulated F595A V617F (unphosphorylated). (c) The SH2-JH2 linker conformations visited during the simulations. JH2-JH1 in the first time frame (t = 0) of each trajectory is shown in ribbon representation and colored as in Fig. 1. The Cα trace of the SH2-JH2 linker (residues 522–536) for each time frame is shown in green, after aligning JH2 in each frame with JH2 at t = 0. As shown, the linker in V617F is least stable in the binding groove between JH2 and JH1. (d) RMSD for Cα atoms in JH1 as a function of simulation time, after aligning Cα atoms in JH2 for each time frame with the Cα atoms in their initial positions. Removal of pSer523 and pTyr570 (WT versus WT pSpY) led to an increase in the overall conformational heterogeneity of JH1 and thus destabilization of the JH2-JH1 complex. V617F caused a further increase in JH1 heterogeneity, and addition of F595A to V617F restored JH1 heterogeneity back to the level of phosphorylated JH2-JH1 (WT pSpY). (e) RMSD for Cα atoms in αC of JH1 (residues 889–904) relative to the active conformation, after aligning all the Cα atoms in JH1. As shown, the active conformation of αC is most stable in V617F.
(a) Atomic models of JAK1 JH2 (PDB 4L00 (ref. 31)) and JH1 (PDB 4E5W (Kulagowski, J.J., et al. J. Med. Chem. 55, 5901–5921 (2012)) were placed by superposition into the positions of JH2 and JH1 of JAK2 (Fig. 2a). The SH2-JH2 and JH2-JH1 linkers were added, and an MD simulation was run for 12 µs. Shown is a representative pose near the end of the simulation. JH2 (residues 575–850) is colored orange, JH1 (residues 866–1154) is colored cyan, with the activation loop (residues 1021–1043) colored red, the SH2-JH2 linker (residues 563–574) is colored green, and the JH2-JH1 linker (residues 851–865) is colored gray. Mapped activating mutations (Hornakova, T., et al. Haematologica 96, 845–853 (2011)) are shown in stick representation, colored pink (carbon atoms), and labeled. Red, oxygen atoms; blue, nitrogen atoms. Other residues of interest are shown in stick representation and labeled. The N-terminus (residue 563) is labeled N, and the C-terminus (residue 1154) is labeled C. (b) Ribbon diagram of the JAK2 JH2-JH1 model from the current study (1), from Lindauer et al.29 (2), and from Wan et al.23 (3). Coloring is the same as in Fig. 1. The side chains of Val617, Arg683, and Asp873 are shown in sphere representation and colored pink (carbon atoms). The models are aligned with one another based on a superposition of JH2. The N- and C-termini for each model are labeled N and C.
For each blot (labeled accordingly), boxes mark the borders of the cropped images shown in Fig. 3.
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Shan, Y., Gnanasambandan, K., Ungureanu, D. et al. Molecular basis for pseudokinase-dependent autoinhibition of JAK2 tyrosine kinase. Nat Struct Mol Biol 21, 579–584 (2014). https://doi.org/10.1038/nsmb.2849
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