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. 2010 Jun 16;5(6):e11157.
doi: 10.1371/journal.pone.0011157.

JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors

Alexandra Dusa  1 , Céline Mouton, Christian Pecquet, Murielle Herman, Stefan N Constantinescu
Affiliations

JAK2 V617F constitutive activation requires JH2 residue F595: a pseudokinase domain target for specific inhibitors

Alexandra Dusa et al. PLoS One. .

Abstract

The JAK2 V617F mutation present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients renders the kinase constitutively active. In the absence of a three-dimensional structure for the full-length protein, the mechanism of activation of JAK2 V617F has remained elusive. In this study, we used functional mutagenesis to investigate the involvement of the JH2 alphaC helix in the constitutive activation of JAK2 V617F. We show that residue F595, located in the middle of the alphaC helix of JH2, is indispensable for the constitutive activity of JAK2 V617F. Mutation of F595 to Ala, Lys, Val or Ile significantly decreases the constitutive activity of JAK2 V617F, but F595W and F595Y are able to restore it, implying an aromaticity requirement at position 595. Substitution of F595 to Ala was also able to decrease the constitutive activity of two other JAK2 mutants, T875N and R683G, as well as JAK2 K539L, albeit to a lower extent. In contrast, the F595 mutants are activated by erythropoietin-bound EpoR. We also explored the relationship between the dimeric conformation of EpoR and several JAK2 mutants. Since residue F595 is crucial to the constitutive activation of JAK2 V617F but not to initiation of JAK2 activation by cytokines, we suggest that small molecules that target the region around this residue might specifically block oncogenic JAK2 and spare JAK2 wild-type.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Sequence homology and distances between homologous JAK2 V617 and F595 positions in other kinases and a model of the relative positions of the two residues in JAK2.
(A) Alignment of part of the pseudokinase domain of JAK2 with other kinases containing a GVCV-like motif. (B) Distances between the nearest atoms of homologous F595 and V617 residues in the other kinases. Distances were estimated based on the PDB coordinates with UCSF Chimera program . (C) Predicted relative position of the JAK2 JH1 and JH2 αC helices based on the homology model of Lindauer et al . In this predicted arrangement, residue F595 in the JH2 αC helix is in the proximity of mutated V617F residue, also located in the pseudokinase domain.
Figure 2
Figure 2. Effect of the substitution of residue F595 in the JH2 αC helix on the constitutive activity of JAK2 V617F and on the ligand-induced activation of JAK2 wild-type measured by luciferase assays.
(A) Mutation of F595 to some residues induces up to an 86% decrease in the constitutive activity of JAK2 V617F, depending on the particular substitution, while mutation of F595 to aromatic residues rescues the constitutive activity of JAK2 V617F. The activity of wild-type JAK2 and of JAK2 F595A were similar and amounted to 15-20% of the activity of JAK2 V617F (not shown). (B) The single substitution mutants at position 595 display Epo responses similar to JAK2 wild-type at various concentrations of Epo and do not induce constitutive activation of JAK2 wild-type. (C) The JAK2 F595X/V617F double mutants also respond to Epo in a manner analogous to JAK2 wild-type.
Figure 3
Figure 3. Role of JH2 αC helix residue F595 in the proliferation and activation of Ba/F3 cells stably expressing the Epo receptor and individual JAK2 mutants.
(A) Proliferation assay of sorted Ba/F3-EpoR cells stably expressing each mutant and wild-type, in medium without growth factors for 7 days. JAK2 V617F is able to proliferate constitutively in this minimal medium starting from the first day (black triangles). The F595A/V617F double mutant lost most of its proliferative advantage (green stars), while the Ba/F3-EpoR parental cell line, or expressing either wild-type or F595A alone could not proliferate in the absence of growth factors. Substitution of F595 to an aromatic residue (F595W) restored autonomous growth (red circles). (B) Luciferase assay in sorted Ba/F3 cells stably expressing the Epo receptor and each individual JAK2 mutant or wild-type. Each cell line was individually electroporated with the pGRR5-Luc and pRLTK-Luc reporters and the STAT5 transcriptional activity was measured. The Ba/F3-EpoR cells expressing JAK2 V617F previously selected for autonomous growth show a typical increase in STAT5 transcriptional activity both in the presence and absence of Epo. (C) Immunoblot analysis of sorted Ba/F3-EpoR cells expressing individual mutants or wild-type, in presence of absence of Epo stimulation. pJAK2, pSTAT5 and pErk1/2 levels of JAK2 V617F, probed with specific antibodies revealed a constitutive signal in the absence of stimulation. This signal was absent in the cells expressing F595A/V617F but partially rescued in cells expressing F595W/V617F.
Figure 4
Figure 4. Substitution of F595 to Ala has an inhibitory effect on the constitutive activity of other JAK2 oncogenic mutants.
(A) JAK2 K539L, T875N and R683G all induce constitutive activation of JAK2, however all three mutants display a marked decrease in STAT5 transcriptional activity in γ-2A cells when the F595A mutation is also introduced. (B) Proliferation assay of sorted Ba/F3-EpoR cells stably expressing each constitutive mutant and wild-type, in medium without growth factors for 7 days. Cells expressing JAK2 V617F (black triangles), K539L (blue lines), T875N (green stars) and R683G (yellow circles) can proliferate constitutively in this minimal medium starting from the first day. Substitution of F595 to Ala individually in the context of each active mutant causes a marked decrease in autonomous growth. The JAK2 K539L/F595A mutant (red lines) lost most of its proliferative advantage during the first 4 days, but by day 7 had regained the same level of autonomous growth as its single counterpart (blue lines). (C) Predicted locations (red spheres) of mutants K539L, R683G, V617F (left panel) and T875N (right panel) within the model structure of JAK2, based on coordinates of the homology model proposed by Lindauer et al . The right panel is rotated 180° relative to the left panel. The kinase domain is depicted in yellow and the pseudokinase domain in blue. The location of F595 is shown as a green sphere. Circular arrows placed between helices C of JH2 and JH1 indicate potential conformational changes originating in JH2 and transmitted to JH1. Solid double-headed arrow placed near the R683G mutation suggests increased flexibility induced by this JH2 hinge mutant. Green star (right panel) suggests T875N mutation changes JH1-JH2 linker segment conformation, which is transmitted to JH2 helix C F595.
Figure 5
Figure 5. The F595A homolog in JAK1, F636A, blocks constitutive activity of JAK1 V658F.
Transient transfection in the JAK1-deficient U4C cell line indicates a decrease in the STAT3 transcriptional activity in the JAK1 F636A/V658F, as compared to JAK1 V658F.
Figure 6
Figure 6. The JAK2 V617F mutation can induce constitutive activation regardless of the dimeric conformation of the receptor it is bound to, while JAK2 wild-type requires a ligand-activated EpoR dimer in order to signal.
(A) The coiled-coil EpoR constructs were made by replacing the extracellular domain of EpoR with the dimeric coiled-coil domain of the yeast transcription factor Put3. (B) Luciferase assay in γ-2A cells transfected with each JAK2 and with previously engineered EpoR dimers containing coiled-coil replacements of their extracellular domains. JAK2 wild-type signals best from the EpoR dimeric conformations imposed by cc-EpoR-III and cc-EpoR-VI, previously shown to correspond to activated dimers. V617F has acquired the ability to signal constitutively from both active (cc-EpoR-III, cc-EpoR-VI) and inactive (cc-EpoR-II, cc-EpoR-V) dimeric interfaces. JAK2 F595A/V617F and F595V/V617F have lost the ability to signal constitutively from active and inactive dimeric conformations, suggesting that F595 may play a role in the constitutive activity of JAK2 V617F. Mutating F595 to Ala or Val forces the double mutant to prefer signaling from the active dimeric EpoR orientations (cc-EpoR-III and VI), akin to wild-type. (C) Activation model for cytokine-activated JAK2 versus JAK2 autoactivation caused by the V617F mutation. Only the pseudokinase domain is shown for simplicity. In wild-type JAK2, residues V617 (grey circle) and F595 (red circle in pseudokinase domain helix C, depicted as grey cylinder) do not play a key role in activation that is brought about by rotation of receptors and scissors-like movements, bringing JAK2 molecules in close proximity, and leading to activation of the kinase domain. In the case of the JAK2 V617F mutant, residues F617 (red circle) and F595 (red circle in pseudokinase domain helix C, depicted as a grey cylinder) are crucial for initiation activation, which is transmitted to the kinase domain of JAK2, in the absence of the rotation and scissors-like movement of the receptors.
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