The constitutive activation of Jak2-V617F is mediated by a π stacking mechanism involving phenylalanines 595 and 617

Abstract

Somatic mutations in the Jak2 allele that lead to constitutive kinase activation of the protein have been identified in human disease conditions such as the myeloproliferative neoplasms (MPNs). The most common mutation in these patients is a V617F substitution mutation, which is believed to play a causative role in the MPN pathogenesis. As such, identifying the molecular basis for the constitutive activation of Jak2-V617F is important for understanding its clinical implications and potential treatment. Here, we hypothesized that conversion of residue 617 from Val to Phe resulted in the formation of novel π stacking interactions with neighboring Phe residues. To test this, we first examined the Jak2 structure via molecular modeling and identified a potential π stacking interaction between F594, F595, and F617. Disruption of this interaction through site-directed mutagenesis impaired Jak2 autophosphorylation, Jak2-dependent gene transcription, and in vitro kinase activity of the Jak2-V617F protein. Further, substitution of F594 and F595 with Trp did not affect Jak2 function significantly, but replacement with charged residues did, showing the importance of aromaticity and hydropathy index conservation at these positions. Using molecular dynamics (MD) simulations, we found that the π stacking interaction between residues 595 and 617 in the Jak2-V617F protein was of much greater energy and conformed to the properties of π stacking, relative to the Jak2-WT or Jak2-V617F/F594A/F595A. In summary, we have identified a π stacking interaction between F595 and F617 that is specific to and is critical for the constitutive activation of Jak2-V617F.

Figure 1. The V617F mutation induces a possible π stacking interaction between F594, F595 and F617 in Jak2

(A, B) Cartoon representations of the Jak2-WT and Jak2-V617F JH2 domain homology models, respectively, generated using SWISS MODEL and visualized with Chimera. (C, D) The distances between F594 (white), F595 (yellow) and 617 (magenta) were lesser in Jak2-V617F when compared to Jak2-WT. All the distances were calculated using the Structural Analysis tools in Chimera.

Figure 2. A potential π stacking interaction between F595 and F617 in Jak2-V617F weakens the interaction between F617 and V1000

Snapshots of the MD simulations for Jak2-WT (A), Jak2-V617F (B), and Jak2-V617F/F594A/F595A (C) at 20 ns focusing on the JH1 (orange) - JH2 (cyan) interface. Amino acid 594 is highlighted in light green, 595 in dark green, 617 in mauve and 1000 in white. The activation loop is shown in yellow and the residues Y1007 and Y1008 are shown in blue. (D) Comparison of the non-bonded energy calculated between the residues 595 and 617 amongst Jak2-WT (black), Jak2-V617F (red) and Jak2-V617F/F594A/F595A (blue) using NAMD energy. (E) Comparison of the non-bonded energy calculated amongst Jak2-WT (black), Jak2-V617F (red) and Jak2-V617F/F594A/F595A (blue) using NAMD energy. (F) Comparison of the distance calculated between the specific carbon atoms of 617 and 1000 amongst Jak2-WT (black, V617 CB and V1000 CB), Jak2-V617F (red, F617 CZ and V1000 CB) and Jak2-V617F/F594A/F595A (blue, F617 CZ and V1000 CB) using the VMD graphics tools.

Figure 3. A potential π stacking interaction between F595 and F617 alters the JH1-JH2 interaction and active site conformation in Jak2-V617F

(A) Snap shots of the surface representations of the JH1 (orange) and JH2 (cyan) domains generated in VMD viewer from the MD simulations of Jak2-WT, Jak2-V617F, and Jak2-V617F/F594A/F595A at 20 ns. The activation loop is shown in yellow, with the residues Y1007/Y1008 in blue, V1000 in white, V/F617 in mauve, and F595 in green. Distance between the JH1 and JH2 domains is represented by the white double-headed arrow. (B) Cartoon representation of the JH1 kinase domain for Jak2-WT, Jak2-V617F and Jak2-V617F/F594A/F595A, displaying the active site conformation. N859 is shown in magenta, catalytic loop is shown in cyan (K970-N981), activation loop (D994-E1024) in yellow and the unique Jak2 insertion loop (S1056-I1078) is shown in ice blue (top). The change in distance between the two is indicated by a white double-headed arrow in a magnified view of the active site (bottom). (C) Comparison of the distance calculated between the specific carbon atoms of 859 and 1064 amongst Jak2-WT (black, N859 CG and M1064 CE), Jak2-V617F (red, N859 CG and M1064 CE) and Jak2-V617F/F594A/F595A (blue, N859 CG and M1064 CE) using the VMD graphics tools.

Figure 4. Jak2 sequence conservation at F594, F595 and V617

Multiple sequence alignment of the human Jak2 primary amino acid sequence with different species (A) and with other human JAK family members (B). Sequence conservation at F594 and F595 are highlighted in blue and that at position V617 is shown in orange. The non-conserved amino acid at 595 is highlighted in pink and that at 617 is shown in green. The reference sequence positions are indicated for human Jak2 protein.

Figure 5. Mutation of F594 and F595 to Ala impairs the autophosphorylation and kinase activity of Jak2-V617F

A) COS-7 cells were transfected with 10 μg of the indicated plasmids and the following day, Jak2 protein was immunoprecipitated from the transfected cells and western blotted for phospho-Jak2 to detect autophosphorylation at Y1007 and Y1008 (Top). The membrane was stripped and re-probed for total Jak2 (bottom). (B) Jak2 autophosphorylation was quantified using densitometry for at least five independent experiments and plotted as a function of Jak2 mutation status. (C) Immunoprecipitated Jak2 from COS-7 cells transfected with the indicated plasmids was allowed to phosphorylate 1 μg of GST-STAT1 in vitro. Phospho-STAT1 and phospho-Jak2 levels were detected by Western blot analysis. The membranes were stripped and re-probed for total STAT1 and total Jak2. (D) Phosphorylation of GST-STAT1 was quantified using densitometry from at least three independent experiments and the levels for each mutant were plotted as a function of Jak2 mutation status. Values are expressed as mean ± S.D, * p < 0.05, **p < 0.005 (Student’s t-test).

Figure 6. Mutation of F594 and F595 to Ala impairs STAT mediated gene transcription downstream of Jak2-V617F

COS-7 cells were co-transfected with 5 μg of the indicated plasmids along with 5 μg of luciferase plasmid. Luciferase activity was measured from the cell lysates using the Reporter Lysis Buffer. Relative Luminescence Units (RLU) were averaged from at least three independent experiments and plotted as a function Jak2 mutation status, ** p < 0.005.

Figure 7. Mutation of F594 and F595 to Ala does not affect Jak2-WT autophosphorylation

(A) COS-7 cells were transfected with 10 μg of the indicated Jak2 expression plasmids and Jak2 protein was subsequently immunoprecipitated from the transfected cells and autophosphorylation was assessed by western blot analysis (top). The same membrane was stripped and re-probed for total Jak2 (bottom) (B) Jak2 autophosphorylation was quantified using densitometry and the average values from at least five independent experiments were plotted as a function of Jak2 mutation status, * p < 0.05.

Figure 8. Side chain structure and hydrophobicity of amino acids at 594, 595 and 617 are important for the constitutive activation of Jak2-V617F

(A) Autophosphorylation of the indicated Jak2 plasmids was assessed by western blot analysis as described previously (top). The same membrane was stripped and re-probed for total Jak2 (bottom). (B) Jak2 autophosphorylation was quantified using densitometry and the average values from at least three independent experiments were plotted as a function of Jak2 mutation status, * p < 0.05. (C) Luciferase assays were conducted using COS-7 cells transfected with the indicated Jak2 expression plasmids. The average luciferase activity (RLU) for each construct from at least three independent experiments was plotted as a function of Jak2 mutation status, * p < 0.05.

Figure 9. Characterization of the π stacking interaction between F595 and F617 by geometrical analysis

(A, B) Definition of the vectors required for the geometrical characterization of the π stacking interaction in Jak2-WT and Jak2-V617F. The centroid vector between the planes of amino acid side chains being compared (F595 and V/F617) is shown in orange and the normal vector that is perpendicular to the plane of side chain is shown in red for F595 and green for V/F617. Theta (θ) is defined as the angle between the normal for the reference amino acid, which is F595, and the centroid vector. Gamma (γ) is defined as the angle between the two normal vectors for the amino acids being compared (F595 and V/F617). (C) Comparison of the centroid distance, theta and gamma angles between 595 and 617 amongst Jak2-WT, Jak2-V617F and Jak2-V617F/F594A/F595A. The indicated parameters were calculated over the period of the individual MD simulations using VMD viewer and the probability density at each measured value was plotted using MATLAB with a bin width of 5°.