A regulating role of the JAK2 FERM domain in hyperactivation of JAK2(V617F)

Abstract

JAK2 (Janus tyrosine kinase 2) is important for signalling through many cytokine receptors, and a gain-of-function JAK2 mutation in its pseudokinase domain, V617F, has been implicated in Philadelphia chromosome-negative myeloproliferative neoplasms. How this mutation hyperactivates JAK2 is poorly understood. In the present paper we report our findings that the V617F mutation has little effect on the Vmax of JAK2 kinase activity, but lowers the Km value for substrates. Therefore under physiological conditions where the concentration level of substrates is presumably below saturation, JAK2(V617F) exhibits hyperactivation compared with wild-type JAK2. This lower Km of JAK2(V617F) towards substrates requires the JAK2 FERM (4.1/ezrin/radixin/moesin) domain, as deletion of the FERM domain abolished this effect. We also show that, in contrast with its positive role in JAK2(V617F) hyperactivation, the FERM domain in wild-type JAK2 is inhibitory. Deletion or mutations of the FERM domain resulted in increased basal JAK2 kinase activity. The results of the present study provide the biochemical basis for how V617F hyperactivates JAK2, and identifies novel regulating roles of the JAK2 FERM domain to control kinase activity at different activation states.

Figure 1. JAK2(V617F) has a lower K_m towards a STAT5-derived peptide than JAK2 or JAK2 kinase domain

(A) Immunoprecipitated HA–JAK2, HA–JAK2(V617F) or HA–JH1 were immunoblotted with anti-phospho-JAK2 antibodies. The same blot was subsequently probed with antibodies against HA. IB, immunoblot; IP, immunoprecipitate; VF, V617F; WT, wild-type. (B) Kinetic analysis of the catalytic activity of HA–JAK2, HA–JAK2(V617F) and HA–JH1. Immunoprecipitated proteins were subjected to an in vitro kinase assay using a STAT5-derived peptide (1 mM) as a substrate. The reactions were stopped at various time points and the reaction mixtures were spotted on to P80 filter paper and counted with a scintillation counter. (C) Immunoprecipitated proteins were subjected to an in vitro kinase assay with various concentrations of a STAT5-derived peptide substrate. The reaction time was 30 min. The peptides were separated on SDS/PAGE followed by quantification using a PhosphorImager. V_max and K_m for each construct were shown. (D) Comparison of V_max values for HA–JAK2, HA–JAK2(V617F) and HA–JH1. Data were normalized to maximal wild-type JAK2 activity. (E) Comparison of K_m values for HA–JAK2, HA–JAK2(V617F) and HA–JH1. The activity of the kinases was normalized to the maximal activity of each construct.

Figure 2. JAK2(V617F) has a lower K_m value towards a JAK2-derived peptide than JAK2 or JAK2 kinase domain

Kinetic analysis of HA–JAK2 (A), HA–JAK2(V617F) (B) or HA–JH1 (C) in in vitro kinase assays using a JAK2-derived peptide as the substrate as described in Figure 1. (D) Comparison of K_m values between constructs.

Figure 3. The JAK2 FERM domain is required for the lower K_m value in JAK2(V617F)

(A) Schematic diagram of the various JAK2 constructs. (B) Immunoprecipitated kinases were immunoblotted with anti-phospho-JAK2 or anti-HA antibodies. (C) Immunoprecipitated HA–ΔFERM or HA–ΔFERM(V617F) were subjected to an in vitro kinase assay using a STAT5-derived peptide (1 mM) as a substrate. The reactions were stopped at various time points and the reaction mixtures were spotted on to P80 filter paper and counted with a scintillation counter. (D) Immunoprecipitated proteins were subjected to an in vitro kinase assay with various concentrations of a STAT5-derived peptide substrate. The reaction time was 30 min. The peptides were separated by SDS/PAGE followed by quantification using a PhosphorImager. (E) HA–JAK2, HA–JAK2(V617F), HA–ΔFERM and HA–ΔFERM(V617F) bound STAT5(Y649F)–Myc. Cell lysates expressing STAT5(Y694F)–Myc with HA–JAK2, HA–JAK2(V617F), HA–ΔFERM, HA–ΔFERM(V617F) or vector alone (−) were subjected to immunoprecipitation with HA-affinity resin and probed with anti-Myc antibodies. IB, immunoblot; IP, immunoprecipitate; VF, V617F; WT, wild-type.

Figure 4. Mutations in the JAK2 FERM domain affect JAK2 catalytic activity

(A) Y114A, Y114C or Y114F mutations were made on the background of HA–JAK2 (lanes 1–4) or HA–JAK2(V617F) (lanes 5–8). Immunoprecipitated kinases from HEK-293T cells were immunoblotted with anti-phospho-JAK2 or anti-HA antibodies. (B) The JAK2 FERM domain interacts with and inhibits the JAK2 kinase domain. Wild-type GST–FERM or GST–FERM with Y114A, Y114C and Y114F were transiently expressed with HA–JH1 in HEK-293T cells. Cell lysates were probed with anti-phospho-JAK2 or anti-HA antibodies (bottom two panels). GST, GST–FERM or GST–FERM with mutations were pulled down by glutathione resin and the precipitants were probed with antibodies against HA or GST (top two panels). IB, immunoblot; WT, wild-type; YA, Y114A; YC, Y114C; YF, Y114F.

Figure 5. A model of intramolecular interactions in JAK2 and JAK2(V617F)

(A) Wild-type JAK2 in the basal state is auto-inhibited by intramolecular interactions. The interaction between JH2 and JH1 keeps the activation loop folded inside the ATP-binding pocket, and the interaction between the FERM domain and JH1 is inhibitory (indicated by ●). (B) JH1 in isolation is active, but has a high K_m. (C) In JAK2(V617F), the inhibitory JH1–JH2 interaction is disrupted so that the activation loop is released, and the FERM domain participates in stabilizing the JH1 domain in a conformation that has a lower K_m compared with JH1 alone (indicated by a filled star). (D) In the absence of the FERM domain, although the inhibitory JH1–JH2 interaction is disrupted by V617F, JH1 cannot adopt the conformation with the lower K_m towards the substrate.