Janus kinase 2 activation mechanisms revealed by analysis of suppressing mutations

Abstract

These results suggest that therapeutic approaches targeting the JH2 ATP binding site and αC could be effective in inhibiting most pathogenic mutations. JH2 ATP site targeting has the potential for reduced side effects by retaining erythropoietin and IFN-γ functions. Simultaneously, however, we identified the JH2 αC interface as a potential target for pathway-selective JAK inhibitors in patients with diseases with unmutated JAK2, thus providing new insights into the development of novel pharmacologic interventions.

Structural models of full-length JAK2 kinase

“…Consistent with this prediction, our mutagenesis experiments showed that a number of JAK2 mutations at these interfaces ( Figure 5A) modulated the constitutive activity of V617F. Unlike previously identified V617F-rescuing mutations 23,24,33,41 located at the N lobe of JH2 or at the SH2-JH2 linker, the mutations we identified here are novel in that they are located on the FERM unit or C lobe of JH2. The mutagenesis experiments provide compelling evidence for our models, in which FERM plays an important structural role in both the active and inactive conformations.…”

“…This is consistent with a recent analysis of mutations that suppress the activity of V617F. 41 In our active dimer model, the F617 residue is packed with F595 and F537, and E592 forms a salt bridge with K415 (of SH2), while E596 forms a salt bridge with K502 (of SH2). These observations offer a structural explanation for the rescuing effect of the F595A, F537A, E592R, and E596R mutations.…”

“…In view of JH1 needing to be set free for auto-transphosphorylation upon ligand binding and activation, JH2 is likely involved in mediating the dimerization of JAKs. This was previously suggested based on mutagenesis studies, 14,22,40,41 but the structural basis has remained unclear.…”

“…In the active monomer model, E666 (of JH2) forms a salt-bridge with the K497 residue (of SH2) at the SH2-JH2 interface; our experiments showed that E666K and K497E mutations each suppressed the constitutive activity of V617F ( Figure 5B; Figure S6A and S6B; Figure S7) to a level similar to the kinase-dead D976N mutation. 41 The I324, D348, K497, and E666 residues are largely exposed in the inactive model and do not appear to be involved in important and stable inter-residue interactions, and they are unlikely to affect the stability of the inactive conformation.…”

“…These observations offer a structural explanation for the rescuing effect of the F595A, F537A, E592R, and E596R mutations. 23,24,33,41 I682 and R683 are two other sites of prevalent pathogenic mutations. Similarly to V617, these two JH2 residues are located at the inter-domain interface in our model of the inactive dimer, interacting with the β2-β3 loop of JH1.…”