Hyperactivation of Oncogenic JAK3 Mutants Depend on ATP Binding to the Pseudokinase Domain.

Juuli Raivola,Alister C Ward,Vilasha Bulleeraz,Anniina T Virtanen,Henrik M Hammarén,Olli Silvennoinen

doi:10.3389/fonc.2018.00560

Abstract

Janus kinase 3 (JAK3) tyrosine kinase has a central role in the control of lymphopoiesis, and mutations in JAK3 can lead to either severe combined immunodeficiency or leukemia and lymphomas. JAK3 associates with the common gamma chain (γc) receptor and functions in a heteromeric signaling pair with JAK1. In IL-2 signaling JAK1 is the effector kinase for STAT5 phosphorylation but the precise molecular regulatory mechanisms of JAK1 and JAK3 and their individual domains are not known. The pseudokinase domain (JAK homology 2, JH2) of JAK3 is of particular interest as approximately half of clinical JAK3 mutations cluster into it.In this study, we investigated the role of JH2s of JAK1 and JAK3 in IL-2R signaling and show that STAT5 activation requires both JH1 and JH2 of JAK1, while both JH1 and JH2 in JAK3 are specifically required for the cytokine-induction of cellular signaling. Characterization of recombinant JAK3 JH2 in thermal shift assay shows an unstable protein domain, which is strongly stabilized by ATP binding. Unexpectedly, nucleotide binding to JAK3 JH2 was found to be cation-independent. JAK3 JH2 showed higher nucleotide binding affinity in MANT-ATP and fluorescent polarization competition assays compared to the other JAK JH2s. Analysis of the functional role of ATP binding in JAK3 JH2 in cells and in zebrafish showed that disruption of ATP binding suppresses ligand-independent activation of clinical JAK3 gain-of-function mutations residing in either JH2 or JH1 but does not inhibit constitutive activation of oncogenic JAK1. ATP-binding site mutations in JAK3 JH2 do not, however, abrogate normal IL-2 signaling making them distinct from JH2 deletion or kinase-deficient JAK3. These findings underline the importance of JAK3 JH2 for cellular signaling in both ligand-dependent and in gain-of-function mutation-induced activation. Furthermore, they identify the JH2 ATP-binding site as a key regulatory region for oncogenic JAK3 signaling, and thus a potential target for therapeutic modulation.

Highlights

The family of Janus kinases (JAK1, JAK2, Janus kinase 3 (JAK3), and TYK2) are key mediators of cytokine signaling that regulate hematopoietic cell development, cellular metabolism and immune responses
Immunoblot analysis of STAT5 Y694 phosphorylation from U4Cγβ cells transfected with JAK1 and JAK3 concurred with STAT5 transcriptional activity showing IL-2independent phosphorylation by JAK1 alone, lack of pSTAT5 by JAK3 alone and requirement for both JAK1 and JAK3 for IL-2-responsiveness (Supplementary Figure 1B)
We focused on characterization of JAK3 JH2 and its function in the context of both normal IL-2 receptor (IL-2R) signaling and oncogenic signaling mediated via activating JAK3 mutations

Summary

Introduction

The family of Janus kinases (JAK1, JAK2, JAK3, and TYK2) are key mediators of cytokine signaling that regulate hematopoietic cell development, cellular metabolism and immune responses. JAK3 functions in a heterodimeric pair with JAK1, which binds to the cytokine specific receptor β chains. In IL-2 receptor (IL-2R) signaling, JAK1 has been shown to play a dominant role in early cytoplasmic activation events where JAK1 is primarily driving STAT5 phosphorylation [11], while JAK3 is required for sustained activation during T-cell proliferation [11, 12]. Both functions are dependent on the catalytic activity of the JH1 domains in JAK1 and JAK3, respectively. Several aspects of IL-2R signal regulation are still elusive, especially with respect to the roles of the pseudokinase domains

Methods

Results

Conclusion