Abstract
JAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed in vitro using a Cbl ubiquitination assay. Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.
Highlights
Janus kinase 2 (JAK2) is a member of the Janus kinase family, which belongs to the non-receptor tyrosine kinase superfamily
We demonstrated that GMRβ interacts with intersectin 2, and this interaction is required for ligand-induced JAK2 activation[35]
We observed that ubiquitination of JAK2 was clearly increased when JAK2 was phosphorylated upon granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, but was not elevated for GMRβ and STAT5 (Fig. 1a)
Summary
Janus kinase 2 (JAK2) is a member of the Janus kinase family, which belongs to the non-receptor tyrosine kinase superfamily. In 2005, a gain-of-function somatic JAK2 mutation, V617F, was identified to be highly prevalent in myeloproliferative disorders[4] Patients with this gain-of-function mutation have frequently been identified in polycythemia vera (PV; 95%), essential thrombocythemia (ET; 20–40%), and primary myelofibrosis (PMF; 50%)[4,5,6,7]. These findings extend the importance of JAK2 dysregulation to include hematopoietic malignancies, in addition to the conventionallyrecognized inflammatory and immunological disorders. The architecture of JAK family proteins has been highly conserved through evolution These proteins contain four conserved domains: FERM, SH2, JH2 pseudo-kinase, and JH1 kinase. Cbl contains a tyrosine kinase-binding (TKB) domain at its N-terminus, followed by a linker region, a central zinc-binding C3HC4 RING finger motif, and a number of proline-rich motifs at the polypeptide C-terminus[24,25,26]
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