Mpl is activated by dimers of MPN-linked calreticulin mutants stabilized by disulfide bonds and ionic interactions

Abstract

Myeloproliferative neoplasms (MPNs) are frequently driven by insertions and deletions within the gene encoding calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Although oncogenic transformation requires both mutated CRT and the myeloproliferative leukemia protein (Mpl), the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. Our studies reveal that the novel C-domain of CRTDel52 encodes specificity both for Mpl binding and for disulfide-mediated CRT dimerization. Disulfide-stabilized CRTDel52 dimers and multimers are observed in MPN patient-derived platelet lysates and in transfected mammalian cells. Cysteine mutations within both the novel C-domain (C400A and C404A) and the conserved N-domain (C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of crystalized CRT oligomers, we tested the relevance of ionic interactions between charged residues proximal to C163 at the N-domain dimerization interface. Charge alteration at these residues affected dimerization and multimerization of both wild type and CRTDel52. Elimination of intermolecular disulfides and disruption of ionic interactions at both proposed dimerization interfaces was required to abrogate the ability of CRTDel52 to induce cytokine-independent cell proliferation via Mpl. Based on these findings, we propose a structural model of the Mpl-activating CRTDel52 unit as a covalently-linked dimer that is stabilized by disulfides and ionic interactions at both the C-domain and N-domain. MPNs exploit a natural dimerization interface of CRT combined with C-domain gain-of-functions to achieve cell transformation.

Functional Consequences of Mutations in Myeloproliferative Neoplasms

“… 21 , 22 Dimerization/oligomerization of mutant CALR contributes to the activation of MPL. 22 , 77 , 78 The interaction between the mutant CALR proteins and MPL leads to thermal stabilization of MPL and can be interrupted by a small molecule that binds to the N-glycan binding domain of CALR. 79 While intracellular complexes of MPL and CALR mutants do induce JAK-STAT signaling, this is not sufficient to transform to autonomous growth cytokine-dependent hematopoietic cells.…”

“…Activation of MPL requires both the N‐terminal glycan‐binding domain of CALR mutants and their positively charged tails 21,22 . Dimerization/oligomerization of mutant CALR contributes to the activation of MPL 22,77,78 . The interaction between the mutant CALR proteins and MPL leads to thermal stabilization of MPL and can be interrupted by a small molecule that binds to the N‐glycan binding domain of CALR 79 .…”