skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 11:00 PM ET on Thursday, June 13 until 2:00 AM ET on Friday, June 14 due to maintenance. We apologize for the inconvenience.


Title: Mechanism of mutant calreticulin-mediated activation of the thrombopoietin receptor in cancers

Myeloproliferative neoplasms (MPNs) are frequently driven by mutations within the C-terminal domain (C-domain) of calreticulin (CRT). CRTDel52 and CRTIns5 are recurrent mutations. Oncogenic transformation requires both mutated CRT and the thrombopoietin receptor (Mpl), but the molecular mechanism of CRT-mediated constitutive activation of Mpl is unknown. We show that the acquired C-domain of CRTDel52 mediates both Mpl binding and disulfide-linked CRTDel52 dimerization. Cysteine mutations within the novel C-domain (C400A and C404A) and the conserved N-terminal domain (N-domain; C163A) of CRTDel52 are required to reduce disulfide-mediated dimers and multimers of CRTDel52. Based on these data and published structures of CRT oligomers, we identify an N-domain dimerization interface relevant to both WT CRT and CRTDel52. Elimination of disulfide bonds and ionic interactions at both N-domain and C-domain dimerization interfaces is required to abrogate the ability of CRTDel52 to mediate cell proliferation via Mpl. Thus, MPNs exploit a natural dimerization interface of CRT combined with C-domain gain of function to achieve cell transformation.

 
more » « less
Award ID(s):
1855425
NSF-PAR ID:
10224058
Author(s) / Creator(s):
; ; ; ; ; ; ;
Publisher / Repository:
DOI PREFIX: 10.1083
Date Published:
Journal Name:
Journal of Cell Biology
Volume:
220
Issue:
7
ISSN:
0021-9525
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK–STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)‐localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN‐linked mutant CRT proteins have novel frameshifted carboxy‐domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+T cells. Mutant CRT‐linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK–STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT‐linked oncogenesis. In this review, we highlight recent findings related to the role of MPN‐linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

     
    more » « less
  2. Stochastic cell-surface expression of α-, β-, and γ-clustered protocadherins (Pcdhs) provides vertebrate neurons with single-cell identities that underlie neuronal self-recognition. Here we report crystal structures of ectodomain fragments comprising cell-cell recognition regions of mouse γ-Pcdhs γA1, γA8, γB2, and γB7 revealing trans-homodimers, and of C-terminal ectodomain fragments from γ-Pcdhs γA4 and γB2, which depict cis-interacting regions in monomeric form. Together these structures span the entire γ-Pcdh ectodomain. The trans-dimer structures reveal determinants of γ-Pcdh isoform-specific homophilic recognition. We identified and structurally mapped cis-dimerization mutations to the C-terminal ectodomain structures. Biophysical studies showed that Pcdh ectodomains from γB-subfamily isoforms formed cis dimers, whereas γA isoforms did not, but both γA and γB isoforms could interact in cis with α-Pcdhs. Together, these data show how interaction specificity is distributed over all domains of the γ-Pcdh trans interface, and suggest that subfamily- or isoform-specific cis-interactions may play a role in the Pcdh-mediated neuronal self-recognition code. 
    more » « less
  3. Summary

    Chronic infection withHelicobacter pylorican lead to the development of gastric ulcers and stomach cancers. The helical cell shape ofH. pyloripromotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non‐enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N‐terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C‐terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi‐protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.

     
    more » « less
  4. Abstract

    KdpD/KdpE two‐component signaling system regulates expression of a high affinity potassium transporter responsible for potassium homeostasis. The C‐terminal module of KdpD consists of a GAF domain linked to a histidine kinase domain. Whereas certain GAF domains act as regulators by binding cyclic nucleotides, the role of the juxtamembrane GAF domain in KdpD is unknown. We report the high‐resolution crystal structure of KdpD GAF domain (KdpDG) consisting of five α‐helices, four β‐sheets and two large loops. KdpDGforms a symmetry‐related dimer, wherein parallelly arranged monomers contribute to a four‐helix bundle at the dimer‐interface, SAXS analysis of KdpD C‐terminal module reveals an elongated structure that is a dimer in solution. Substitution of conserved residues with various residues that disrupt the dimer interface produce a range of effects on gene expression demonstrating the importance of the interface in inactive to active transitions during signaling. Comparison of ligand binding site of the classic cyclic nucleotide‐binding GAF domains to KdpDGreveals structural differences arising from naturally occurring substitutions in primary sequence of KdpDGthat modifies the canonical NKFDE sequence motif required for cyclic nucleotide binding. Together these results suggest a structural role for KdpDGin dimerization and transmission of signal to the kinase domain.

     
    more » « less
  5. Abstract

    The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion.

     
    more » « less