Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformationReportar como inadecuado

Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Biology

, 14:76

First Online: 08 September 2016Received: 31 March 2016Accepted: 12 August 2016DOI: 10.1186-s12915-016-0298-6

Cite this article as: Buetow, L., Tria, G., Ahmed, S.F. et al. BMC Biol 2016 14: 76. doi:10.1186-s12915-016-0298-6


BackgroundCasitas B-lineage lymphoma Cbl or c-Cbl is a RING ubiquitin ligase that negatively regulates protein tyrosine kinase PTK signalling. Phosphorylation of a conserved residue Tyr371 on the linker helix region LHR between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available, they do not reveal how Tyr371 mutations affect Cbl’s conformation. Here, we investigate how Tyr371 mutations affect Cbl’s conformation in solution and how this relates to Cbl’s ability to potentiate transformation in cells.

ResultsTo explore how Tyr371 mutations affect Cbl’s properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin E2–Ub, small angle X-ray scattering studies to investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation potential in cells. Cbl Tyr371 mutants enhance E2–Ub binding and cause Cbl to adopt extended conformations in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are those that perturb the LHR-substrate-binding domain interaction.

Conclusionsc-Cbl’s LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl.

KeywordsUbiquitin Cbl Myeloproliferative neoplasms SAXS Transformation potential AbbreviationsCblCasitas B-lymphoma lineage

DmaxMaximum particle distance

E2 ~ UbE2 conjugated to ubiquitin via a thioester

EOMEnsemble optimization method

GAGenetic algorithm

GSTGlutathione S-transferase

LHLinker helix

LHRLinker helix region

LL1Linker loop 1

LL2Linker loop 2

MDS-MPNMyelodysplastic syndromes-myeloproliferative neoplasms

MMMolecular mass

N-CblN-terminal fragment of Cbl comprising residues 47–435

PTKProtein tyrosine kinase

RflexQuantification of protein flexibility estimated by EOM

RgRadius of gyration

SAXSSmall angle X-ray scattering

SPRSurface plasmon resonance

TKTyrosine kinase

TKBDTyrosine kinase binding domain


UbcH5B–UbUbiquitin stably conjugated to UbcH5B via an isopeptide bond


Electronic supplementary materialThe online version of this article doi:10.1186-s12915-016-0298-6 contains supplementary material, which is available to authorized users.

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Autor: Lori Buetow - Giancarlo Tria - Syed Feroj Ahmed - Andreas Hock - Hao Dou - Gary J. Sibbet - Dmitri I. Svergun - Danny T


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