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Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation

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ABSTRACT

Background: Casitas 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.

Results: To 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.

Conclusions: c-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.

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

No MeSH data available.


Cartoon ensemble representation of pTyr371-N-Cbl. The TKBD domain is coloured blue. The predicted RING domain conformations are shown in several colours as indicated in the table. The missing loops connecting the RING and TKBD domains are coloured according to the corresponding RING domains but presented as transparent surfaces
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Fig4: Cartoon ensemble representation of pTyr371-N-Cbl. The TKBD domain is coloured blue. The predicted RING domain conformations are shown in several colours as indicated in the table. The missing loops connecting the RING and TKBD domains are coloured according to the corresponding RING domains but presented as transparent surfaces

Mentions: Given the expected flexibility of pTyr371-N-Cbl, structural modelling was performed using an Ensemble Optimization Method (EOM) [45]. Here, ensembles of models with variable conformations are selected from a pool of randomly generated models such that the scattering from the ensemble fits the experimental data, and the distributions of the overall parameters (e.g. Rg) in the selected pool are compared to the original pool. As a negative control, EOM was first used on the data from N-Cbl and the selected ensembles showed predominantly compact conformations (Fig. 2d). In contrast, the selected ensembles for pTyr371-N-Cbl displayed broader distributions of predominantly extended conformations, which were on average more extended than the random pool (Fig. 2d). These data indicate that pTyr371-N-Cbl is extended and flexible in solution, suggesting that phosphorylation of the LH allows the RING domain to adopt multiple conformations in solution and access multiple surfaces of the TKBD (Fig. 4).Fig. 4


Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation
Cartoon ensemble representation of pTyr371-N-Cbl. The TKBD domain is coloured blue. The predicted RING domain conformations are shown in several colours as indicated in the table. The missing loops connecting the RING and TKBD domains are coloured according to the corresponding RING domains but presented as transparent surfaces
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015263&req=5

Fig4: Cartoon ensemble representation of pTyr371-N-Cbl. The TKBD domain is coloured blue. The predicted RING domain conformations are shown in several colours as indicated in the table. The missing loops connecting the RING and TKBD domains are coloured according to the corresponding RING domains but presented as transparent surfaces
Mentions: Given the expected flexibility of pTyr371-N-Cbl, structural modelling was performed using an Ensemble Optimization Method (EOM) [45]. Here, ensembles of models with variable conformations are selected from a pool of randomly generated models such that the scattering from the ensemble fits the experimental data, and the distributions of the overall parameters (e.g. Rg) in the selected pool are compared to the original pool. As a negative control, EOM was first used on the data from N-Cbl and the selected ensembles showed predominantly compact conformations (Fig. 2d). In contrast, the selected ensembles for pTyr371-N-Cbl displayed broader distributions of predominantly extended conformations, which were on average more extended than the random pool (Fig. 2d). These data indicate that pTyr371-N-Cbl is extended and flexible in solution, suggesting that phosphorylation of the LH allows the RING domain to adopt multiple conformations in solution and access multiple surfaces of the TKBD (Fig. 4).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: Casitas 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.

Results: To 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.

Conclusions: c-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.

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

No MeSH data available.