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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.


EOM 2.0 analysis of N-Cbl Tyr371 mutants, N-Cbl Y368F and N-Cbl M222E. Mutants were clustered according to their ability to perturb the LHR-TKBD interaction based on SAXS parameters shown in Table 3. a Strongly perturbing mutants. b Moderately perturbing mutants. c Weakly or non-perturbing mutants
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Fig5: EOM 2.0 analysis of N-Cbl Tyr371 mutants, N-Cbl Y368F and N-Cbl M222E. Mutants were clustered according to their ability to perturb the LHR-TKBD interaction based on SAXS parameters shown in Table 3. a Strongly perturbing mutants. b Moderately perturbing mutants. c Weakly or non-perturbing mutants

Mentions: Comparison of the SAXS-derived parameters as well as quantification of the flexibility (Rflex) estimated by using EOM revealed a link between the conformation of N-Cbl in solution and the extent of perturbation of the Tyr371-TKBD interaction similar to the trend observed in our SPR data (Tables 1 and 3 and Fig. 5). Our N-Cbl Y371 variants were classified into four categories, depending on the extent of LHR-TKBD perturbation observed based on our SPR findings: complete (pTyr371-N-Cbl), strongly-perturbing (Y371S, Y371D, Y371E), moderately perturbing (Y371A, Y371H, Y371C), and non-perturbing (Y371F). pTyr371-N-Cbl, which abolishes the LHR-TKBD interaction, appears the most elongated, whereas unphosphorylated, wild-type N-Cbl appears more compact. Compared to wild-type N-Cbl, the strongly perturbing mutants, N-Cbl Y371S, Y371E and Y371D, are partially elongated (Rgs are 2.6, 2.7 and 2.7 nm, respectively) but not as much as pTyr371-N-Cbl. Conformational rearrangements of these mutants are also confirmed by the corresponding Rflex values of approximately 84 %, 90 % and 88 % compared to approximately 60 % for wild-type N-Cbl. The moderately and non-perturbing mutants have Rg and Dmax values more similar to the wild-type N-Cbl, although with different degrees of flexibility (Table 3 and Fig. 5). Notably, N-Cbl Y371F appears slightly more compact and less flexible than wild-type N-Cbl, with an Rg of 2.38 nm, a Dmax of 7.67 nm, and an Rflex of approximately 56 %. Together with SPR, these data demonstrate that, in solution, RING domain accessibility is connected to N-Cbl conformational rearrangements induced by mutation or modification of Tyr371 in the LHR.Table 3


Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation
EOM 2.0 analysis of N-Cbl Tyr371 mutants, N-Cbl Y368F and N-Cbl M222E. Mutants were clustered according to their ability to perturb the LHR-TKBD interaction based on SAXS parameters shown in Table 3. a Strongly perturbing mutants. b Moderately perturbing mutants. c Weakly or non-perturbing mutants
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: EOM 2.0 analysis of N-Cbl Tyr371 mutants, N-Cbl Y368F and N-Cbl M222E. Mutants were clustered according to their ability to perturb the LHR-TKBD interaction based on SAXS parameters shown in Table 3. a Strongly perturbing mutants. b Moderately perturbing mutants. c Weakly or non-perturbing mutants
Mentions: Comparison of the SAXS-derived parameters as well as quantification of the flexibility (Rflex) estimated by using EOM revealed a link between the conformation of N-Cbl in solution and the extent of perturbation of the Tyr371-TKBD interaction similar to the trend observed in our SPR data (Tables 1 and 3 and Fig. 5). Our N-Cbl Y371 variants were classified into four categories, depending on the extent of LHR-TKBD perturbation observed based on our SPR findings: complete (pTyr371-N-Cbl), strongly-perturbing (Y371S, Y371D, Y371E), moderately perturbing (Y371A, Y371H, Y371C), and non-perturbing (Y371F). pTyr371-N-Cbl, which abolishes the LHR-TKBD interaction, appears the most elongated, whereas unphosphorylated, wild-type N-Cbl appears more compact. Compared to wild-type N-Cbl, the strongly perturbing mutants, N-Cbl Y371S, Y371E and Y371D, are partially elongated (Rgs are 2.6, 2.7 and 2.7 nm, respectively) but not as much as pTyr371-N-Cbl. Conformational rearrangements of these mutants are also confirmed by the corresponding Rflex values of approximately 84 %, 90 % and 88 % compared to approximately 60 % for wild-type N-Cbl. The moderately and non-perturbing mutants have Rg and Dmax values more similar to the wild-type N-Cbl, although with different degrees of flexibility (Table 3 and Fig. 5). Notably, N-Cbl Y371F appears slightly more compact and less flexible than wild-type N-Cbl, with an Rg of 2.38 nm, a Dmax of 7.67 nm, and an Rflex of approximately 56 %. Together with SPR, these data demonstrate that, in solution, RING domain accessibility is connected to N-Cbl conformational rearrangements induced by mutation or modification of Tyr371 in the LHR.Table 3

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.