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

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.


Related in: MedlinePlus

Transformation potential of Cbl variants in focus formation assays. a Immunoblot of lysates from 3T3 fibroblasts infected with FLAG-tagged Cbl variants using α-FLAG antibody (top) and α-actin (bottom) antibody as a loading control. b Sulforhodamine B-stained 3T3 fibroblasts infected with indicated Cbl variants. c Mean number of foci formed by Cbl variant-infected 3T3 fibroblasts shown in a bar graph (n = 3). No foci were present in 3T3 cells infected with wild-type Cbl, Cbl M222E or Cbl Y368F. Double asterisks (**) denote significant differences (P < 0.05) between indicated Cbl variant and wild-type using ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate standard deviation. d As in (c) but for A564 of extracted sulforhodamine B from Cbl-infected 3T3 fibroblasts
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Fig6: Transformation potential of Cbl variants in focus formation assays. a Immunoblot of lysates from 3T3 fibroblasts infected with FLAG-tagged Cbl variants using α-FLAG antibody (top) and α-actin (bottom) antibody as a loading control. b Sulforhodamine B-stained 3T3 fibroblasts infected with indicated Cbl variants. c Mean number of foci formed by Cbl variant-infected 3T3 fibroblasts shown in a bar graph (n = 3). No foci were present in 3T3 cells infected with wild-type Cbl, Cbl M222E or Cbl Y368F. Double asterisks (**) denote significant differences (P < 0.05) between indicated Cbl variant and wild-type using ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate standard deviation. d As in (c) but for A564 of extracted sulforhodamine B from Cbl-infected 3T3 fibroblasts

Mentions: To test the transforming potential of Cbl Y371 mutants in cells, we performed focus formation assays using 3T3 cells stably transfected with N-terminally FLAG-tagged Cbl variants. Relative protein expression levels of each variant were assessed by immunoblotting (Fig. 6a). Foci were visualized with sulforhodamine B staining and counted manually with a DNA Safe Imager (Invitrogen). Afterwards, the sulforhodamine B stain was extracted and the A564 measured to compare relative cell densities [26, 27]. Foci counts and relative cell densities were analyzed by one-way ANOVA followed by Dunnett’s test with wild-type Cbl as the control. Cbl70Z was included among the variants tested as a positive transforming control [22]. Other variants tested included the same set used in our biochemical assays except in a full-length context.Fig. 6


Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation
Transformation potential of Cbl variants in focus formation assays. a Immunoblot of lysates from 3T3 fibroblasts infected with FLAG-tagged Cbl variants using α-FLAG antibody (top) and α-actin (bottom) antibody as a loading control. b Sulforhodamine B-stained 3T3 fibroblasts infected with indicated Cbl variants. c Mean number of foci formed by Cbl variant-infected 3T3 fibroblasts shown in a bar graph (n = 3). No foci were present in 3T3 cells infected with wild-type Cbl, Cbl M222E or Cbl Y368F. Double asterisks (**) denote significant differences (P < 0.05) between indicated Cbl variant and wild-type using ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate standard deviation. d As in (c) but for A564 of extracted sulforhodamine B from Cbl-infected 3T3 fibroblasts
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5015263&req=5

Fig6: Transformation potential of Cbl variants in focus formation assays. a Immunoblot of lysates from 3T3 fibroblasts infected with FLAG-tagged Cbl variants using α-FLAG antibody (top) and α-actin (bottom) antibody as a loading control. b Sulforhodamine B-stained 3T3 fibroblasts infected with indicated Cbl variants. c Mean number of foci formed by Cbl variant-infected 3T3 fibroblasts shown in a bar graph (n = 3). No foci were present in 3T3 cells infected with wild-type Cbl, Cbl M222E or Cbl Y368F. Double asterisks (**) denote significant differences (P < 0.05) between indicated Cbl variant and wild-type using ANOVA followed by Dunnett’s multiple comparisons test. Error bars indicate standard deviation. d As in (c) but for A564 of extracted sulforhodamine B from Cbl-infected 3T3 fibroblasts
Mentions: To test the transforming potential of Cbl Y371 mutants in cells, we performed focus formation assays using 3T3 cells stably transfected with N-terminally FLAG-tagged Cbl variants. Relative protein expression levels of each variant were assessed by immunoblotting (Fig. 6a). Foci were visualized with sulforhodamine B staining and counted manually with a DNA Safe Imager (Invitrogen). Afterwards, the sulforhodamine B stain was extracted and the A564 measured to compare relative cell densities [26, 27]. Foci counts and relative cell densities were analyzed by one-way ANOVA followed by Dunnett’s test with wild-type Cbl as the control. Cbl70Z was included among the variants tested as a positive transforming control [22]. Other variants tested included the same set used in our biochemical assays except in a full-length context.Fig. 6

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&rsquo;s conformation. Here, we investigate how Tyr371 mutations affect Cbl&rsquo;s conformation in solution and how this relates to Cbl&rsquo;s ability to potentiate transformation in cells.

Results: To explore how Tyr371 mutations affect Cbl&rsquo;s properties, we used surface plasmon resonance to measure Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2&ndash;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&ndash;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&rsquo;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.


Related in: MedlinePlus