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Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.

ABSTRACT: The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule regulators of Cbl (RING) domain. The FP assay was used to measure binding affinities and inhibition constants of UbCH7 peptides and small molecule regulators of Cbl (RING) domains, respectively. In order to rule out promiscuous, aggregation-based inhibition, two assay conditions were developed and compared side by side. Under optimized conditions, we screened a 10,000 natural compound library in detergent-free and detergent-present (0.01% Triton X-100) systems. The results indicate that the detergent-present system is more suitable for high-throughput screens. Three potential compounds, methylprotodioscin, leonuride and catalpol, have been identified that bind to Cbl (RING) domain and interfere with the Cbl (RING)-UbCH7 protein-protein interaction.

SUBMITTER: Xie X

PROVIDER: S-EPMC3814989 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.

Xie Xingliang X Sun Lin L Pessetto Ziyan Yuan ZY Zhao Yan Y Zang Zhihe Z Zhong Ling L Wu Min M Su Qing Q Gao Xiurong X Zan Wang W Sun Yiyi Y

PloS one 20131031 10

The E3 ubiquitin protein ligase Casitas B-lineage Lymphoma (Cbl) proteins and their binding partners play an important role in regulating signal transduction pathways. It is important to utilize regulators to study the protein-protein interactions (PPIs) between these proteins. However, finding specific small-molecule regulators of PPIs remains a significant challenge due to the fact that the interfaces involved in PPIs are not well suited for effective small molecule binding. We report the deve ...[more]

PMID: 24205080

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Project description: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.

Dataset Information

Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.

Publications

Development of a fluorescence polarization based high-throughput assay to identify Casitas B-lineage lymphoma RING domain regulators.

Similar Datasets

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