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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
In vitro SCFbeta-Trcp1-mediated IkappaBalpha ubiquitination assay for high-throughput screen
Shuichan Xu1, Palka Patel, Mahan Abbasian
1Celgene Corporation, San Diego, California, USA.
Insights
We developed an in vitro assay to measure IkappaBalpha ubiquitination, a key step in NF-kappaB pathway activation. This assay can identify inhibitors that may offer new treatments for inflammatory diseases and cancer.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Signaling
Background:
- Constitutive activation of NF-kappaB is implicated in tumorigenesis and inflammation.
- Ubiquitination and degradation of IkappaB are crucial for NF-kappaB pathway activation.
Purpose of the Study:
- To establish an in vitro assay system for IkappaBalpha ubiquitination.
- To identify potential inhibitors of IkappaBalpha ubiquitination for therapeutic applications.
Main Methods:
- Utilized purified components: E1, E2, SCF(beta-Trcp1) E3 ligase, IkappaBalpha, IKK2, and ubiquitin.
- Developed a 384-well plate assay using antibody capture of IkappaBalpha and detection of biotinylated ubiquitin with Eu-labeled streptavidin.
- Demonstrated that IkappaBalpha ubiquitination is dependent on IKK2 phosphorylation and the presence of the ubiquitination machinery.
Main Results:
- Successfully generated ubiquitinated IkappaBalpha in vitro.
- Established a robust and sensitive assay for monitoring IkappaBalpha ubiquitination.
- Confirmed the requirement of IKK2 phosphorylation for IkappaBalpha ubiquitination.
Conclusions:
- The developed assay system enables the discovery of inhibitors targeting IkappaBalpha ubiquitination.
- Inhibitors blocking IkappaBalpha ubiquitination could stabilize IkappaB levels, inhibiting NF-kappaB activation.
- This approach offers a potential therapeutic strategy for NF-kappaB-related diseases.
Abstract:
An increasing body of evidence indicates that constitutive activation of NF-kappaB contributes to tumorigenesis and inflammation. Ubiquitination and degradation of IkappaB plays an essential role in NF-kappaB activation. Here we describe an in vitro IkappaBalpha ubiquitination assay system in which purified E1, E2, SCF(beta-Trcp1) E3, IkappaBalpha, IKK2, and Ub were used to generate ubiquitinated IkappaBalpha. The ubiquitination of IkappaBalpha is strictly dependent on its phosphorylation by IKK2, as well as the presence of E1, E2, E3, and Ub. The assay was adapted into 384-well plate format in which an antibody against IkappaBalpha was used to capture IkappaBalpha, and the biotinylated ubiquitin attached to IkappaBalpha was detected with europium (Eu)-labeled streptavidin. This assay can be used to discover inhibitors of IkappaBalpha ubiquitination. Such inhibitors would block NF-kappaB activation by stabilizing IkappaB levels in cells and thus provide a new therapeutic approach to NF-kappaB-related human diseases.
