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Active Cdk5 Immunoprecipitation and Kinase Assay.
Andrew N Bankston1,2, Li Ku1, Yue Feng1
1Department of Pharmacology, Emory University, Atlanta, GA.
Bio-Protocol
|September 5, 2017
Summary
Cyclin-dependent kinase 5 (Cdk5) activity is regulated by p35 and p39 activators. This study details a method to measure total Cdk5 activity, identifying p39 as the main activator in oligodendroglial cells.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Cyclin-dependent kinase 5 (Cdk5) activity is modulated by its regulatory subunits, p35 and p39.
- The distinct properties of p35-Cdk5 and p39-Cdk5 complexes necessitate specific assay conditions.
- Direct measurement of Cdk5 activation requires preserving interactions with its activators.
Purpose of the Study:
- To develop and validate a robust protocol for measuring total Cdk5 activity.
- To preserve both p35-Cdk5 and p39-Cdk5 complexes during cell lysis and immunoprecipitation.
- To identify the primary Cdk5 activator in the oligodendroglial lineage.
Main Methods:
- Cell lysis and protein concentration determination in post-nuclear supernatant.
- Immunoprecipitation of Cdk5 from equal protein amounts, followed by washing to remove non-specific proteins.
- In vitro kinase assay using histone H1 as a substrate and [γ-32P]ATP, with subsequent SDS-PAGE and immunoblotting.
Main Results:
- The developed assay successfully preserves p35- and p39-Cdk5 complexes.
- Histone H1 phosphorylation indicates Cdk5 kinase activity.
- p39 was identified as the predominant activator of Cdk5 in oligodendroglial cells.
Conclusions:
- This protocol provides a reliable method for assessing total Cdk5 activity across different cell types.
- The findings highlight the crucial role of p39 in regulating Cdk5 function within the oligodendroglial lineage.
- The assay is adaptable for investigating Cdk5 regulation in various cellular contexts.
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