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Measuring GSK3 expression and activity in cells
Adam R Cole1, Calum Sutherland
1Pathology and Neurosciences, University of Dundee, Ninewells Hospital, Dundee, Scotland.
Abstract:
Glycogen synthase kinase (GSK)-3 is a key signalling intermediate in the action ofWnts. This protein kinase is ubiquitously expressed and has high inherent activity but is inhibited by activation of Wnt signalling or activation of growth factor receptor tyrosine kinases (e.g. insulin, nerve growth factor [NGF], platelet-derived growth factor [PDGF], etc.). The degree of inhibition of GSK3 in cells treated with such reagents is dependent on the cell type and the stimulus used. Therefore, the ability to accurately measure GSK3 activity in cells is an important aspect of GSK3 research. The activity of GSK3 is reduced by posttranslational modification (phosphorylation) and this can be measured by immunoblot with specific reagents (indirect), or by immunoprecipitation and assay (direct), as long as the modification is protected during these procedures. However, inhibition by phosphorylation is specific to cellular activation by growth factors and nutrients. Wnt inhibition of GSK3 does not involve phosphorylation of these residues on GSK3 and therefore it cannot be measured using this modification. Currently, the simplest way to assess Wnt inhibition of GSK3 is to monitor phosphorylation of specific GSK3 substrates in cells (e.g. beta-catenin). Alternatively, Wnt inhibition of GSK3 can be measured by partial purification of cellular GSK3 by ion exchange chromatography and assay of fractions or possibly by immunoprecipitation and assay. In this chapter, we demonstrate the use of the different approaches to measure GSK3 activity in SH-SY5Y cells, describe the best antibodies currently available, and discuss the potential drawbacks of each method.
Insights
Measuring glycogen synthase kinase (GSK)-3 activity is crucial for understanding Wnt signaling. This study evaluates methods for assessing GSK-3 inhibition, highlighting challenges and best practices for accurate measurement in cells.
Area of Science:
- Cellular biology
- Molecular signaling
Background:
- Glycogen synthase kinase (GSK)-3 is a central regulator in Wnt signaling pathways.
- GSK-3 activity is modulated by various stimuli, including Wnt signaling and growth factors, necessitating accurate measurement techniques.
- Different stimuli inhibit GSK-3 through distinct mechanisms, complicating activity assessment.
Purpose of the Study:
- To evaluate and compare different methods for measuring GSK-3 activity in cells.
- To identify the most effective antibodies for assessing GSK-3 activity.
- To discuss the limitations and advantages of each measurement approach.
Main Methods:
- Immunoblotting with specific antibodies to detect GSK-3 phosphorylation (indirect measurement).
- Immunoprecipitation followed by enzymatic assay (direct measurement).
- Monitoring phosphorylation of GSK-3 substrates, such as beta-catenin, to infer Wnt-mediated inhibition.
- Ion-exchange chromatography for partial purification and subsequent GSK-3 activity assay.
- Application of these methods in SH-SY5Y cells.
Main Results:
- Phosphorylation-based methods are suitable for measuring growth factor-induced GSK-3 inhibition but not Wnt-mediated inhibition.
- Wnt inhibition of GSK-3 cannot be detected by assessing GSK-3 phosphorylation.
- Monitoring substrate phosphorylation (e.g., beta-catenin) offers a viable approach for assessing Wnt-induced GSK-3 inhibition.
- Direct assays via immunoprecipitation or chromatography can measure Wnt-induced GSK-3 activity.
- Specific antibodies for GSK-3 detection and phosphorylation site analysis were identified.
Conclusions:
- Accurate measurement of GSK-3 activity is essential for studying Wnt signaling.
- Different methods are required to assess GSK-3 inhibition depending on the stimulus (Wnt vs. growth factors).
- The study provides a comprehensive guide to GSK-3 activity measurement techniques, including antibody selection and potential pitfalls, using SH-SY5Y cells as a model.
