Measuring GSK3 expression and activity in cells

Adam R. Cole, Calum Sutherland

    Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)

    15 Citations (Scopus)

    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.
    Original languageEnglish
    Title of host publicationWnt signaling
    Subtitle of host publicationpathway methods and mammalian models
    EditorsElizabeth Vincan
    PublisherHumana Press
    Pages45-65
    Number of pages21
    ISBN (Electronic)9781597452496
    ISBN (Print)9781588299123
    DOIs
    Publication statusPublished - 2008

    Publication series

    NameMethods in Molecular Biology
    PublisherHumana Press
    Volume468
    ISSN (Print)1064-3745

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