An assay for glycogen synthase kinase 3 (GSK-3) for use in crude cell extracts

We have developed a specific and sensitive GSK-3 kinase assay which does not require prior purification of GSK-3. Its simplicity allows the rapid analysis of large numbers of samples. We have shown that it can detect GSK-3 activity in both Dictyostelium and mouse cells, and when used to examine the insulin response of 10T1/2 cells our assay method gives results comparable to those previously reported. The evolutionary distance between Dictyostelium and mouse GSK-3 kinases suggests that our assay can be applied to all species. In both of the cell lines investigated in this report, there is no evidence for a lithium-sensitive background of kinase activity which acts on the GSM substrate. This, however, may not be the case for other applications and should be tested using an unphosphorylated substrate control, as in our experiments and in previous assays (16,17). In the experiments described here we have used a novel peptide substrate, GSM; however, we see no reason why this assay should not work with all other GSK-3 peptide substrates and we have successfully carried out assays using both the original GS-2 substrate and the CREB peptide substrate (unpublished results).