Evidence that glycogen synthase kinase-3 isoforms have distinct substrate preference in the brain

Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3α and GSK3β, while a splice variant of GSK3β (GSK3β2), containing a 13 amino acid insert, is enriched in neurons. GSK3α and GSK3β deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, β-catenin, c-Myc, c-Jun and some residues on tau associated with Alzheimer's disease, is altered in cortical tissue lacking both isoforms of GSK3. This confirms that they are physiological targets for GSK3. However, deletion of each GSK3 isoform produces distinct substrate phosphorylation, indicating that each has a different spectrum of substrates (e.g. phosphorylation of Thr509, Thr514 and Ser518 of CRMP is not detectable in cortex lacking GSK3β, yet normal in cortex lacking GSK3α). Furthermore, the neuron-enriched GSK3β2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3β1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3β isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3β splice variant is phosphorylated to a similar extent at the regulatory sites, Ser9 and Tyr216, and exhibit identical sensitivities to the ATP competitive inhibitor CT99021, suggesting upstream regulation and ATP binding properties of GSK3β1 and GSK3β2 are similar.