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

P>Mammalian glycogen synthase kinase-3 (GSK3) is generated from two genes, GSK3 alpha and GSK3 beta, while a splice variant of GSK3 beta (GSK3 beta 2), containing a 13 amino acid insert, is enriched in neurons. GSK3 alpha and GSK3 beta deletions generate distinct phenotypes. Here, we show that phosphorylation of CRMP2, CRMP4, beta-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 GSK3 beta, yet normal in cortex lacking GSK3 alpha). Furthermore, the neuron-enriched GSK3 beta 2 variant phosphorylates phospho-glycogen synthase 2 peptide, CRMP2 (Thr509/514), CRMP4 (Thr509), Inhibitor-2 (Thr72) and tau (Ser396), at a lower rate than GSK3 beta 1. In contrast phosphorylation of c-Myc and c-Jun is equivalent for each GSK3 beta isoform, providing evidence that differential substrate phosphorylation is achieved through alterations in expression and splicing of the GSK3 gene. Finally, each GSK3 beta 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 GSK3 beta 1 and GSK3 beta 2 are similar.