Site-specific Phosphorylation Protects Glycogen Synthase Kinase-3β from Calpain-mediated Truncation of Its N and C Termini

Glycogen synthase kinase-3 beta (GSK-3 beta), a key regulator of neuronal apoptosis, is inhibited by the phosphorylation of Ser-9/Ser-389 and was recently shown to be cleaved by calpain at the N terminus, leading to its subsequent activation. In this study calpain was found to cleave GSK-3 beta not only at the N terminus but also at the C terminus, and cleavage sites were identified at residues Thr-38-Thr-39 and Ile-384-Gln-385. Furthermore, the cleavage of GSK-3 beta occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3 beta by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3 beta at both N and C termini, whereas non-phosphorylatable mutant GSK-3 beta S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3 beta at the C terminus but not the N terminus. Calpain-mediated cleavage resulted in three truncated products, all of which contained an intact kinase domain: Delta N-GSK-3 beta (amino acids 39-420), Delta C-GSK-3 beta (amino acids 1-384), and Delta N/Delta C-GSK-3 beta (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with Delta N/Delta C-GSK-3 beta being the most active form. This observation suggests that the GSK-3 beta C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3 beta by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3 beta at both termini. In contrast, Ser-389 phosphorylation inhibits only C-terminal cleavage but not N-terminal cleavage. These findings also identify a mechanism by which site-specific phosphorylation and calpain-mediated cleavage operate in concert to regulate GSK-3 beta activity.