Site-specific phosphorylation protects glycogen synthase kinase-3ß from calpain-mediated truncation of its N and C termini.

Glycogen synthase kinase-3ß (GSK-3ß), 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-3ß 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-3ß occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3ß by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3ß at both N and C termini, whereas non-phosphorylatable mutant GSK-3ß S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3ß 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: ΔN-GSK-3ß (amino acids 39-420), ΔC-GSK-3ß (amino acids 1-384), and ΔN/ΔC-GSK-3ß (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3ß being the most active form. This observation suggests that the GSK-3ß C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3ß by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3ß 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-3ß activity.