Glycogen synthase kinase-3β (GSK-3β), a key regulator of neuronal apoptosis, is inhibited via phosphorylation on Ser-9, and has also been recently shown to be cleaved by calpain at the N-terminus and subsequently activated.In the present study we show that calpain truncates GSK-3β at not only N-terminus but also C-terminus, which occurs following Ser9 dephosphorylation during neuronal apoptosis.We found that, calpain-mediated cleavage was significantly attenuated by increasing Ser9 phosphorylation of GSK-3β via inhibition of phosphatase 1/2A in vivo or pretreatment with purified active Akt in vitro.Mutating serine 9 to alanine increased GSK-3βs interaction with and sensitivity to calpain.These results demonstrate that Set9 phosphorylation protects GSK-3β from calpain-mediated truncation through interrupting their interaction.Mass spectrometry analysis indicated GSK-3β cleavage occurred at residues Thr38-Thr39 and Ile384-Gln385.Calpain cleavage generated three truncated products all leaving the kinase domain intact, AN-GSK-3β (39-420aa), AC-GSK-3β (1-384aa) and AN/C-GSK-3β (39-384aa), each of them exhibiting increased kinase activity and pro-apoptotic effect.These results suggest that GSK-3β C-terminus,similar to N-terminus, serves as an auto-inhibitory domain.In agreement with this, C-terminal peptide inhibits GSK-3β activity and protects neurons from death in a Ser389 phosphorylation dependent manner.We therefore conclude that an important role of GSK-3β cleavage during apoptosis is the removal of its N-/C-terminal inhibitory domains to increase its activity, whereas an opposing role of Ser9 phosphorylation of GSK-3β during survival is the direct suppression of its activity and the decrease of calpain-mediated cleavage.