title: Site-specific phosphorylation protects glycogen synthase kinase-3β from calpain-mediated truncation of its N and C termini.
abstract: Glycogen synthase kinase-3β (GSK-3β), 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-3β 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-3β occurred in tandem with Ser-9 dephosphorylation during cerebellar granule neuron apoptosis. Increasing Ser-9 phosphorylation of GSK-3β by inhibiting phosphatase 1/2A or pretreating with purified active Akt inhibited calpain-mediated cleavage of GSK-3β at both N and C termini, whereas non-phosphorylatable mutant GSK-3β S9A facilitated its cleavage. In contrast, Ser-389 phosphorylation selectively inhibited the cleavage of GSK-3β 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-3β (amino acids 39-420), ΔC-GSK-3β (amino acids 1-384), and ΔN/ΔC-GSK-3β (amino acids 39-384). All three truncated products showed increased kinase and pro-apoptotic activity, with ΔN/ΔC-GSK-3β being the most active form. This observation suggests that the GSK-3β C terminus acts as an autoinhibitory domain similar to the N terminus. Taken together, these findings demonstrate that calpain-mediated cleavage activates GSK-3β by removing its N- and C-terminal autoinhibitory domains and that Ser-9 phosphorylation inhibits the cleavage of GSK-3β 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-3β activity.
authors.affiliation: Department of Pharmacology and Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080, China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Ma
authors.forename: Shaojun
authors.initials: S
authors.lastname: Liu
authors.forename: Qiaoying
authors.initials: Q
authors.lastname: Huang
authors.forename: Bo
authors.initials: B
authors.lastname: Xie
authors.forename: Bingquan
authors.initials: B
authors.lastname: Lai
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.forename: Bin
authors.initials: B
authors.lastname: Song
authors.forename: Mingtao
authors.initials: M
authors.lastname: Li
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002154:Calpain / Q000378:metabolism*; D002531:Cerebellum / Q000166:cytology; D005973:Glutamine / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000737:chemistry / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D057809:HEK293 Cells; D006801:Humans; D007532:Isoleucine / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D009410:Nerve Degeneration / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation / Q000502:physiology; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
chemical_list: D007532:Isoleucine; D005973:Glutamine; D013912:Threonine; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D002154:Calpain