Department of Pharmacology and Proteomics Center, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou 510080, China.
J Biol Chem. 2012 Jun 29;287(27):22521-32. doi: 10.1074/jbc.M111.321349. Epub 2012 Apr 11.
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.
糖原合酶激酶-3β（GSK-3β）是神经元凋亡的关键调节因子，其活性受到丝氨酸 9/389 位磷酸化的抑制，最近发现它可被钙蛋白酶在 N 端切割，从而被激活。本研究发现钙蛋白酶不仅可在 GSK-3β的 N 端，而且还可在 C 端切割该酶，其切割位点位于 Thr-38-Thr-39 和 Ile-384-Gln-385 残基。此外，在小脑颗粒神经元凋亡过程中，GSK-3β的切割与 Ser-9 去磷酸化同时发生。通过抑制磷酸酶 1/2A 或用纯化的活性 Akt 预处理，增加 GSK-3β Ser-9 的磷酸化可抑制钙蛋白酶介导的 GSK-3β 在 N 和 C 端的切割，而非磷酸化突变体 GSK-3β S9A 则促进其切割。相反，Ser-389 磷酸化选择性地抑制 GSK-3β 在 C 端而非 N 端的切割。钙蛋白酶介导的切割导致三个截断产物，它们均含有完整的激酶结构域：ΔN-GSK-3β（残基 39-420）、ΔC-GSK-3β（残基 1-384）和 ΔN/ΔC-GSK-3β（残基 39-384）。所有三个截断产物的激酶和促凋亡活性均增加，其中 ΔN/ΔC-GSK-3β 活性最强。这一观察结果表明，GSK-3β 的 C 端类似于 N 端，充当一个自身抑制结构域。综上所述，这些发现表明钙蛋白酶介导的切割通过去除 GSK-3β 的 N 和 C 端自身抑制结构域而激活 GSK-3β，而 Ser-9 磷酸化抑制 GSK-3β 在两端的切割。相反，Ser-389 磷酸化仅抑制 C 端切割而不抑制 N 端切割。这些发现还确定了一种机制，通过该机制，特定部位的磷酸化和钙蛋白酶介导的切割协同作用来调节 GSK-3β 的活性。
