Laboratory of Molecular Neuroscience, Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo, Japan.
Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo, Japan.
Sci Rep. 2017 Aug 17;7(1):8602. doi: 10.1038/s41598-017-09239-5.
Glycogen synthase kinase 3β (GSK3β) is a multifunctional protein kinase involved in many cellular activities including development, differentiation and diseases. GSK3β is thought to be constitutively activated by autophosphorylation at Tyr216 and inactivated by phosphorylation at Ser9. The GSK3β activity has previously been evaluated by inhibitory Ser9 phosphorylation, but it does not necessarily indicate the kinase activity itself. Here, we applied the Phos-tag SDS-PAGE technique to the analysis of GSK3β phosphoisotypes in cells and brains. There were three phosphoisotypes of GSK3β; double phosphorylation at Ser9 and Tyr216, single phosphorylation at Tyr216 and the nonphosphorylated isotype. Active GSK3β with phosphorylation at Tyr216 represented half or more of the total GSK3β in cultured cells. Although levels of phospho-Ser9 were increased by insulin treatment, Ser9 phosphorylation occurred only in a minor fraction of GSK3β. In mouse brains, GSK3β was principally in the active form with little Ser9 phosphorylation, and the phosphoisotypes of GSK3β changed depending on the regions of the brain, age, sex and disease conditions. These results indicate that the Phos-tag SDS-PAGE method provides a simple and appropriate measurement of active GSK3β in vivo, and the activity is regulated by the mechanism other than phosphorylation on Ser9.
糖原合酶激酶 3β（GSK3β）是一种多功能蛋白激酶，参与许多细胞活动，包括发育、分化和疾病。GSK3β 被认为通过 Tyr216 的自身磷酸化而持续激活，并通过 Ser9 的磷酸化而失活。GSK3β 的活性以前曾通过抑制性 Ser9 磷酸化来评估，但它并不一定表明激酶活性本身。在这里，我们将 Phos-tag SDS-PAGE 技术应用于细胞和大脑中 GSK3β 磷酸化异构体的分析。GSK3β 有三种磷酸化异构体；Ser9 和 Tyr216 的双重磷酸化、Tyr216 的单一磷酸化和非磷酸化同工型。在培养细胞中，具有 Tyr216 磷酸化的活性 GSK3β 代表总 GSK3β 的一半或更多。虽然胰岛素处理会增加磷酸化 Ser9 的水平，但 Ser9 磷酸化仅发生在 GSK3β 的一小部分中。在小鼠大脑中，GSK3β 主要处于活性形式，Ser9 磷酸化程度较低，并且 GSK3β 的磷酸化异构体根据大脑区域、年龄、性别和疾病状况而变化。这些结果表明，Phos-tag SDS-PAGE 方法为体内活性 GSK3β 提供了一种简单而合适的测量方法，其活性受 Ser9 磷酸化以外的机制调节。
