From the Department of Medicine (S.-B.W., V.V., T.L., R.H., M.R., D.A.K., B.O'R., J.E.V.E.).
Johns Hopkins University, Baltimore, MD; Department of Medicine, Advanced Clinical Biosystems Research Institute, The Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., R.H., J.E.V.E.).
Circ Res. 2018 May 25;122(11):1517-1531. doi: 10.1161/CIRCRESAHA.118.312789. Epub 2018 Mar 21.
GSK-3β (glycogen synthase kinase 3β) is a multifunctional and constitutively active kinase known to regulate a myriad of cellular processes. The primary mechanism to regulate its function is through phosphorylation-dependent inhibition at serine-9 residue. Emerging evidence indicates that there may be alternative mechanisms that control GSK-3β for certain functions.
Here, we sought to understand the role of protein -nitrosylation (SNO) on the function of GSK-3β. SNO-dependent modulation of the localization of GSK-3β and its ability to phosphorylate downstream targets was investigated in vitro, and the network of proteins differentially impacted by phospho- or SNO-dependent GSK-3β regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied.
We found that GSK-3β undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes, as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. -nitrosylation of GSK-3β significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. -nitrosylation of GSK-3β promotes its nuclear translocation and access to novel downstream phosphosubstrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phosphoproteomics pathway analysis reveals that nuclear GSK-3β plays a central role in cell cycle control, RNA splicing, and DNA damage response.
The results indicate that SNO has a differential effect on the location and activity of GSK-3β in the cytoplasm versus the nucleus. SNO modification of GSK-3β occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.
GSK-3β（糖原合酶激酶 3β）是一种多功能且组成性激活的激酶，已知可调节多种细胞过程。调节其功能的主要机制是通过丝氨酸-9 残基的磷酸化依赖性抑制。新出现的证据表明，对于某些功能，可能存在控制 GSK-3β 的替代机制。
本研究旨在了解蛋白质硝化（SNO）对 GSK-3β功能的作用。在体外研究了 SNO 依赖性调节 GSK-3β的定位及其磷酸化下游靶标的能力，并研究了在心力衰竭发展过程中受磷酸化或 SNO 依赖性 GSK-3β调节以及体内关键信号激酶的 SNO 修饰影响的差异蛋白网络。
我们发现 GSK-3β在体外（在 HEK293 细胞、H9C2 成肌细胞和原代新生大鼠心室肌细胞中）以及体内（在心力衰竭和心脏性猝死动物模型的心脏中）均发生特异性 SNO。GSK-3β 的硝化作用显著抑制其激酶活性，而不依赖于典型的磷酸化抑制途径。GSK-3β 的硝化作用促进其核易位，并使其能够进入新型的下游磷酸化底物，这些底物富含新型氨基酸共识序列基序。定量磷酸蛋白质组学通路分析表明，核 GSK-3β在细胞周期控制、RNA 剪接和 DNA 损伤反应中起核心作用。
结果表明，SNO 对细胞质和核内 GSK-3β的位置和活性有不同的影响。GSK-3β 的 SNO 修饰发生在体内，可能导致心力衰竭和心脏性猝死的病理生物学发生。
