Protein S-Nitrosylation Controls Glycogen Synthase Kinase 3 beta Function Independent of Its Phosphorylation State

Rationale: GSK-3 beta (glycogen synthase kinase 3 beta) 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-3 beta for certain functions. Objectives: Here, we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK-3 beta. SNO-dependent modulation of the localization of GSK- 3 beta and its ability to phosphorylate downstream targets was investigated in vitro, and the network of proteins differentially impacted by phospho- or SNO-dependent GSK-3 beta regulation and in vivo SNO modification of key signaling kinases during the development of heart failure was also studied. Methods and Results: We found that GSK-3 beta 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. S-nitrosylation of GSK-3 beta significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK-3 beta 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-3 beta plays a central role in cell cycle control, RNA splicing, and DNA damage response. Conclusions: The results indicate that SNO has a differential effect on the location and activity of GSK-3 beta in the cytoplasm versus the nucleus. SNO modification of GSK-3 beta occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.