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

Rationale: Glycogen synthase kinase 3β (GSK3β) 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 GSK3β for certain functions. Objective: Here we sought to understand the role of protein S-nitrosylation (SNO) on the function of GSK3β. SNO-dependent modulation of the localization of GSK3β and its ability to phosphorylate downstream targets was investigated in vitro and the network of proteins differentially impacted by phospho- or SNO-dependent GSK3β 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 GSK3β undergoes site-specific SNO both in vitro, in HEK293 cells, H9C2 myoblasts, and primary neonatal rat ventricular myocytes (NRVM), as well as in vivo, in hearts from an animal model of heart failure and sudden cardiac death. S-nitrosylation of GSK3β significantly inhibits its kinase activity independent of the canonical phospho-inhibition pathway. S-nitrosylation of GSK3β promotes its nuclear translocation and access to novel downstream phospho-substrates which are enriched for a novel amino acid consensus sequence motif. Quantitative phospho-proteomics pathway analysis reveals that nuclear GSK3β 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 GSK3β in the cytoplasm versus the nucleus. SNO modification of GSK3β occurs in vivo and could contribute to the pathobiology of heart failure and sudden cardiac death.