Glycogen synthase kinase 3-β phosphorylates novel S/T-P-S/T domains in Notch1 intracellular domain and induces its nuclear localization

► Novel S/T-P-S/T domains were identified in NICD. ► Phosphorylation of NICD on the S/T-P-S/T domains induced nuclear localization. ► GSK-3β phosphorylated S and T residues in NICD S/T-P-S/T domains. We identified two S/T-P-S/T domains (2122–2124, 2126–2128) inducing Notch intracellular domain (NICD) nuclear localization. The GFP-NICD (1963–2145) fusion protein deletion mutant without classical NLS was localized in the nucleus like the full length GFP-NICD. However, quadruple substitution mutant (T2122A T2124A S2126A T2128A) showed increased cytoplasmic localization. GSK-3β enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3β phosphorylated S and T residues in NICD S/T-P-S/T domains. These results suggest that the novel S/T-P-S/T domain, phosphorylated by GSK-3β is also involved in the nuclear localization of NICD as well as classical NLS.