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-3 beta enhanced nuclear localization and transcriptional activity of WT NICD but not of quadruple substitution mutant. In vitro kinase assays revealed that GSK-3 beta phosphorylated S and T residues in NICD S/T-p-S/T domains. These results suggest that the novel SIT-P-SIT domain, phosphorylated by GSK-3 beta is also involved in the nuclear localization of NICD as well as classical NLS. (C) 2012 Elsevier Inc. All rights reserved.