Identification and characterization of novel S/T-P-S/T domains and their involvement in nuclear localization of Notch intracellular domain

Abstract

Notch proteins mediate important cell fate decisions such as differentiation, proliferation, and apoptosis. Following proteolysis, it is thought that the intracellular portion of Notch translocates to the nucleus, where it is involved in regulating gene expression. Recent studies have shown that phosphorylated S/T-P-S/T acts as a general nuclear translocation signal (NTS) for nuclear localization signal (NLS)-lacking proteins that shuttle to the nucleus upon stimulation. In this study, we identified two S/T-P-S/T domains (379-381, 383-385) that are responsible for inducing nuclear translocation of NICD. The green fluorescent protein (GFP)-NICD1 (220-402) fusion protein deletion mutant which do not contain any NLS was localized in the nucleus like the full length GFP-NICD1 with three NLS and GFP-NICD1 (1-244) with a single NLS. Mutation of a serine or threonine residues in the S/T-P-S/T domain to alanine (T379A T381A), (S383A T385A) still resulted in the nuclear localization of GFP-NICD1 (220-402). However, quadruple mutant (T379A T381A S383A T385A) of GFP-NICD1 (220-402) resulted in both cytoplasmic and nuclear localization. These results may suggest that the novel S/T-P-S/T domain is also involved in the nuclear localization of NICD as well as classical NLS.