Minimal requirements for the nuclear localization of p27(Kip1), a cyclin-dependent kinase inhibitor

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localizat ion is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa ce lls and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively l ocalized into nucleus, while GFP alone localized in both cytosol and nucleu s. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is r equired to achieve a full function of NLS. The site-directed mutation of th e full-length p27(Kip1) therefore showed that four basic residues (K153, R1 54, K165, R166), especially R166, play a critical role in the nuclear local ization of p27(Kip1). (C) 2000 Academic Press.