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 localization 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 cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. 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 required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).
Zeng
Y
Y
Department of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
Hirano
K
K
Hirano
M
M
Nishimura
J
J
Kanaide
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000378:metabolism; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013552:Swine; D014162:Transfection; D025521:Tumor Suppressor Proteins
D000596:Amino Acids; D018797:Cell Cycle Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27