Sequence requirement for nuclear localization and growth inhibition of p27Kip1R, a degradation-resistant isoform of p27Kip1.
p27(Kip1R) is an isoform of p27(Kip1), having a distinct C-terminus. The sequences of p27(Kip1R) required for nuclear localization and growth inhibition were determined in HeLa cells using a green fluorescence protein (GFP) as a reporter molecule. Region 153-168 and residues K168 and I169 were determined to play a critical role in the nuclear localization of p27(Kip1R). Aliphatic amino acid was found to be a substitute for the basic residue in the typical nuclear localization signal, while its functional substitution was incomplete, thereby causing a significant cytoplasmic retention of p27(Kip1R). p27(Kip1R) is thus the first example of an atypical bipartite nuclear localization signal with aliphatic amino acid as a functional residue. Despite cytoplasmic retention, p27(Kip1R) inhibited the cell growth as well as p27(Kip1), while GFP alone had no effect. The mutants lacking an N-terminus containing the binding regions for cyclins and cyclin-dependent kinases also showed a significant degree of nuclear localization, but failed to inhibit cell growth. The growth inhibition by p27(Kip1R) as well as p27(Kip1) was thus suggested to originate in the common N-terminal region.
Hirano
Katsuya
K
Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Zeng
Ying
Y
Hirano
Mayumi
M
Nishimura
Junji
J
Kanaide
Hideo
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D049452:Green Fluorescent Proteins; D006131:Growth Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018797:Cell Cycle Proteins; D006131:Growth Inhibitors; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27