Sequence requirement for nuclear localization and growth inhibition of p27^Kip1R, a degradation-resistant isoform of p27^Kip1

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.