32. Analysis of Functional Regions for the Nuclear Localization of NPAT
Masashi Sagara, Yasuharu Ninomiya, Hiroko Ito and Takashi Imai
Keywords: NPAT, nuclear localization signal, basic amino acid clusters
NPAT plays a role in S phase entry as a substrate of cyclin E-CDK2 and activation of histone gene transcription. Although previous amino acid sequence analysis suggested that NPAT contains three clusters of basic residues, 1368KKRK1371, 1397KKKK1400 and 1402KKKK1405 at the carboxyl terminus, resembling the classical nuclear localization signal (NLS) motifs, no experimental data have showed these predictive NLS motifs are functional. To investigate whether these basic amino acid clusters are effective for nuclear transport of the protein, a green fluorescent protein-NPAT (GF-NP) was constructed. The full coding region of NPAT was amplified by PCR and fused in-frame to pCMX-SAH/Y145F to generate GF-NP. The fusion gene was transfected into cultured mammalian cells and the cells fixed with 4% paraformaldehyde. The image of the GF-NP was obtained using a conventional microscope equipped with a fluorescein isothiocyanate filter set. After transfection of the fusion gene containing the full coding region of NPAT into the cells, the GF-NP product was detected clearly in the nucleus. When the basic amino acid clusters at the carboxyl terminus were mutated by the PCR-based site directed mutagenesis method, 1368KKRK1371 to 1368KELK1371, 1397KKKK1400 to 1397KELK1400 and 1402KKKK1405 to 1402KLKK1405 respectively, most of the mutants could not be retained in nucleus. A truncated fusion protein, which lacked all of the three basic amino acid-clusters, i.e. 1368KKRstop1371, was also distributed throughout the cytoplasm and nucleus. These data suggest that all three clusters of basic residues in NPAT are essential for targeting the nucleus.
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