title: Characterization of functional regions for nuclear localization of NPAT.
abstract: NPAT plays a role in S phase entry as a substrate of cyclin E-CDK2 and activation of histone gene transcription. Although analysis of its sequence indicates that NPAT contains typical nuclear localization signals (NLS) comprising segments of positively charged amino acids, there are currently no experimental data to show that these predictive NLS are functional. To investigate whether these sequences are effective for nuclear transport of NPAT, an NPAT-green fluorescent protein fusion (NP-GFP) was constructed. After transfection of the fusion gene containing the full coding region of NPAT into cultured cells, the NP-GFP product was found exclusively in the nucleus. As expected, some deletion mutants that retained the basic amino acid clusters at the carboxyl terminus also localize the fusion protein in the nucleus. However, other fusions that lacked one of the three basic amino acid-clusters were distributed throughout the nucleus and cytoplasm. Therefore all three clusters of basic residues are necessary for localization of NPAT to the nucleus. However, another sequence outside the carboxyl terminal region functions similarly to NLS. Construction of GFP fusions with a series of truncated forms of NPAT indicated that a short peptide sequence consisting of mainly hydrophobic amino acids near the central domain of NPAT also contributes to localizing the protein in the nucleus.
authors.affiliation: Frontier Research Center, National Institute of Radiological Sciences, Anagawa, Inage-ku, Chiba 263-8555, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Sagara
authors.forename: Eri
authors.initials: E
authors.lastname: Takeda
authors.forename: Akiyo
authors.initials: A
authors.lastname: Nishiyama
authors.forename: Syunsaku
authors.initials: S
authors.lastname: Utsumi
authors.forename: Yoshiroh
authors.initials: Y
authors.lastname: Toyama
authors.forename: Shigeki
authors.initials: S
authors.lastname: Yuasa
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Ninomiya
authors.forename: Takashi
authors.initials: T
authors.lastname: Imai
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D006801:Humans; D051379:Mice; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; C101693:NPAT protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C543824:p220(NPAT) protein, mouse