title: The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization sequence by importin.
abstract: The mechanism by which phosphorylation regulates nuclear localization sequence (NLS)-dependent nuclear protein import is largely unclear. Whereas nuclear accumulation of SV40 large tumor antigen (T-ag) fusion proteins is completely dependent on the T-ag NLS (amino acids 126-132), the rate of nuclear import is increased 50-fold by amino acid residues 111-125 and in particular a site for the protein kinase CK2 (CK2) at serine 111/112. Because the first step of nuclear protein import involves the binding of the NLS by an NLS-receptor complex such as the importin 58/97 heterodimer, we established a novel enzyme-linked immunosorbent assay to test whether NLS recognition is influenced by amino acids amino-terminal to the NLS and the CK2 site. We found that recognition of the T-ag NLS by importin 58/97 was enhanced 10-fold in the presence of amino acid residues 111-125 and strongly dependent on importin 97. A T-ag fusion protein in which the spacer between the CK2 site and the NLS was decreased showed 30% reduced binding by importin 58/97. Maximal nuclear accumulation of this protein was reduced by more than 50%, indicating the physiological importance of the correctly positioned CK2 site. Phosphorylation by CK2 increased the T-ag NLS binding affinity for importin 58/97 by a further 40%. We conclude that flanking sequences and in particular phosphorylation at the CK2 site are mechanistically important in NLS recognition and represent the basis of their enhancement of T-ag nuclear import. This study thus represents the first elucidation of the mechanistic basis of the regulation of nuclear protein import through phosphorylation within a phosphorylation-regulated NLS.
authors.affiliation: Nuclear Signaling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, ACT 2601, Australia.
authors.forename: S
authors.initials: S
authors.lastname: Hübner
authors.forename: C Y
authors.initials: CY
authors.lastname: Xiao
authors.forename: D A
authors.initials: DA
authors.lastname: Jans
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000378:metabolism*; D001665:Binding Sites; D047390:Casein Kinase II; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D028884:Karyopherins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013539:Simian virus 40 / Q000276:immunology*; D014407:Tumor Cells, Cultured
chemical_list: D000957:Antigens, Viral, Tumor; D004268:DNA-Binding Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases