2. Author's Information
    Marcos R. M. Fontes
    Structural Biology Laboratory, St. Vincent`s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia

    Trazel Teh
    Structural Biology Laboratory, St. Vincent`s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia

    Gabor Toth
    Institute for Medical Chemistry, Szeged Medical University, Szeged, Hungary

    Anna John
    Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia

    Imre Pavo
    Institute for Medical Chemistry, Szeged Medical University, Szeged, Hungary

    David A. Jans
    Institute for Medical Chemistry, Szeged Medical University, Szeged, Hungary

    Bostjan Kobe
    Structural Biology Laboratory, St. Vincent`s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia
  3. Abstract
    The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser112 in the vicinity of the NLS (nuclear localization sequence). To determine the structural basis of the effect of the sequences flanking the basic cluster KKKRK, and the effect of phosphorylation on the recognition of the NLS by the nuclear import factor importin-? (Imp?), we co-crystallized non-autoinhibited Imp? with peptides corresponding to the phosphorylated and non-phosphorylated forms of the NLS, and determined the crystal structures of the complexes. The structures show that the amino acids N-terminally flanking the basic cluster make specific contacts with the receptor that are distinct from the interactions between bipartite NLSs and Imp?. We confirm the important role of flanking sequences using binding assays. Unexpectedly, the regions of the peptides containing the phosphorylation site do not make specific contacts with the receptor. Binding assays confirm that phosphorylation does not increase the affinity of the T-antigen NLS to Imp?. We conclude that the sequences flanking the basic clusters in NLSs play a crucial role in nuclear import by modulating the recognition of the NLS by Imp?, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Imp?.
    Keywords
    importin-? (karyopherin-?), nuclear localization sequence, recognition (NLS recognition), phosphorylation, simian-virus-40 (SV40) large tumour-antigen nuclear localization sequence, X-ray crystal structure
    DOI: 10.1042/bj20030510
    ADLID: 55615-v4
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  1. Keywords
    importin-? (karyopherin-?) nuclear localization sequence recognition (NLS recognition) phosphorylation simian-virus-40 (SV40) large tumour-antigen nuclear localization sequence X-ray crystal structure
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