Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-α

Marcos R.M. Fontes, Trazel Teh, Gabor Toth, Anna John, Imre Pavo, David A. Jans, Bostjan Kobe

Research output: Contribution to journalArticle

92 Citations (Scopus)

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-α (Impa), we co-crystallized non-autoinhibited Impa 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 Impα. 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 Impα. 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 Impα, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impα.

Original languageEnglish
Pages (from-to)339-349
Number of pages11
JournalBiochemical Journal
Volume375
Issue number2
DOIs
Publication statusPublished - Oct 15 2003

Keywords

  • Importin-α (karyopherin-α)
  • Nuclear localization sequence recognition (NLS recognition)
  • Phosphorylation
  • Simian-virus-40 (SV40) large tumour-antigen nuclear localization sequence
  • X-ray crystal structure

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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