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

Fontes, R. M., Teh, T., Toth, G., John, A.I., Pavo, I. D. A., Jans, D. A. and Kobe, B. (2003) Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-a. Biochemical Journal, 375 2: 339-349. doi:10.1042/BJ20030510


Author Fontes, R. M.
Teh, T.
Toth, G.
John, A.I.
Pavo, I. D. A.
Jans, D. A.
Kobe, B.
Title Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-a
Journal name Biochemical Journal   Check publisher's open access policy
ISSN 0264-6021
Publication date 2003-10-15
Sub-type Article (original research)
DOI 10.1042/BJ20030510
Volume 375
Issue 2
Start page 339
End page 349
Total pages 11
Editor P. J. Parker
Place of publication London
Publisher Portland Press Ltd.
Collection year 2003
Language eng
Subject C1
270103 Protein Targeting and Signal Transduction
780105 Biological sciences
Abstract The nuclear import of simian-virus-40 large T-antigen (tumour antigen) is enhanced via phosphorylation by the protein kinase CK2 at Ser(112) 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-alpha (Impalpha), we co-crystallized non-autoinhibited Impalpha 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 Impalpha. 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 Impalpha. 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 Impalpha, whereas phosphorylation of the T-antigen enhances nuclear import by a mechanism that does not involve a direct interaction of the phosphorylated residue with Impalpha.
Keyword Importin-alpha (karyopherin-alpha)
Nuclear Localization Sequence Recognition (nls Recognition)
Phosphorylation
Simian-virus-40 (sv40) Large Tumour-antigen Nuclear Localization Sequence
X-ray Crystal Structure
Amino-acid Hydroxylases
Protein-transport
Karyopherin-alpha
Structural Basis
Crystallographic Analysis
Enhances Recognition
Negative Charge
Chlorin E(6)
Signal
Site
Q-Index Code C1

 
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Created: Wed, 15 Aug 2007, 02:09:54 EST