1. Antibodies against the SV40 large T antigen nuclear localization sequence
B Wolff, M K Park, E Klima, J A Hanover Arch Biochem Biophys. 1991 Jul;288(1):131-40. doi: 10.1016/0003-9861(91)90174-h.
Transport of large proteins into the nucleus requires both a nuclear localization signal (NLS) and exposure of that signal to components of the transport machinery. In this report, polyclonal and monoclonal antibodies were generated against the NLS of SV40 large T antigen. Several of these antibodies immunoprecipitated large T antigen produced by in vitro transcription-translation and recognized T antigen expressed in cultured cells. Binding of the antibodies to T antigen was quantified using an indirect radioimmunoassay and found to be specifically inhibited by peptides corresponding to the T antigen NLS. The ability of NLS-specific antibodies to recognize large T antigen suggests that the NLS is exposed on the surface of T antigen. When one of the NLS-specific monoclonal antibodies was introduced into the cytoplasm of cells expressing T antigen, the antibody remained cytoplasmic. These results suggested either that cytoplasmic components compete for binding to the NLS or that the antibody dissociates from T antigen during transport into the nucleus. When an antibody directed against an epitope distinct from the NLS was microinjected into the cytoplasm of cells expressing large T antigen, both the antibody and antigen were transported into the nucleus. The observed stability of the antigen-antibody complex strongly suggest protein unfolding is not required for nuclear protein transport.
2. Role of flanking sequences and phosphorylation in the recognition of the simian-virus-40 large T-antigen nuclear localization sequences by importin-alpha
Marcos R M Fontes, Trazel Teh, Gabor Toth, Anna John, Imre Pavo, David A Jans, Bostjan Kobe Biochem J. 2003 Oct 15;375(Pt 2):339-49. doi: 10.1042/BJ20030510.
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-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.
3. The fluorinated and chlorinated nuclear localization sequence of the SV 40 T antigen
Alexander Sturzu, Stefan Heckl Chem Biol Drug Des. 2009 Jan;73(1):127-31. doi: 10.1111/j.1747-0285.2008.00736.x.
Nuclear uptake of the simian virus (SV) 40 T antigen is triggered by a specific nuclear localization sequence. However, such a nuclear localization sequence is only poorly taken up by the cytoplasm of cells when administered to the culture medium. Our aim was to improve the cytoplasmic uptake of the SV 40 T antigen nuclear localization sequence. Consequently, we synthesized novel fluorescein isothiocyanate-labelled conjugates containing the nuclear localization sequences of the SV 40 T antigen and either trichlorobenzoic or trifluorobenzoic acid. Applied at 260 microM such halogenated NLS conjugates were nuclearly taken up by 75-85% of U373 and LN18 glioma cells and resulted in cell death. Nuclear staining and cell death were also found at lower concentrations (130 and 65 microM) of halogenated nuclear localization sequence conjugates. By contrast only a low cellular staining rate and no cell death could be observed after co-incubation with a trichlorobenzoic acid or trifluorobenzoic acid-lacking nuclear localization sequence conjugate and free, unbound trichlorobenzoic acid or trifluorobenzoic acid at the high concentration (260 microM). Such small non-radioactive fluorinated and chlorinated nuclear localization sequences may be used as important components for future antiglioma drug development.
