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

2003 ◽  
Vol 375 (2) ◽  
pp. 339-349 ◽  
Author(s):  
Marcos R. M. FONTES ◽  
Trazel TEH ◽  
Gabor TOTH ◽  
Anna JOHN ◽  
Imre PAVO ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Binding Assays ◽  
Importin Α ◽  
Flanking Sequences

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 (Impα), we co-crystallized non-autoinhibited Impα 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.

scholarly journals Normal Telomere Length Maintenance in Saccharomyces cerevisiae Requires Nuclear Import of the Ever Shorter Telomeres 1 (Est1) Protein via the Importin Alpha Pathway

2014 ◽  
Vol 13 (8) ◽  
pp. 1036-1050 ◽  
Author(s):  
Charlene Hawkins ◽  
Katherine L. Friedman
Keyword(s):  
Telomere Length ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Protein Import ◽  
Repetitive Sequences ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Importin Α

ABSTRACT The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component ( TLC1 ) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening—phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the TLC1 RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin α. The reduction in telomere length observed at the semipermissive temperature in a srp1 mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast.

The role of large T antigen in simian virus 40-induced reactivation of silent rRNA genes in human-mouse hybrid cells

Virology ◽  
1980 ◽  
Vol 102 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Kenneth J. Soprano ◽  
Mara Rossini ◽  
Carlo Croce ◽  
Renato Baserga
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rrna Genes ◽  
Hybrid Cells ◽  
Large T Antigen

scholarly journals Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport

1996 ◽  
Vol 315 (1) ◽  
pp. 33-39 ◽  
Author(s):  
Ulrich SEYDEL ◽  
David A. JANS
Keyword(s):  
Nuclear Import ◽  
Feedback Loop ◽  
Protein Import ◽  
Nuclear Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Accumulation ◽  
Large T Antigen ◽  
Inhibitory Feedback

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled β-galactosidase fusion protein carrying amino acids 111–135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 °C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 °C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 °C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.

scholarly journals RanBP3 Contains an Unusual Nuclear Localization Signal That Is Imported Preferentially by Importin-α3

1999 ◽  
Vol 19 (12) ◽  
pp. 8400-8411 ◽  
Author(s):  
Katie Welch ◽  
Jacqueline Franke ◽  
Matthias Köhler ◽  
Ian G. Macara
Keyword(s):  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Consensus Sequence ◽  
Full Range ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Nuclear Localization Signals ◽  
Importin Α

ABSTRACT The full range of sequences that constitute nuclear localization signals (NLSs) remains to be established. Even though the sequence of the classical NLS contains polybasic residues that are recognized by importin-α, this import receptor can also bind cargo that contains no recognizable signal, such as STAT1. The situation is further complicated by the existence of six mammalian importin-α family members. We report the identification of an unusual type of NLS in human Ran binding protein 3 (RanBP3) that binds preferentially to importin-α3. RanBP3 contains a variant Ran binding domain most similar to that found in the yeast protein Yrb2p. Anti-RanBP3 immunofluorescence is predominantly nuclear. Microinjection of glutathione S-transferase–green fluorescent protein–RanBP3 fusions demonstrated that a region at the N terminus is essential and sufficient for nuclear localization. Deletion analysis further mapped the signal sequence to residues 40 to 57. This signal resembles the NLSs of c-Myc and Pho4p. However, several residues essential for import via the c-Myc NLS are unnecessary in the RanBP3 NLS. RanBP3 NLS-mediated import was blocked by competitive inhibitors of importin-α or importin-β or by the absence of importin-α. Binding assays using recombinant importin-α1, -α3, -α4, -α5, and -α7 revealed a preferential interaction of the RanBP3 NLS with importin-α3 and -α4, in contrast to the simian virus 40 T-antigen NLS, which interacted to similar extents with all of the isoforms. Nuclear import of the RanBP3 NLS was most efficient in the presence of importin-α3. These results demonstrate that members of the importin-α family possess distinct preferences for certain NLS sequences and that the NLS consensus sequence is broader than was hitherto suspected.

scholarly journals Activation of the Tumor-Specific Death Effector Apoptin and Its Kinase by an N-Terminal Determinant of Simian Virus 40 Large T Antigen

2004 ◽  
Vol 78 (18) ◽  
pp. 9965-9976 ◽  
Author(s):  
Ying-Hui Zhang ◽  
Klaas Kooistra ◽  
Alexandra Pietersen ◽  
Jennifer L. Rohn ◽  
Mathieu H. M. Noteborn
Keyword(s):  
Nuclear Localization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Chicken Anemia Virus ◽  
Nuclear Accumulation ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Normal Cells ◽  
J Domain

ABSTRACT Apoptin, a viral death protein derived from chicken anemia virus, displays a number of tumor-specific behaviors. In particular, apoptin is phosphorylated, translocates to the nucleus, and induces apoptosis specifically in tumor or transformed cells, whereas it is nonphosphorylated and remains primarily inactive in the cytoplasm of nontransformed normal cells. Here, we show that in normal cells apoptin can also be activated by the transient transforming signals conferred by ectopically expressed simian virus 40 (SV40) large T antigen (LT), which rapidly induces apoptin's phosphorylation, nuclear accumulation, and the ability to induce apoptosis. Further analyses with mutants of LT showed that the minimum domain capable of inducing all three of apoptin's tumor-specific properties resided in the N-terminal J domain, a sequence which is largely shared by SV40 small t antigen (st). Interestingly, the J domain in st, which lacks its own nuclear localization signal (NLS), required nuclear localization to activate apoptin. These results reveal the existence of a cellular pathway shared by conditions of transient transformation and the stable cancerous or precancerous state, and they support a model whereby a transient transforming signal confers on apoptin both the upstream activity of phosphorylation and the downstream activity of nuclear accumulation and apoptosis induction. Such a pathway may reflect a general lesion contributing to human cancers.

Evidence against a role for simian virus 40 in malignant mesothelioma in Japan

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 10040-10040
Author(s):  
A. Hiraki ◽  
K. Aoe ◽  
T. Murakami ◽  
S. Toyooka ◽  
N. Shivapurkar ◽  
...  
Keyword(s):  
Malignant Mesothelioma ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Positive Control ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Taqman Technology ◽  
Antigen Antibody

10040 Background: Malignant mesothelioma is a highly aggressive tumor arising from serosal membranes, most commonly the pleura. Worldwide incidence is increasing because of widespread exposure to asbestos, the major causal agent. Incidence of this disease also is increasing dramatically in Japan. Association of simian virus 40 (SV40) with malignant mesothelioma has been reported, suggesting that SV40 plays an important role in the origin of a subset of these tumors. Most recently, evidence against any role for SV40 in this disease has been also reported. The role of SV40 in malignant mesothelioma remains still controversial. In addition, it has been argued that differences in the reported frequency of SV40 detection in malignant mesothelioma may be related to geographic variation in populations exposed to the virus. Whether SV40 is associated with malignant mesothelioma in Japan therefore is an important issue. However, no study concerning SV40 in malignant mesothelioma has been reported from Japan. Methods: To address this, we studied 35 malignant mesotheliomas including 32 men and 3 women with a median age of 61 years (ranges 34 to 85) and examined the presence of SV40 large T antigen DNA with real time PCR based on TaqMan technology using primers that PCR amplified a specific 156-bp region of the large Tag of SV40 as well as its expression with immnohistological methods using anti-SV40 large T antigen antibody (pAb101, Santa Cruz Biotechnology, Inc., CA). Results: Two of 35 mesotheliomas were considered positive for the presence of SV40 large T antigen DNA, showing ratios of 36.0 and 4.9. The ratio in the positive control was 199.0. The two positive cases consisted of one epithelioid tumor and one biphasic tumor. In addition, none of 35 malignant mesothelioma specimens were positve for staining with SV40 large T antigen antibody; in contrast, diffuse staining for SV40 large T antigen was observed in the cytoplasm and on the cell membranes in the positive control. Conclusions: Taken together, these findings strongly argue against any role of SV40 in the etiology of the majority of malignant mesothelioma in Japan. No significant financial relationships to disclose.

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