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 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.

scholarly journals p34cdc2-mediated phosphorylation at T124 inhibits nuclear import of SV-40 T antigen proteins.

1991 ◽  
Vol 115 (5) ◽  
pp. 1203-1212 ◽  
Author(s):  
D A Jans ◽  
M J Ackermann ◽  
J R Bischoff ◽  
D H Beach ◽  
R Peters
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
T Antigen ◽  
Regulatory Proteins ◽  
Nuclear Accumulation ◽  
Nuclear Localization Sequence ◽  
Kinetic Measurements ◽  
Nuclear Protein Import

The nuclear import of transcription regulatory proteins appears to be used by the cell to trigger transitions in cell cycle, morphogenesis, and transformation. We have previously observed that the rate at which SV-40 T antigen fusion proteins containing a functional nuclear localization sequence (NLS; residues 126-132) are imported into the nucleus is enhanced in the presence of the casein kinase II (CK-II) site S111/112. In this study purified p34cdc2 kinase was used to phosphorylate T antigen proteins specifically at T124 and kinetic measurements at the single-cell level performed to assess its effect on nuclear protein import. T124 phosphorylation, which could be functionally simulated by a T-to-D124 substitution, was found to reduce the maximal extent of nuclear accumulation whilst negligibly affecting the import rate. The inhibition of nuclear import depended on the stoichiometry of phosphorylation. T124 and S111/112 could be phosphorylated independently of one another. Two alternative mechanisms were considered to explain the inhibition of nuclear import by T124 phosphorylation: inactivation of the NLS and cytoplasmic retention, respectively. Furthermore, we speculate that in vivo T124 phosphorylation may regulate the small but functionally significant amount of cytoplasmic SV-40 T antigen. A sequence comparison showed that many transcription regulatory proteins contain domains comprising potential CK-II-sites, cdc2-sites, and NLS. This raises the possibility that the three elements represent a functional unit regulating nuclear protein import.

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 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.

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