Regulation of protein transport to the nucleus: central role of phosphorylation

1996 ◽  
Vol 76 (3) ◽  
pp. 651-685 ◽  
Author(s):  
D. A. Jans ◽  
S. Hubner
Keyword(s):  
Nuclear Localization ◽  
Protein Transport ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Nuclear Accumulation ◽  
Control Of Gene Expression ◽  
Nuclear Localization Signals

Nuclear protein transport is integral to eukaryotic cell processes such as differentiation, transformation, and the control of gene expression. Although the targeting role of nuclear localization signals (NLSs) has been known for some time, more recent results indicate that NLS-dependent nuclear protein import is precisely regulated. Phosphorylation appears to be the main mechanism controlling the nuclear transport of a number of proteins, including transcription factors such as NFkappaB, c-rel, dorsal, and SWI5 from yeast. Cytoplasmic retention factors, intra- and intermolecular NLS masking, and NLS masking by phosphorylation are some of the mechanisms by which phosphorylation specifically regulates nuclear transport. Even nuclear localization of the archetypal NLS-containing simian virus 40 large tumor antigen (T-ag) is regulated, namely by the "CcN motif," which comprises the T-ag NLS ("N") determining ultimate subcellular destination, a casein kinase II site ("C") 13 amino acids NH2-terminal to the NLS modulating the rate of nuclear import, and a cyclin-dependent kinase site ("c") adjacent to the NLS regulating the maximal level of nuclear accumulation. The CcN motif appears to be a special form of phosphorylation-regulated NLS (prNLS), where phosphorylation at site(s) close to the NLS specifically regulates NLS function. The regulation of nuclear transport through phosphorylation and prNLSs appears to be common in eukaryotic cells from yeast and plants to higher mammals.

scholarly journals A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

1991 ◽  
Vol 113 (6) ◽  
pp. 1243-1254 ◽  
Author(s):  
U Stochaj ◽  
M Osborne ◽  
T Kurihara ◽  
P Silver
Keyword(s):  
Nuclear Localization ◽  
Binding Proteins ◽  
Protein Import ◽  
Nuclear Protein ◽  
Binding Protein ◽  
Protein Localization ◽  
Binding Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Localization Signals

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

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.

Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis

1990 ◽  
Vol 10 (12) ◽  
pp. 6565-6577
Author(s):  
G Shaulsky ◽  
N Goldfinger ◽  
A Ben-Ze'ev ◽  
V Rotter
Keyword(s):  
Nuclear Localization ◽  
Cell Nucleus ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Nuclear Accumulation ◽  
Mutant P53 ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Wild Type P53

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish

1997 ◽  
Vol 75 (5) ◽  
pp. 633-640 ◽  
Author(s):  
Philippe Collas ◽  
Peter Aleström
Keyword(s):  
Nuclear Localization ◽  
Plasmid Dna ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Transport ◽  
Nuclear Targeting ◽  
Nuclear Localization Signals ◽  
Expression Frequency ◽  
Cytoplasmic Injection

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

scholarly journals Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis.

1990 ◽  
Vol 10 (12) ◽  
pp. 6565-6577 ◽  
Author(s):  
G Shaulsky ◽  
N Goldfinger ◽  
A Ben-Ze'ev ◽  
V Rotter
Keyword(s):  
Nuclear Localization ◽  
Cell Nucleus ◽  
P53 Protein ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Nuclear Accumulation ◽  
Mutant P53 ◽  
Wild Type ◽  
Nuclear Localization Signals ◽  
Wild Type P53

The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.

Novel properties of the protein kinase CK2-site-regulated nuclear- localization sequence of the interferon-induced nuclear factor IFI 16

2000 ◽  
Vol 353 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Lyndall J. BRIGGS ◽  
Ricky W. JOHNSTONE ◽  
Rachel M. ELLIOT ◽  
Chong-Yun XIAO ◽  
Michelle DAWSON ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Protein Kinase Ck2 ◽  
Protein Import ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Kinase Ck2

Members of the interferon-induced class of nuclear factors possess a putative CcN motif, comparable with that within proteins such as the simian virus 40 large tumour antigen (T-ag), which confers phosphorylation-mediated regulation of nuclear-localization sequence (NLS)-dependent nuclear import. Here we examine the functionality of the interferon-induced factor 16 (IFI 16) CcN motif, demonstrating its ability to target a heterologous protein to the nucleus, and to be phosphorylated specifically by the CcN-motif-phosphorylating protein kinase CK2 (CK2). The IFI 16 NLS, however, has novel properties, conferring ATP-dependent nuclear import completely independent of cytosolic factors, as well as binding to nuclear components. The IFI 16 NLS is not recognized with high affinity by the NLS-binding importin heterodimer, and transport mediated by it is insensitive to non-hydrolysable GTP analogues. The IFI 16 NLS thus mediates nuclear import through a pathway completely distinct from that of conventional NLSs, such as that of T-ag, but intriguingly resembling that of the NLS of the HIV-1 transactivator protein Tat. Since the IFI 16 CK2 site enhances nuclear import through facilitating binding to nuclear components, this represents a novel mechanism by which the site regulates nuclear-protein import, and constitutes a difference between the IFI 16 and Tat NLSs that may be of importance in the immune response.

A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA

1999 ◽  
Vol 112 (12) ◽  
pp. 2033-2041
Author(s):  
J.J. Ludtke ◽  
G. Zhang ◽  
M.G. Sebestyen ◽  
J.A. Wolff
Keyword(s):  
Active Transport ◽  
Nuclear Localization ◽  
Transport Process ◽  
Nuclear Transport ◽  
Passive Diffusion ◽  
Viral Gene ◽  
Nuclear Localization Signals ◽  
Size Limit ◽  
Cytosolic Extract ◽  
Active Transport Process

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310–1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

scholarly journals Evolution of molecular determinants for SUMO-activating enzyme subcellular localization in plants

2020 ◽  
Author(s):  
Abraham Más ◽  
Laura Castaño-Miquel ◽  
Lorenzo Carretero-Paulet ◽  
Núria Colomé ◽  
Francesc Canals ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Regulatory Mechanism ◽  
Large Subunit ◽  
Post Translational Modification ◽  
Nuclear Localization Signals ◽  
Sumo Conjugation ◽  
Phylogenetic Studies ◽  
Molecular Determinants ◽  
Evolutionary Role

AbstractPost-translational modification by Small Ubiquitin-related Modifier (SUMO) is an essential regulatory mechanism in eukaryotes. In the cell, SUMO conjugates are highly enriched in the nucleus and, consistently, SUMOylation machinery components are mainly nuclear. Nonetheless, cytosolic SUMO targets also exist and the mechanisms that facilitate SUMO conjugation in the cytosol are unknown. Here, we show that the nuclear localization of the Arabidopsis SUMO activating enzyme large subunit SAE2 is dependent on two nuclear localization signals, the canonical NLS1 and the non-canonical NLS2 identified and validated here. NLS2 is proteolytic processed from SAE2 during seed development, facilitating SAE2 enrichment in the cytosol. Results obtained using transgenic plants expressing different SAE2 proteoforms suggest that SAE2 cytosolic enrichment could constitute a rapid signal for growth arrest. Phylogenetic studies indicated that the Arabidopsis NLS1-NLS2 structural organization is conserved only in seed plants, providing a potential evolutionary role of cytosolic SUMOylation in seed appearance.

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