Recent progress indicates that there are multiple pathways of nucleocytoplasmic transport which involve specific targeting sequences, such as nuclear localization sequences (NLSs), and cytosolic receptor molecules of the importin/karyopherin superfamily which recognise and dock the NLS‐containing proteins at the nuclear pore. This first step of nuclear import/export is of central importance, with the affinity of the importin‐targeting sequence interaction a critical parameter in determining transport efficiency. Different importins possess distinct NLS‐binding specificities, which allows the system to be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same protein, and between different proteins for the same importin. The targeting sequence‐importin interaction can also be influenced directly by phosphorylation increasing the affinity of the interaction with importins or by targeting sequence masking through phosphorylation or specific protein binding. Targeting sequence recognition thus appears to represent a key control point in the regulation of nuclear transport. BioEssays 22:532–544, 2000. © 2000 John Wiley & Sons, Inc.
The potential dangers of using viruses to deliver and integrate DNA into host cells in gene therapy have been poignantly highlighted in recent clinical trials. Safer, non-viral gene delivery approaches have been largely ignored in the past because of their inefficient delivery and the resulting transient transgene expression. However, recent advances indicate that efficient, long-term gene expression can be achieved by non-viral means. In particular, integration of DNA can be targeted to specific genomic sites without deleterious consequences and it is possible to maintain transgenes as small episomal plasmids or artificial chromosomes. The application of these approaches to human gene therapy is gradually becoming a reality.
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.
The mechanism by which phosphorylation regulates nuclear localization sequence (NLS)-dependent nuclear protein import is largely unclear. Whereas nuclear accumulation of SV40 large tumor antigen (T-ag) fusion proteins is completely dependent on the T-ag NLS (amino acids 126 -132), the rate of nuclear import is increased 50-fold by amino acid residues 111-125 and in particular a site for the protein kinase CK2 (CK2) at serine 111/112. Because the first step of nuclear protein import involves the binding of the NLS by an NLS-receptor complex such as the importin 58/97 heterodimer, we established a novel enzyme-linked immunosorbent assay to test whether NLS recognition is influenced by amino acids amino-terminal to the NLS and the CK2 site. We found that recognition of the T-ag NLS by importin 58/97 was enhanced 10-fold in the presence of amino acid residues 111-125 and strongly dependent on importin 97. A T-ag fusion protein in which the spacer between the CK2 site and the NLS was decreased showed 30% reduced binding by importin 58/97. Maximal nuclear accumulation of this protein was reduced by more than 50%, indicating the physiological importance of the correctly positioned CK2 site. Phosphorylation by CK2 increased the T-ag NLS binding affinity for importin 58/97 by a further 40%. We conclude that flanking sequences and in particular phosphorylation at the CK2 site are mechanistically important in NLS recognition and represent the basis of their enhancement of T-ag nuclear import. This study thus represents the first elucidation of the mechanistic basis of the regulation of nuclear protein import through phosphorylation within a phosphorylation-regulated NLS.Nuclear protein transport is dependent on specific targeting signals called nuclear localization sequences (NLSs), 1 defined as the sequences sufficient and necessary for nuclear targeting (1, 2). They are typically short sequences of a single series of basic residues (resembling the NLS of the SV40 large tumor antigen (T-ag)) or of two clusters of basic residues interrupted by a 10 -12-amino acid spacer (bipartite NLSs), and they appear to be functional in various protein contexts, suggesting that NLS function is largely independent of secondary/tertiary structure (see .NLS-dependent nuclear protein import can be divided into two steps, the first of which is energy-independent and involves recognition and targeting of the NLS-bearing protein to the nuclear pore complex (NPC) by a heterodimeric protein complex. The NLS is specifically recognized by the smaller subunit of the complex or NLS receptor (6), known variously as importin 58 (7), importin ␣ (8), hSRP1/NP-1 (9), or karyopherin ␣ (10). The larger protein subunit, importin 97 (11), importin  (12), karyopherin  (13), p97 (14), or Kap95p (15), binds importin 58 specifically but cannot bind the NLS. Its role is to target the importin-NLS carrying protein complex to the NPC through its affinity for NPC components such as nucleoporins (13, 16 -18). The second, energy-dependent step of translocation of th...
We have previously demonstrated [Rihs, H.‐P. and Peters, R. (1989) EMBO J., 8, 1479–1484] that the nuclear transport of recombinant proteins in which short fragments of the SV40 T‐antigen are fused to the amino terminus of Escherichia coli beta‐galactosidase is dependent on both the nuclear localization sequence (NLS, T‐antigen residues 126–132) and a phosphorylation‐site‐containing sequence (T‐antigen residues 111–125). While the NLS determines the specificity, the rate of transport is controlled by the phosphorylation‐site‐containing sequence. The present study furthers this observation and examines the role of the various phosphorylation sites. Purified, fluorescently labeled recombinant proteins were injected into the cytoplasm of Vero or hepatoma (HTC) cells and the kinetics of nuclear transport measured by laser microfluorimetry. By replacing serine and threonine residues known to be phosphorylated in vivo, we identified the casein kinase II (CK‐II) site S111/S112 to be the determining factor in the enhancement of the transport. Either of the residues 111 or 112 was sufficient to elicit the maximum transport enhancement. The other phosphorylation sites (S120, S123, T124) had no influence on the transport rate. Examination of the literature suggested that many proteins harboring a nuclear localization sequence also contain putative CK‐II sites at a distance of approximately 10–30 amino acid residues from the NLS. CK‐II has been previously implicated in the transmission of growth signals to the nucleus. Our results suggest that CK‐II may exert this role by controlling the rate of nuclear protein transport.
Transport of macromolecules into and out of the nucleus is generally effected by targeting signals that are recognized by specific members of the importin/exportin transport receptor family. The latter mediate passage through the nuclear envelope-embedded nuclear pore complexes (NPCs) by conferring interaction with NPC constituents, as well as with other components of the nuclear transport machinery, including the guanine nucleotide-binding protein Ran. Importantly, nuclear transport is regulated at multiple levels via a diverse range of mechanisms, such as the modulation of the accessibility and affinity of target signal recognition by importins/exportins, with phosphorylation/dephosphorylation as a major mechanism. Alteration of the level of the expression of components of the nuclear transport machinery also appears to be a key determinant of transport efficiency, having central importance in development, differentiation and transformation.
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