Two signals mediate nuclear localization of influenza virus (A/WSN/33) polymerase basic protein 2.

Polymerase basic protein 2 (PB2), a component of the influenza virus polymerase complex, when expressed alone from cloned cDNA in the absence of other influenza virus proteins, is transported into the nucleus. In this study, we have examined the nuclear translocation signal of PB2 by making deletions and mutations in the PB2 sequence. Our studies showed that two distant regions in the polypeptide sequence were involved in the nuclear translocation of PB2. In one region, four basic residues (K-736 R K R) played a critical role in the nuclear translocation of PB2, since the deletion or mutation of these residues rendered the protein totally cytoplasmic. However, seven residues (M K R K R N S) of this region, including the four basic residues, failed to translocate a cytoplasmic reporter protein into the nucleus, suggesting that these sequences were necessary but not sufficient for nuclear translocation. Deletion of another region (amino acids 449 to 495) resulted in a mutant protein which was cytoplasmic with a perinuclear distribution. This novel phenotype suggests that a perinuclear binding step was involved prior to translocation of PB2 across the nuclear pore and that a signal might be involved in perinuclear binding. Possible involvement of these two signal sequences in the nuclear localization of PB2 is discussed.     

Coupling cellular localization and function of checkpoint kinase 1 (Chk1) in checkpoints and cell viability

Chk1 plays a key role in regulating the replication checkpoint and DNA damage response. Recent evidence suggests that mammalian Chk1 regulates both the nuclear and cytoplasmic checkpoint events. However, mechanisms regulating cellular mobilization of Chk1 were not well understood. Here, we report the identification of regions of human Chk1 that regulate its protein cellular localization and checkpoint function. We demonstrate that the two highly conserved motifs (CM1 and CM2) at the C terminus of Chk1 function as a nuclear export signal and nuclear localization signal, respectively. Mutating five highly conserved residues within these two motifs of Chk1 resulted in its accumulation mainly in the cytoplasm. These cytoplasmic Chk1 mutants were less stable and exhibited significantly reduced phosphorylation by DNA damage treatment, yet they retained, at least partially, checkpoint function. Using an adenovirus-mediated gene targeting technique, we attempted to create an HCT116 cell line in which endogenous Chk1 is mutated so that it is expressed exclusively in the cytoplasm. However, we failed to obtain homozygous mutant cell lines. We found that even the heterozygous mutant cell lines showed cell survival defects accompanied by spontaneous cell death. Together, these results reveal novel regulatory mechanisms that couple protein cellular localization with the checkpoint response and cell viability of Chk1.     

Analysis of nuclear transport signals in the human apurinic/apyrimidinic endonuclease (APE1/Ref1)

The mammalian abasic-endonuclease1/redox-factor1 (APE1/Ref1) is an essential protein whose subcellular distribution depends on the cellular physiological status. However, its nuclear localization signals have not been studied in detail. We examined nuclear translocation of APE1, by monitoring enhanced green fluorescent protein (EGFP) fused to APE1. APE1's nuclear localization was significantly decreased by deleting 20 amino acid residues from its N-terminus. Fusion of APE1's N-terminal 20 residues directed nuclear localization of EGFP. An APE1 mutant lacking the seven N-terminal residues (ND7 APE1) showed nearly normal nuclear localization, which was drastically reduced when the deletion was combined with the E12A/D13A double mutation. On the other hand, nearly normal nuclear localization of the full-length E12A/D13A mutant suggests that the first 7 residues and residues 8–13 can independently promote nuclear import. Both far-western analyses and immuno-pull-down assays indicate interaction of APE1 with karyopherin alpha 1 and 2, which requires the 20 N-terminal residues and implicates nuclear importins in APE1's nuclear translocation. Nuclear accumulation of the ND7 APE1(E12A/D13A) mutant after treatment with the nuclear export inhibitor leptomycin B suggests the presence of a previously unidentified nuclear export signal, and the subcellular distribution of APE1 may be regulated by both nuclear import and export.     

Cross-species sequence analysis reveals multiple charged residue-rich domains that regulate nuclear/cytoplasmic partitioning and membrane localization of a kinase anchoring protein 12 (SSeCKS/Gravin)

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.     

Cyclin F regulates the nuclear localization of cyclin B1 through a cyclin-cyclin interaction

The key regulator of G(2)-M transition of the cell cycle is M-phase promoting factor (MPF), a complex composed of cdc2 and a B-type cyclin. Cyclin B1 nuclear localization involves phosphorylation within a region called the cytoplasmic retention signal, which also contains a nuclear export signal. The mechanism of MPF nuclear localization remains unclear since it contains no functional nuclear localization signal (NLS). We exploited the yeast two-hybrid screen to find protein(s) potentially mediating localization of cyclin B1 and identified a novel interaction between cyclin B1 and cyclin F. We found that cdc2, cyclin B1 and cyclin F form a complex that exhibits histone H1 kinase activity. Cyclin B1 and cyclin F also colocalize through immunofluorescence studies. Additionally, deletion analysis revealed that each putative NLS of cyclin F is functional. Taken together, the data suggest that the NLS regions of cyclin F regulate cyclin B1 localization to the nucleus. The interaction between cyclin B1 and cyclin F represents the first example of direct cyclin-cyclin binding, and elucidates a novel mechanism that regulates MPF localization and function.     

Analysis of a developmentally regulated nuclear localization signal in Xenopus

The 289 residue nuclear oncoprotein encoded by the adenovirus 5 Ela gene contains two peptide sequences that behave as nuclear localization signals (NLS). One signal, located at the carboxy terminus, is like many other known NLSs in that it consists of a short stretch of basic residues (KRPRP) and is constitutively active in cells. The second signal resides within an internal 45 residue region of E1a that contains few basic residues or sequences that resemble other known NLSs. Moreover, this internal signal functions in injected Xenopus oocytes, but not in transfected Xenopus A6 cells, suggesting that it could be regulated developmentally (Slavicek et al. 1989. J. Virol. 63:4047). In this study, we show that the activity of this signal is sensitive to ATP depletion in vivo, efficiently directs the import of a 50 kD fusion protein and can compete with the E1a carboxy-terminal NLS for nuclear import. In addition, we have delineated the precise amino acid residues that comprise the second E1a NLS, and have assessed its utilization during Xenopus embryogenesis. Using amino acid deletion and substitution analyses, we show that the signal consists of the sequence FV(X)7-20MXSLXYM(X)4MF. By expressing in Xenopus embryos a truncated E1a protein that contains only the second NLS and by monitoring its cytoplasmic/nuclear distribution during development with indirect immunofluorescence, we find that the second NLS is utilized up to the early neurula stage. In addition, there appears to be a hierarchy among the embryonic germ layers as to when the second NLS becomes nonfunctional. For this reason, we refer to this NLS as the developmentally regulated nuclear localization signal (drNLS). The implications of these findings for early development are discussed.     

Characterization of RanBPM Molecular Determinants that Control Its Subcellular Localization

RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. In particular, RanBPM comprises two motifs that can confer nuclear localization, one proline/glutamine-rich motif in the extreme N-terminus which has a dominant effect on RanBPM localization, and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise, deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus, suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm, RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally, in the nucleus, a significant fraction of RanBPM is associated with chromatin. Altogether, these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly, likely through interactions with other proteins and by intramolecular folding.     

An unusual pattern of protein expression and localization of yeast alanyl-tRNA synthetase isoforms

Previous studies have shown that in Saccharomyces cerevisiae the mitochondrial and cytoplasmic forms of alanyl-tRNA synthetase are encoded by a single nuclear gene, ALA1, through alternative use of in-frame successive ACG triplets and a downstream AUG triplet. Here we show that despite the obvious participation of the non-AUG-initiated leader peptide in mitochondrial localization, the leader peptide per se cannot target a cytoplasmic passenger protein into mitochondria under normal conditions. Functional mapping further shows that an efficient targeting signal is composed of the leader peptide and an 18-residue sequence downstream of Met1. Consistent to this observation, overexpression of the cytoplasmic form enables it to overcome the compartmental barrier and function in the mitochondria as well, but deletion of as few as eight amino acid residues from its amino-terminus eliminates such a potential. Thus, the sequence upstream of the first in-frame AUG initiator not only carries an unusual initiation site, but also contributes to a novel pattern of protein expression and localization.     

A Nuclear Targeting Determinant for SATB1, a Genome Organizer in the T Cell Lineage

SATB1 is a nuclear protein which acts as a cell-type specific genome organizer and gene regulator essential for T cell differentiation and activation. Several functional domains of SATB1 have been identified. However, the region required for nuclear localization remains unknown. To delineate this region, we employed sequence analysis to identify phylogenetically diverse members of the SATB1 protein family, and used hidden Markov model (HMM)-based analysis to define conserved regions and motifs in this family. One of the regions conserved in SATB1- and SATB2-like proteins in mammals, fish, frog and bird, is located near the N-terminus of family members. We found that the N-terminus of human SATB1 was essential for the nuclear localization of the protein. Furthermore, fusing residues 20-40 to a cytoplasmic green fluorescence protein (GFP) fused to pyruvate kinase (PK) was sufficient to quantitatively translocate the pyruvate kinase into the nucleus. The nuclear targeting sequence of human SATB1 (residues 20-40) is novel and does not contain clusters of basic residues, typically found in ‘classical’ nuclear localization signals (NLSs). We investigated the importance of four well-conserved residues (Lys29, Arg32, Glu34, and Asn36) in this nuclear targeting sequences. Remarkably, full-length SATB1 harboring a single point mutation at either Lys29 or Arg32, but not Glu34 or Asn36, did not enter the nucleus. Our results indicate that SATB1 N-terminal residues 20-40 represent a novel determinant of nuclear targeting.     

Yeast PIAS-type Ull1/Siz1 is composed of SUMO ligase and regulatory domains

SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization.     

The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.     

Loss of cytoplasmic retention ability of mutant LKB1 found in Peutz-Jeghers syndrome patients.

LKB1 Serine/Threonine (ST) kinase (also called STK11) originally identified in our novel protein kinase search project has recently been recognized as a susceptibility gene of Peutz-Jeghers Syndrome (PJS; MIM 175200). PJS is a dominantly inherited human disorder which is characterized by gastrointestinal hamartomatous polyposis and mucocutaneous melanin pigmentation. Since PJS patients also show a predisposition to a wide spectrum of cancers, it is speculated that LKB1 has a tumor suppressor function. In the present study we have characterized the basic biochemical property of LKB1. In the analysis of mutant LKB1 identified in PJS patients, it was found that one of the mutants, SL26, does not lose its kinase function, but alters its subcellular distribution to accumulate in the nucleus only, whereas wild type LKB1 shows both nuclear and cytoplasmic localization. Domain mapping of the nuclear targeting signal of LKB1 assigned it to its amino terminal side. Furthermore, it was shown that LKB1 also has a cytoplasmic retention ability which is considered defective and pathogenic in the SL26 mutant. It is speculated that subcellular distribution of LKB1 is regulated in the balance of these two forces, importation into the nucleus and retention within the cytoplasm; and the cytoplasmic retention ability is necessary for LKB1 to fulfil its normal function.     

Discrete domains mediate the light-responsive nuclear and cytoplasmic localization of Arabidopsis COP1

The Arabidopsis CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) protein plays a critical role in the repression of photomorphogenesis during Arabidopsis seedling development. We investigated the control of COP1 partitioning between nucleus and cytoplasm, which has been implicated in the regulation of COP1 activity, by using fusion proteins between COP1 and beta-glucuronidase or the green fluorescent protein. Transient expression assays using onion epidermal cells and data from hypocotyl cells of stably transformed Arabidopsis demonstrated that COP1 carries a single, bipartite nuclear localization signal that functions independently of light. Nuclear exclusion was mediated by a novel and distinct signal, bordering the zinc-finger and coiled-coil motifs, that was able to redirect a heterologous nuclear protein to the cytoplasm. The cytoplasmic localization signal functioned in a light-independent manner. Light regulation of nuclear localization was reconstituted by combining the individual domains containing the nuclear localization signal and the cytoplasmic localization signal; the WD-40 repeat domain of COP1 was not required. However, phenotypic analysis of transgenic seedlings suggested that the constitutively nuclear-localized WD-40 repeat domain was able to mimic aspects of COP1 function, as indicated by exaggerated hypocotyl elongation under light conditions.     

Minimal requirements for the nuclear localization of p27(Kip1), a cyclin-dependent kinase inhibitor

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).     

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.