Identification of a bipartite nuclear localization sequence necessary for nuclear import of 5-lipoxygenase.

5-Lipoxygenase catalyzes the synthesis of leukotrienes from arachidonic acid. This enzyme can reside either in the cytoplasm or the nucleus; its subcellular distribution is influenced by extracellular factors, and its nuclear import correlates with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of native 5-lipoxygenase in primary alveolar macrophages. Within the 5-lipoxygenase protein is a sequence (Arg(638)-Lys(655)) that closely resembles a bipartite nuclear localization signal. Studies using deletion mutants indicated that this region was necessary for nuclear import of 5-lipoxygenase. Analysis of mutants containing specific amino acid substitutions within this sequence confirmed that it was this sequence that was necessary for nuclear import of 5-lipoxygenase and that a specific arginine residue was critical for this function. As nuclear import of 5-lipoxygenase may regulate leukotriene production, natural or induced mutations in this bipartite nuclear localization sequence may also be important in affecting leukotriene synthesis.     

The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal

The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.     

A novel fragmentation of human myelin basic protein: identification of phosphorylated domains

Human myelin basic protein (MBP) was fragmented into three major polypeptides comprised of a NH2-terminal domain (residues 1-83), a middle domain (residues 84-119) which contains an experimental allergic encephalitogenic determinant and a highly conserved triproline sequence, and a COOH-terminal domain (residues 120-170) by Staphylococcus aureus V8 protease at pH 4.0. These three polypeptides could be identified and purified by reversed-phase high-performance liquid chromatography. Analysis of the sites of phosphorylation of the component 1 of human MBP, the most cationic species, catalyzed by a purified Ca2+-activated and phospholipid-dependent protein kinase and cAMP-dependent protein kinase revealed that although these protein kinases could incorporate approximately 6 and 4 mol 32P, respectively, into MBP, none of the potential sites were located within the middle domain.     

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.     

The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen

The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus. The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAGQAKKKKLD, and the sequence AVKRPAATKKAGQAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism.     

Two domains in the terminal inverted-repeat sequence of transposon Tn3

Tn3 and related transposons have terminal inverted repeats (IR) of about 38 bp that are needed as sites for transposition. We made mini-Tn3 derivatives which had a wild-type IR of Tn3 at one end and either the divergent IR of the Tn3-related transposon, gamma delta or IS101, or a mutant IR of Tn3 at the other end. We then examined both in vivo transposition (cointegration between transposition donor and target molecules) of these mini-Tn3 elements and in vitro binding of Tn3-encoded transposase to their IRs. None of the elements with an IR of gamma delta or IS101 mediated cointegration efficiently. This was due to inefficient binding of transposase to these IR. Most mutant IR also interfered with cointegration, even though transposase bound to some mutant IR as efficiently as it did to wild type. This permitted the Tn3 IR sequence to be divided into two domains, named A and B, with respect to transposase binding. Domain B, at positions 13-38, was involved in transposase binding, whereas domain A, at positions 1-10, was not. The A domain may contain the sequence recognized by some other (e.g., host) factor(s) to precede the actual cointegration event.     

Interaction domains and nuclear targeting signals in subunits of the U2 small nuclear ribonucleoprotein particle-associated splicing factor SF3a

Human splicing factor SF3a is a component of the mature U2 small nuclear ribonucleoprotein particle (snRNP) and its three subunits of 60, 66, and 120 kDa are essential for splicing in vitro and in vivo. The SF3a heterotrimer forms in the cytoplasm and enters the nucleus independently of the U2 snRNP. Here, we have analyzed domains required for in vitro interactions between the SF3a subunits. Our results indicate that the SF3a66-SF3a120 interaction is mediated by a 27-amino acid region in SF3a120 C-terminal to the second suppressor-of-white-apricot and prp21/spp91 domain and amino acids 108-210 of SF3a66. Neither of these sequences contains known structural motifs, suggesting that the interaction domains are novel. Moreover, an ～100-amino acid region, including the SURP2 domain of SF3a120 but extending into neighboring regions, is sufficient for binding to SF3a60. Analysis of determinants for nuclear import of SF3a demonstrates that SF3a120 provides the major nuclear localization signal and SF3a60 contributes to nuclear import.     

Activation mechanism of the nuclear chaperone nucleoplasmin: role of the core domain

Nucleoplasmin (NP) mediates nucleosome assembly by removing basic proteins from sperm chromatin and exchanging them with histones. This function is modulated by phosphorylation of NP at multiple sites. NP is pentameric, each monomer consisting of two domains: a core, which forms a stable ring-like pentamer, and a tail, that holds a polyglutamic tract and the nuclear localization signal. In the present study, we have explored the role of the core domain in the functionality of NP. Despite lacking the poly-Glu region, a putative binding site for basic proteins, the isolated core domain of the hyperphosphorylated protein isolated from eggs of Xenopus laevis is able to bind sperm basic proteins and decondense chromatin, in contrast to the inactive, non-phosphorylated recombinant core. This activity can be reproduced artificially in the recombinant core domain through mutation of putative phosphorylation sites to aspartate, thus mimicking the charge effect of phosphorylation. The mutated residues locate in flexible or loop regions exposed on the "distal face" of the core pentamer, where a short acidic region is also found, indicating that phosphorylation might activate the core domain of NP by generating a strong localized negative potential. Our results show that the phosphorylated core domain of NP is active in chromatin decondensation, thus it could contribute together with the poly-Glu containing tail in displaying a binding surface for sperm basic proteins on the NP pentamer.     

A monopartite nuclear localization sequence regulates nuclear targeting of the actin binding protein myopodin

Myopodin is an actin bundling protein that shuttles between nucleus and cytoplasm in response to cell stress or during differentiation. Here, we show that the myopodin sequence 58KKRRRRARK66, when tagged to either enhanced green fluorescent protein (EGFP) or to enhanced cyan fluorescent protein-CapG (ECFPCapG), is able to target these proteins to the nucleolus in HeLa or HEK293T cells. By contrast, 58KKRR61-ECFP-CapG accumulates in the nucleus. Mutation of 58KKRRRRARK66 into alanine residues blocks myopodin nuclear import and promotes formation of cytoplasmic actin filaments. A second putative nuclear localization sequence, 612KTSKKKGKK620, displays much weaker activity in a heterologous context, and appears not to be functional in the full length protein. Thus myopodin nuclear translocation is dependent on a monopartite nuclear localization sequence.     

Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence

We have shown previously that protein kinase Cdelta (PKCdelta) is required for mitochondrial-dependent apoptosis. Here we show that PKCdelta is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCdelta. Mutation of the caspase cleavage site of PKCdelta inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCdelta catalytic fragment (CFdelta) in transfected cells results in nuclear localization and apoptosis. We show that the PKCdelta NLS is required for nuclear import of both full-length PKCdelta and CFdelta, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFdelta prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFdelta) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFdelta to protect against etoposide-induced apoptosis. These results indicate that PKCdelta regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.     

LIM domain protein Trip6 has a conserved nuclear export signal, nuclear targeting sequences, and multiple transactivation domains

Trip6 is a member of a subfamily of LIM domain proteins, including also zyxin, LPP, Ajuba, and Hic-5, which localize primarily to focal adhesion plaques. However, in this report, we demonstrate that Trip6 is largely in the nucleus in cells treated with leptomycin B, suggesting that Trip6 shuttles between nuclear and cytoplasmic compartments and that nuclear export of Trip6 is dependent on Crm1. Consistent with this finding, we have identified a nuclear export signal (NES) in Trip6, and mutation of this NES also results in sequestration of Trip6 in the nucleus. Addition of the Trip6 NES to the nuclear v-Rel oncoprotein redirects v-Rel to the cytoplasm. Trip6 also has at least two sequences that can direct cytoplasmic beta-galactosidase to the nucleus. Using GAL4 fusion proteins and reporter gene assays, we demonstrate that Trip6 has multiple transactivation domains, including one that appears to overlap with sequences of the NES. In vitro- or in vivo-synthesized Trip6, however, does not bind to DNA-cellulose. Taken together, these results are consistent with Trip6, and other members of this LIM protein family, having a role in relaying signals between focal adhesion plaques and the nucleus.     

Importins fulfil a dual function as nuclear import receptors and cytoplasmic chaperones for exposed basic domains

Many nuclear transport pathways are mediated by importin beta-related transport receptors. Here, we identify human importin (Imp) 4b as well as mouse Imp4a, Imp9a and Imp9b as novel family members. Imp4a mediates import of the ribosomal protein (rp) S3a, while Imp9a and Imp9b import rpS7, rpL18a and apparently numerous other substrates. Ribosomal proteins, histones and many other nuclear import substrates are very basic proteins that aggregate easily with cytoplasmic polyanions such as RNA. Imp9 effectively prevents such precipitation of, for example, rpS7 and rpL18a by covering their basic domains. The same applies to Imp4, Imp5, Imp7 and Impbeta and their respective basic import substrates. The Impbeta-Imp7 heterodimer appears specialized for the most basic proteins, such as rpL4, rpL6 and histone H1, and is necessary and sufficient to keep them soluble in a cytoplasmic environment prior to rRNA or DNA binding, respectively. Thus, just as heat shock proteins function as chaperones for exposed hydrophobic patches, importins act as chaperones for exposed basic domains, and we suggest that this represents a major and general cellular function of importins.     

Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle-regulated dynamics.

The nuclear lamin proteins assemble at the nuclear membrane into a highly dynamic network whose integrity is exquisitely controlled by the major cell cycle regulators. Although the actual cellular functions of the lamins remain elusive, the processes of targeted lamin assembly and phosphorylation-induced disassembly have come to light recently. These processes require functionally interacting lamin domains to execute targeting to the nucleus and the nuclear membranes, lamina assembly, and the disassembly response to cell-cycle-dependent phosphorylation.     

Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.

The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.     

Statistical limits to the identification of ion channel domains by sequence similarity

The study of ion channel function is constrained by the availability of structures for only a small number of channels. A commonly used bioinformatics technique is to assert, based on sequence similarity, that a domain within a channel of interest has the same structure as a reference domain for which the structure is known. This technique, while useful, is often employed when there is only a slight similarity between the channel of interest and the domain of known structure. In this study, we exploit recent advances in structural genomics to calculate the sequence-based probability of the presence of putative domains in a number of ion channels. We find strong support for the presence of many domains that have been proposed in the literature. For example, eukaryotic and prokaryotic CLC proteins almost certainly share a common structure. A number of proposed domains, however, are not as well supported. In particular, for the COOH terminus of the BK channel we find a number of literature proposed domains for which the assertion of common structure based on common sequence has a nontrivial probability of error.     

Two domains are critical for the nuclear localization of soluble adenylyl cyclase

Soluble adenylyl cyclase (sAC) is a newly identified source of cyclic adenosine 3',5'-monophosphate (cAMP). Unlike the well-known transmembrane adenylyl cyclases (tmACs), sAC locates to the nucleus, mitochondria and microtubules. For most cAMP-signaling microdomains, there is always an AC nearby, for example tmAC. But it was until the discovery of sAC that there was not known cAMP resource in the nucleus. sAC associates with nuclear cAMP-signaling microdomains, which were once considered to depend on the diffusion of cAMP produced by tmAC. In this report, we focus on the truncated soluble adenylyl cyclase (tsAC), the most common existence form of sAC in tissues. Two domains (145-200 aa and 257-318 aa) related with sAC nuclear localization were present here. The findings provide evidence that these two domains are critical for the nuclear localization of sAC and they collocated with the catalytic domains.