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PubMed1999

Two well-defined motifs in the cAMP-dependent protein kinase inhibitor (PKIalpha) correlate with inhibitory and nuclear export function.

The heat stable inhibitor of cAMP-dependent protein kinase (PKIalpha) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIalpha to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CaH chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKIalpha was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIalpha (1-25), contains an alpha-helix from residues 1-13. This helix extends by one turn the helix observed in the crystal structure of a PKIalpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35-47, overlaps with the nuclear export signal in the PKIalpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

Amino Acid Sequence↗
PubMed2003

Association of the herpes simplex virus type 1 Us11 gene product with the cellular kinesin light-chain-related protein PAT1 results in the redistribution of both polypeptides.

The herpes simplex virus type 1 (HSV-1) Us11 gene encodes a multifunctional double-stranded RNA (dsRNA)-binding protein that is expressed late in infection and packaged into the tegument layer of the virus particle. As a tegument component, Us11 associates with nascent capsids after its synthesis late in the infectious cycle and is delivered into newly infected cells at times prior to the expression of viral genes. Us11 is also an abundant late protein that regulates translation through its association with host components and contains overlapping nucleolar retention and nuclear export signals, allowing its accumulation in both nucleoli and the cytosol. Thus, at various times during the viral life cycle and in different intracellular compartments, Us11 has the potential to execute discrete tasks. The analysis of these functions, however, is complicated by the fact that Us11 is not essential for viral replication in cultured cells. To discover new host targets for the Us11 protein, we searched for cellular proteins that interact with Us11 and have identified PAT1 as a Us11-binding protein according to multiple, independent experimental criteria. PAT1 binds microtubules, participates in amyloid precursor protein trafficking, and has homology to the kinesin light chain (KLC) in its carboxyl terminus. The carboxyl-terminal dsRNA-binding domain of Us11, which also contains the nucleolar retention and nuclear export signals, binds PAT1, whereas 149 residues derived from the KLC homology region of PAT1 are important for binding to Us11. Both PAT1 and Us11 colocalize within a perinuclear area in transiently transfected and HSV-1-infected cells. The 149 amino acids derived from the KLC homology region are required for colocalization of the two polypeptides. Furthermore, although PAT1 normally accumulates in the nuclear compartment, Us11 expression results in the exclusion of PAT1 from the nucleus and its accumulation in the perinuclear space. Similarly, Us11 does not accumulate in the nucleoli of infected cells that overexpress PAT1. These results establish that Us11 and PAT1 can associate, resulting in an altered subcellular distribution of both polypeptides. The association between PAT1, a cellular trafficking protein with homology to KLC, and Us11, along with a recent report demonstrating an interaction between Us11 and the ubiquitous kinesin heavy chain (R. J. Diefenbach et al., J. Virol. 76:3282-3291, 2002), suggests that these associations may be important for the intracellular movement of viral components.

Amyloid beta-Protein Precursor↗
PubMed2006

Coordinated intracellular translocation of phosphoinositide-specific phospholipase C-delta with the cell cycle.

The delta family phosphoinositide (PI)-specific phospholipase C (PLC) are most fundamental forms of eukaryotic PI-PLCs. Despite the presence of lipid targeting domains such as the PH domain and C2 domain, the isoforms are also found in the cytoplasm and nucleus as well as at the plasma membrane. The isoforms have sequences or regions that can serve as a nuclear localization signal (NLS) and a nuclear export signal (NES). Their intracellular localization differs from one isoform to another, presumably due to the difference in the transport equilibrium balanced by the strength of the two signals of each isoform. Even for a particular isoform, its intracellular localization seems to vary during the cell cycle. As an example, PLCdelta(1), which is generally found at the plasma membrane and in the cytoplasm of quiescent cells, localizes to discrete nuclear structures in the G(1)/S boundary of the cell cycle. This may be at least partly due to an increase in intracellular Ca(2+), since Ca(2+) facilitates the formation of a nuclear transport complex comprised of PLCdelta(1) and importin beta1, a carrier molecule for the nuclear import. PLCdelta(1) as well as PLCdelta(4) may play a pivotal role in controlling the initiation of DNA synthesis in S phase. Spatio-temporal changes in the levels of PtdIns(4,5)P(2) seem to be another major determinant for the localization and regulation of the delta isoforms. High nuclear PtdIns(4,5)P(2) levels are associated with the G(1)/S phases. After entering M phase, PtdIns(4,5)P(2) synthesis at sites of cell division occurs and PLCs seem to localize to the cleavage furrow during cytokinesis. Coordinated translocation of PLCs with the cell cycle or with stress responses may result in changes in intra-nuclear environments and local membrane architectures that modulate proliferation and differentiation. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling as well as roles in the cell cycle progression of the delta isoforms of PLC will be discussed.

Animals↗
PubMed2006

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein.

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.

Active Transport, Cell Nucleus↗
PubMed2000

Nucleocytoplasmic shuttling signals: two for the price of one.

It has been appreciated for some time that basic-amino-acid-type nuclear localization signals control nuclear uptake of proteins and that leucine-rich nuclear export signals mediate export back into the cytoplasm. The machinery that recognizes and escorts these well-defined protein transport signals through the nuclear pore complex has been identified and characterized. Does this mean that the nuclear transport field knows all it needs to about transport signals? Not quite, as several recent publications have expanded the membership of a growing family of transport signals, known as nucleocytoplasmic shuttling (NS) signals. All proteins currently known to contain this type of signal also associate with mRNA. This article reviews what is currently known about mediators of NS signal transport and discusses the link between NS signal-containing proteins and RNA export.

Biological Transport↗
PubMed2004

Nuclear efflux of heterogeneous nuclear ribonucleoprotein C1/C2 in apoptotic cells: a novel nuclear export dependent on Rho-associated kinase activation.

Using a proteomic approach, we searched for protein changes dependent on Rho-associated kinase (ROCK) during phorbol-12-myristate-13-acetate (PMA)-induced apoptosis. We found that heterogeneous nuclear ribonucleoprotein C1 and C2 (hnRNP C1/C2), two nuclear restricted pre-mRNA binding proteins, are translocated to the cytosolic compartment in a ROCK-dependent manner in PMA-induced pro-apoptotic cells, where nuclear envelopes remain intact. The subcellular localization change of hnRNP C1/C2 appears to be dependent on ROCK-mediated cytoskeletal change and independent of caspase execution and new protein synthesis. Such a ROCK-dependent translocation is also seen in TNFalpha-induced apoptotic NIH3T3 cells. By overexpressing the dominant active form of ROCK, we showed that a ROCK-mediated signal is sufficient to induce translocation of hnRNP C1/C2. Deletion experiments indicated that the C-terminal 40-amino-acid region of hnRNP C1/C2 is required for ROCK-responsive translocation. By using nuclear yellow fluorescent protein (YFP) fusion, we determined that the C-terminal 40-amino-acid region of hnRNP C1/C2 is a novel nuclear export signal responsive to ROCK-activation. We conclude that a novel nuclear export is activated by the ROCK signaling pathway to exclude hnRNP C1/C2 from nucleus, by which the compartmentalization of specific hnRNP components is disturbed in apoptotic cells.

3T3 Cells↗
PubMed2003

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates.

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.

Active Transport, Cell Nucleus↗
PubMed2006

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.

Animals↗
PubMed2004

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes.

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

Journal Article↗
PubMed2005

Structural determinants of the BRCA1 : estrogen receptor interaction.

Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-alpha), resulting in the inhibition of estradiol (E2)-stimulated ER-alpha transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1-302) and the ligand-binding domain/activation function-2 (LBD/AF-2) region (within aa 282-420) of ER-alpha. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-alpha interaction. We found that the N-terminal RING domain (aa 20-64) is not required for the BRCA1 : ER-alpha interaction. We identified two separate contact points for ER-alpha, one within aa 1-100 and the other within aa 100-200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 N-terminus, we found that aa 67-100 and 101-133 are required for the interaction with ER-alpha, but that aa 1-67 and 134-302 are dispensible. Previously, we showed that BRCA1 aa 1-302 does not inhibit E2-stimulated ER-alpha transcriptional activity but does bind to ER-alpha and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1-100 and BRCA1 aa 101-200 (but not aa 201-300) each inhibited ER-alpha activity, although not as efficiently as full-length BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86-95) impaired the ability of both truncated (aa 1-100) and full-length (aa 1-1863) BRCA1 proteins to interact with and/or repress ER-alpha activity. Based on these findings, a partial BRCA1 : ER-alpha three-dimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-alpha interaction are discussed.

Amino Acid Sequence↗
PubMed2002

Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export.

MAPK-activated protein kinase 2 (MAPKAPK2), one of several kinases directly phosphorylated and activated by p38 MAPK, plays a central role in the inflammatory response. The activated MAPKAPK2 phosphorylates its nuclear targets CREB/ATF1, serum response factor, and E2A protein E47 and its cytoplasmic targets HSP25/27, LSP-1, 5-lipoxygenase, glycogen synthase, and tyrosine hydroxylase. The crystal structure of unphosphorylated MAPKAPK2, determined at 2.8 A resolution, includes the kinase domain and the C-terminal regulatory domain. Although the protein is inactive, the kinase domain adopts an active conformation with aspartate 366 mimicking the missing phosphorylated threonine 222 in the activation loop. The C-terminal regulatory domain forms a helix-turn-helix plus a long strand. Phosphorylation of threonine 334, which is located between the kinase domain and the C-terminal regulatory domain, may serve as a switch for MAPKAPK2 nuclear import and export. Phosphorylated MAPKAPK2 masks the nuclear localization signal at its C terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.

Amino Acid Sequence↗
PubMed2003

Rat inositol 1,4,5-trisphosphate 3-kinase C is enzymatically specialized for basal cellular inositol trisphosphate phosphorylation and shuttles actively between nucleus and cytoplasm.

The calcium-liberating second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by Ins(1,4,5)P3 3-kinases (IP3Ks) that add a fourth phosphate group to the 3-position of the inositol ring. Two isoforms of IP3Ks (named A and B) from different vertebrate species have been well studied. Recently the cloning and examination of a human full-length cDNA encoding a novel isoform, termed human IP3K-C (HsIP3K-C), has been reported. In the present study we report the cloning of a full-length cDNA encoding a rat homologue of HsIP3K-C with a unique mRNA expression pattern, which differs remarkably from the tissue distribution of HsIP3K-C. Of the rat tissues examined, rat IP3K-C (RnIP3K-C) is mainly present in heart, brain, and testis and shows the strongest expression in an epidermal tissue, namely tongue epithelium. RnIP3K-C has a calculated molecular mass of approximately 74.5 kDa and shows an overall identity of approximately 75% with HsIP3K-C. A bacterially expressed, enzymatically active and Ca2+-calmodulin-regulated fragment of this isoform displays remarkable enzymatic properties like a very low Km for Ins(1,4,5)P3 ( approximately 0.2 microm), substrate inhibition by high concentrations of Ins(1,4,5)P3, allosteric product activation by Ins(1,3,4,5)P4 in absence of Ca2+-calmodulin (Ka(app) 0.52 microm), and the ability to efficiently phosphorylate a second InsP3 substrate, inositol 2,4,5-trisphosphate, to inositol 2,4,5,6-tetrakisphosphate in the presence of Ins(1,3,4,5)P4. Furthermore, the RnIP3K-C fused with a fluorescent protein tag is actively transported into and out of the nucleus when transiently expressed in mammalian cells. A leucine-rich nuclear export signal and an uncharacterized nuclear import activity are localized in the N-terminal domain of the protein and determine its nucleocytoplasmic shuttling. These findings point to a particular role of RnIP3K-C in nuclear inositol trisphosphate phosphorylation and cellular growth.

Animals↗
PubMed2006

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.

Animals↗
PubMed1997

Rapid targeting of nuclear proteins to the cytoplasm.

BACKGROUND: The transcription factor NF-ATc plays a key role in the activation of many early immune response genes and is regulated by subcellular localization. NF-ATc translocates from the cytoplasm to the nucleus in response to a rise in intracellular calcium, and immediately returns to the cytoplasm when intracellular calcium levels fall. The rapid nuclear exit of NF-ATc is thought to be one mechanism by which cells distinguish between sustained and transient calcium signals. RESULTS: To study the nuclear export of NF-ATc, we have developed a general, non-invasive assay for the identification and study of nuclear export signals (NESs). The NES is defined by its ability to translocate a protein from the nucleus to the cytoplasm when the two are tethered by a membrane-permeable ligand. This procedure has allowed us to identify a NES within NF-ATc that functions in concert with a glycogen synthase kinase-regulated process to direct the rapid nuclear exit of NF-ATc. CONCLUSIONS: The rapid nuclear export of NF-ATc via its NES and a glycogen synthase kinase-regulated event may be an important mechanism for insulating cells from transient spikes in intracellular calcium which might otherwise lead to inappropriate activation. The assay we have developed allows the rapid identification of NESs and can be used as a general method for the inducible cytoplasmic export of nuclear proteins.

Amino Acid Sequence↗
PubMed2003

The ins and outs of STAT1 nuclear transport.

There is an inherent elegance in being in the right place at the right time. The STAT1 transcription factor possesses regulatory signals that ensure its distribution to the right cellular location at the right time. Latent STAT1 resides primarily in the cytoplasm, and there it responds to hormone signaling through tyrosine phosphorylation by Janus kinases or growth factor receptors. After phosphorylation, STAT1 dimerizes, and this conformational change reveals a nuclear import signal that is recognized by a specific nuclear import carrier. In the nucleus, the STAT1 dimer dissociates from the import carrier and binds to specific DNA target sites in the promoters of regulated genes. STAT1 is subsequently dephosphorylated in the nucleus by a constitutively active tyrosine phosphatase, leading to its dissociation from DNA. A nuclear export signal of STAT1 appears to be masked when dimers are bound to DNA, but it becomes accessible to the CRM1 export carrier after dissociation from DNA. CRM1 binds STAT1 and transports the transcription factor back to the cytoplasm. Studies show that the regulatory trafficking signals that guide the nuclear import and export of STAT1 reside within its DNA binding domain. The location of these signals indicates that their function has coevolved with the ability of STAT1 to bind DNA and regulate gene expression. The nuclear import and subsequent recycling of STAT1 to the cytoplasm are integral to its function as a signal transducer and activator of transcription.

Active Transport, Cell Nucleus↗
PubMed2002

Nuclear import and export signals in control of the p53-related protein p73.

The p53-family of proteins, including p53, p63, and p73, shares a high degree of structural similarity and can carry out some redundant functions. However, mechanisms that regulate the localization and activity of these proteins have not been fully clarified. In this study, a nuclear localization signal (NLS) was identified in p73, which is required for p73 nuclear import and which could promote the nuclear import of a heterologous, cytoplasmic protein. Mutants lacking the NLS localized to the cytoplasm and displayed diminished transcriptional activity. A nuclear export signal (NES) was also recognized in p73s C terminus, the deletion of which caused p73 to display a more nuclear localization pattern. This NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Interestingly, p73 mutant proteins lacking the NLS or the NES were more stable than wild-type p73, suggesting that nuclear import and nuclear export are required for efficient p73 degradation. Our results indicate that p73 localization is controlled by both nuclear import and export and suggest that the overall distribution of p73 is likely to result from the balance between these two processes. Proper control of nuclear import and export is likely to be an important regulatory determinant of p73.

Animals↗
PubMed1998

Mta has properties of an RNA export protein and increases cytoplasmic accumulation of Epstein-Barr virus replication gene mRNA.

The Epstein-Barr virus (EBV) Zta and Mta regulatory proteins were previously found to be required for efficient replication of oriLyt in cotransfection-replication assays, but the contribution of Mta to the replication process was unknown. We now demonstrate that Mta regulates replication gene expression. Using the polymerase processivity factor BMRF1 as an example, we found that in transfected cells, total BMRF1 mRNA levels were unaffected by Mta but that the amounts of cytoplasmic BMRF1 RNA and protein were greatly increased in the presence of Mta. Mta also increased cytoplasmic accumulation of the BALF2, BALF5, BSLF1, and BBLF4 replication gene mRNAs but did not affect cytoplasmic levels of BBLF2/3 mRNA. Thus, five of the six core replication genes require Mta for efficient accumulation of cytoplasmic RNA. The contribution of Mta to posttranscriptional RNA processing was examined. Examination of Mta localization in transfected cells by indirect immunofluorescence revealed that Mta colocalized with the splicing factor SC35. We also found that Mta has RNA binding activity. Glutathione S-transferase-Mta bound to BMRF1 and BMLF1 transcripts but not to a control cellular gene RNA. Mta contains a consensus leucine-rich nuclear export signal. Such signal sequences are characteristic of proteins that undergo nuclear export. Examination of Mta localization in a heterokaryon assay provided evidence that Mta shuttles between the nucleus and the cytoplasm. Our experiments indicate that Mta functions in RNA processing and transport and mediates cytoplasmic accumulation of a number of EBV early mRNAs.

Amino Acid Sequence↗
PubMed2002

Nuclear localization of the tight junction protein ZO-2 in epithelial cells.

The tight junction constitutes the major barrier to solute and water flow through the paracellular space of epithelia and endothelia. It is formed by transmembrane proteins and submembranous molecules such as the MAGUKs ZOs. We have previously found that several MAGUKs, including those of the tight (ZO-1, ZO-2, and ZO-3) and septate junction (tamou and Dlg), contain one or two nuclear sorting signals located at their first PDZ and GK domains. Now we show that these proteins also contain a nuclear export signal and focus our study on the nuclear membrane shuttling of ZO-2. In sparse cultures this molecule concentrates at the nucleus in clusters, where it partially colocalizes with splicing factor SC35. Nuclear staining diminishes as the monolayer acquires confluence through a process sensitive to the nuclear export inhibitor leptomycin B. Nuclear localization can be induced by impairing cell-cell contacts, by mechanical injury. ZO-2 that shuttles from the cell periphery into the nucleus is not newly synthesized but originates from a preexistent pool. The movement of this protein is mediated by the actin cytoskeleton.

Active Transport, Cell Nucleus↗