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PubMed2006

Inhibition of transforming growth factor-beta1-induced signaling and epithelial-to-mesenchymal transition by the Smad-binding peptide aptamer Trx-SARA.

Overexpression of the inhibitory Smad, Smad7, is used frequently to implicate the Smad pathway in cellular responses to transforming growth factor beta (TGF-beta) signaling; however, Smad7 regulates several other proteins, including Cdc42, p38MAPK, and beta-catenin. We report an alternative approach for more specifically disrupting Smad-dependent signaling using a peptide aptamer, Trx-SARA, which comprises a rigid scaffold, the Escherichia coli thioredoxin A protein (Trx), displaying a constrained 56-amino acid Smad-binding motif from the Smad anchor for receptor activation (SARA) protein. Trx-SARA bound specifically to Smad2 and Smad3 and inhibited both TGF-beta-induced reporter gene expression and epithelial-to-mesenchymal transition in NMuMG murine mammary epithelial cells. In contrast to Smad7, Trx-SARA had no effect on the Smad2 or 3 phosphorylation levels induced by TGF-beta1. Trx-SARA was primarily localized to the nucleus and perturbed the normal cytoplasmic localization of Smad2 and 3 to a nuclear localization in the absence of TGF-beta1, consistent with reduced Smad nuclear export. The key mode of action of Trx-SARA was to reduce the level of Smad2 and Smad3 in complex with Smad4 after TGF-beta1 stimulation, a mechanism of action consistent with the preferential binding of SARA to monomeric Smad protein and Trx-SARA-mediated disruption of active Smad complexes.

Amino Acid Sequence↗
PubMed2003

Regulation of PRAK subcellular location by p38 MAP kinases.

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. p38 regulated/activated protein kinase (PRAK, also known as mitogen-activated protein kinase activated protein kinase 5 [MAPKAPK5]) functions downstream of p38alpha and p38beta in mediating the signaling of the p38 pathway. Immunostaining revealed that endogenous PRAK was predominantly localized in the cytoplasm. Interestingly, ectopically expressed PRAK was localized in the nucleus and can be redistributed by coexpression of p38alpha or p38beta to the locations of p38alpha and p38beta. Mutations in the docking groove on p38alpha/p38beta, or the p38-docking site in PRAK, disrupted the PRAK-p38 interaction and impaired the ability of p38alpha and p38beta to redistribute ectopically expressed PRAK, indicating that the location of PRAK could be controlled by its docking interaction with p38alpha and p38beta. Although the majority of PRAK molecules were detected in the cytoplasm, PRAK is consistently shuttling between the cytoplasm and the nucleus. A sequence analysis of PRAK shows that PRAK contains both a putative nuclear export sequence (NES) and a nuclear localization sequence (NLS). The shuttling of PRAK requires NES and NLS motifs in PRAK and can be regulated through cellular activation induced by stress stimuli. The nuclear content of PRAK was reduced after stimulation, which resulted from a decrease in the nuclear import of PRAK and an increase in the nuclear export of PRAK. The nuclear import of PRAK is independent from p38 activation, but the nuclear export requires p38-mediated phosphorylation of PRAK. Thus, the subcellular distribution of PRAK is determined by multiple factors including its own NES and NLS, docking interactions between PRAK and docking proteins, phosphorylation of PRAK, and cellular activation status. The p38 MAPKs not only regulate PRAK activity and PRAK activation-related translocation, but also dock PRAK to selected subcellular locations in resting cells.

Genes, Reporter↗
PubMed2006

E6AP mediates regulated proteasomal degradation of the nuclear receptor coactivator amplified in breast cancer 1 in immortalized cells.

The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein.

Breast Neoplasms↗
PubMed2006

Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta stimulate extracellular signal-regulated kinase 1/2 signaling by accelerating recycling through the endocytic recycling compartment.

We demonstrate that recycling through the endocytic recycling compartment (ERC) is an essential step in Fc epsilonRI-induced activation of extracellular signal-regulated kinase (ERK)1/2. We show that ERK1/2 acquires perinuclear localization and colocalizes with Rab 11 and internalized transferrin in Fc epsilonRI-activated cells. Moreover, a close correlation exists between the amount of ERC-localized ERK1/2 and the amount of phospho-ERK1/2 that resides in the nucleus. We further show that by activating phosphatidylinositol 4-kinase beta (PI4Kbeta) and increasing the cellular level of phosphatidylinositol(4) phosphate, neuronal calcium sensor-1 (NCS-1), a calmodulin-related protein, stimulates recycling and thereby enhances Fc epsilonRI-triggered activation and nuclear translocation of ERK1/2. Conversely, NCS-1 short hairpin RNA, a kinase dead (KD) mutant of PI4Kbeta (KD-PI4Kbeta), the pleckstrin homology (PH) domain of FAPP1 as well as RNA interference of synaptotagmin IX or monensin, which inhibit export from the ERC, abrogate Fc epsilonRI-induced activation of ERK1/2. Consistently, NCS-1 also enhances, whereas both KD-PI4Kbeta and FAPP1-PH domain inhibit, Fc epsilonRI-induced release of arachidonic acid/metabolites, a downstream target of ERK1/2 in mast cells. Together, our results demonstrate a novel role for NCS-1 and PI4Kbeta in regulating ERK1/2 signaling and inflammatory reactions in mast cells. Our results further identify the ERC as a crucial determinant in controlling ERK1/2 signaling.

1-Phosphatidylinositol 4-Kinase↗
PubMed2001

Nuclear shuttling of mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase (ERK) 2) was dynamically controlled by MAP/ERK kinase after antigen stimulation in RBL-2H3 cells.

The mitogen-activated protein kinase (MAPK) cascade consists of the MAPK (extracellular signal-regulated kinase 2; ERK2) and its activator, MAPK kinase (MAP/ERK kinase; MEK). However, the mechanisms for activation of ERK2 have not been defined yet in cells. Here, we used fluorescent protein-tagged ERK2 and MEK to examine the localization of ERK2 and MEK in living rat basophilic leukemia (RBL-2H3) cells. ERK2 was mainly in the cytoplasm in resting cells but translocated into the nucleus after the ligation of IgE receptors. The import of ERK2 reached the maximum at 6--7 min, and then the imported ERK2 was exported from the nucleus. MEK mainly resided in the cytoplasm, and no significant MEK translocation was detected statically after ligation of IgE receptors. However, analysis of the dynamics of ERK2 and MEK suggested that both of them rapidly shuttle between the cytoplasm and the nucleus and that MEK regulates the nuclear shuttling of ERK2, whereas MEK remains mainly in the cytoplasm. In addition, the data suggested that the sustained calcium increase was required for the optimal translocation of ERK2 into the nucleus in RBL-2H3 cells. These results gave a new insight of the dynamics of ERK2 and MEK in the nuclear shuttling of RBL-2H3 cells after the ligation of IgE receptors.

Active Transport, Cell Nucleus↗
PubMed2001

Protein export from the nucleus.

The evolution of the nucleus imposed on eukaryotic cells the necessity to strictly control exchange of molecules between the nucleus and the remainder of the cell, not only to protect and correctly transmit genetic information, but also to coordinate nuclear and cytoplasmic functions. Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for transport of molecules between the nucleus and the cytoplasm. In addition, regulation of the nucleocytoplasmic distribution of diverse cellular factors has emerged as one of the most efficient mechanism to adapt gene expression to the cell environment, for example by controlling the access of transcriptional regulators to their target genes. In this review, we focus on the molecular basis of protein nuclear export that relies on interactions between targeting sequences present on the cargoes, specific export receptors or exportins and nuclear pore proteins, with special emphasis on the role of the Ran GTPase and associated proteins in this process.

Active Transport, Cell Nucleus↗
PubMed1999

p53 regulation by post-translational modification and nuclear retention in response to diverse stresses.

p53 activation by diverse stresses involves post-translational modifications that alter its structure and result in its nuclear accumulation. We will discuss several unresolved topics regarding p53 regulation which are currently under investigation. DNA damage is perhaps the best-studied stress which activates p53, and recent data implicate phosphorylation at N-terminal serine residues as critical in this process. We discuss recent data regarding the potential kinases which modify p53 and the possible role of the resulting phosphorylation events. By contrast, much less is understood about agents which disrupt the mitotic spindle. The cell cycle phase, induction signal, and biochemical mechanism of the reversible arrest induced by microtubule disruption are currently under investigation. Finally, a key event in response to any genotoxic stress is the accumulation of p53 in the nucleus. The factors which determine the steady state level of p53 are starting to be elucidated, but the mechanisms responsible for nuclear accumulation and nuclear export remain controversial. We discuss new studies revealing a mechanism for nuclear retention of p53, and the potential contributions of MDM2 to this process.

Animals↗
PubMed2005

Phosphorylation of MdmX by CDK2/Cdc2(p34) is required for nuclear export of Mdm2.

Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Intriguingly, the activities of these proteins are interdependent; MdmX stabilizes Mdm2, enabling its activities towards p53, but it also requires Mdm2 for its nuclear localization. Here we demonstrate that via its phosphorylation by CDK2/Cdc2p34, MdmX regulates nuclear export of Mdm2. Cdc2p34 phosphorylates MdmX on Ser 96 in vitro. Mutation within this site (MdmX(S96A)) impairs, whereas phosphomimic substitution (MdmX(S96D)) increases the cytoplasmic localization of MdmX, suggesting that CDK2/Cdc2p34 phosphorylation is required for export of MdmX from the nucleus. Consequently, cells that express MdmX(S96A) retain Mdm2 in their nuclei, suggesting that export of Mdm2 to the cytoplasm is MdmX-dependent. Similarly, treatment of cells with the pharmacological inhibitor of CDK2/Cdc2p34 or with a dominant-negative Cdc2 results in nuclear localization of MdmX and Mdm2 and decreases the level of Mdm2 expression. Since Cdc2p34 is active in nonstressed conditions, our finding provides a novel insight into the signaling cascade involved in the regulation of MdmX localization and for regulation of Mdm2 localization and stability.

Active Transport, Cell Nucleus↗
PubMed2002

Regulation of Gli1 transcriptional activity in the nucleus by Dyrk1.

To investigate the cellular role of dual specificity Yak1-related kinase (Dyrk) 1, a nuclear localized dual specificity protein kinase, we examined its effect on transcriptional regulation using reporter gene assays. We found that Dyrk1 can substantially enhance Gli1-dependent, but not LEF-1-, c-Jun-, or Elk-dependent, gene transcription. In part, Dyrk1 does this through retaining Gli1 in the nucleus. However, we also demonstrate that Dyrk1 can enhance the transcriptional activity of Gli1-AHA, a nuclear export mutant, suggesting that Dyrk1 may be more directly involved in regulating the transcriptional activity of Gli1. In addition, Dyrk1 acted synergistically with Sonic hedgehog (Shh) to induce gene transcription and differentiation in mouse C3H10T1/2 cells. The failure of Shh to stimulate Dyrk1 kinase activity suggests that Dyrk1 may not be directly regulated by the Shh signaling pathway but functionally interacts with it. Thus, Gli1 transcriptional activity may be subjected to further regulation in the cell nucleus by a pathway distinct from Shh signaling, one mediated by Dyrk1.

3T3 Cells↗
PubMed2000

MondoA, a novel basic helix-loop-helix-leucine zipper transcriptional activator that constitutes a positive branch of a max-like network.

Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix-leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

Amino Acid Sequence↗
PubMed2001

The docking of kinesins, KIF5B and KIF5C, to Ran-binding protein 2 (RanBP2) is mediated via a novel RanBP2 domain.

The Ran-binding protein 2 (RanBP2) is a vertebrate mosaic protein composed of four interspersed RanGTPase binding domains (RBDs), a variable and species-specific zinc finger cluster domain, leucine-rich, cyclophilin, and cyclophilin-like (CLD) domains. Functional mapping of RanBP2 showed that the domains, zinc finger and CLD, between RBD1 and RBD2, and RBD3 and RBD4, respectively, associate specifically with the nuclear export receptor, CRM1/exportin-1, and components of the 19 S regulatory particle of the 26 S proteasome. Now, we report the mapping of a novel RanBP2 domain located between RBD2 and RBD3, which is also conserved in the partially duplicated isoform RanBP2L1. Yet, this domain leads to the neuronal association of only RanBP2 with two kinesin microtubule-based motor proteins, KIF5B and KIF5C. These kinesins associate directly in vitro and in vivo with RanBP2. Moreover, the kinesin light chain and RanGTPase are part of this RanBP2 macroassembly complex. These data provide evidence of a specific docking site in RanBP2 for KIF5B and KIF5C. A model emerges whereby RanBP2 acts as a selective signal integrator of nuclear and cytoplasmic trafficking pathways in neurons.

Animals↗
PubMed1997

Viral RNA export.

Viruses can express intron-containing and intronless mRNAs, which are exported by alternative pathways. The study of the nuclear export of these unconventional mRNAs can provide key insights into the normal process of nuclear export and the alternative pathways provide an attractive target for the development of specific antiviral therapies.

Amino Acid Sequence↗
PubMed2002

Two parallel pathways mediate cytoplasmic localization of the dioxin (aryl hydrocarbon) receptor.

The dioxin receptor is a ligand-dependent transcription factor that mediates the biological effects of dioxin and related environmental pollutants. In the absence of ligand the receptor is present in the cytoplasmic compartment of the cell associated with the hsp90-dependent chaperone complex. This complex regulates several functions of the receptor such as ligand binding and nuclear import. Furthermore, intracellular localization of the receptor is modulated by multiple factors such as the export protein CRM-1 and the hsp90-associated immunophilin XAP-2. We have identified the mechanism of XAP-2-induced cytoplasmic localization of the receptor and studied the potential cross-talk between CRM-1 and XAP-2. We show that XAP-2 anchors the ligand-free receptor to cytoskeletal structures. This effect is blocked upon treatment with the actin inhibitor cytochalasin B, whereas the tubulin inhibitor colchicine had no effect on receptor localization. In addition, we show that the receptor interacts with CRM-1 both in the presence and absence of ligand. CRM-1-mediated nuclear export occurs independently of XAP-2. Our data provide evidence that CRM-1 and XAP-2 act in parallel through different mechanisms and target different interfaces of the receptor. These results suggest that two pathways cooperate to localize the non-activated receptor in the cytoplasmic compartment of the cell.

Colchicine↗
PubMed2001

Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1.

Proteinase inhibitor 9 (PI-9) is a human serpin present in the cytoplasm of cytotoxic lymphocytes and epithelial cells. It inhibits the cytotoxic lymphocyte granule proteinase granzyme B (graB) and is thought to protect cytotoxic lymphocytes and bystander cells from graB-mediated apoptosis. Following uptake into cells, graB promotes DNA degradation, rapidly translocating to the nucleus, where it binds a nuclear component. PI-9 should therefore be found in cytotoxic lymphocyte and bystander cell nuclei to ensure complete protection against graB. Here we demonstrate by microscopy and subcellular fractionation experiments that PI-9 is present in the nuclei of human cytotoxic cells, endothelial cells, and epithelial cells. We also show that the related serpins, PI-6, monocyte neutrophil elastase inhibitor (MNEI), PI-8, plasminogen activator inhibitor 2 (PAI-2), and the viral serpin CrmA exhibit similar nucleocytoplasmic distributions. Because these serpins lack classical nuclear localization signals and are small enough to diffuse through nuclear pores, we investigated whether import occurs actively or passively. Large (approximately 70 kDa) chimeric proteins comprising PI-9, PI-6, PI-8, MNEI, or PAI-2 fused to green fluorescent protein (GFP) show similar nucleocytoplasmic distributions to the parent proteins, indicating that nuclear import is active. By contrast, CrmA-GFP is excluded from nuclei, indicating that CrmA is not actively imported. In vitro nuclear transport assays show that PI-9 accumulates at a rate above that of passive diffusion, that it requires cytosolic factors but not ATP, and that it does not bind an intranuclear component. Furthermore, PI-9 is exported from nuclei via a leptomycin B-sensitive pathway, implying involvement of the export factor Crm1p. We conclude that the nucleocytoplasmic distribution of PI-9 and related serpins involves a nonconventional nuclear import pathway and Crm1p.

Amino Acid Sequence↗
PubMed1996

A novel receptor-mediated nuclear protein import pathway.

Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.

Amino Acid Sequence↗
PubMed2000

Nascent 60S ribosomal subunits enter the free pool bound by Nmd3p.

Nmd3p from yeast is required for the export of the large (60S) ribosomal subunit from the nucleus (Ho et al., 2000). Here, we show that Nmd3p forms a stable complex with free 60S subunits. Using an epitope-tagged Nmd3p, we show that free 60S subunits can be coimmunoprecipitated with Nmd3p. The interaction was specific for 60S subunits; 40S subunits were not coimmunoprecipitated. Using this coprecipitation technique and pulse-chase labeling of ribosomal subunit proteins we showed that Nmd3p bound nascent subunits, consistent with its role in export. However, under conditions in which ribosome biogenesis was inhibited (e.g., inhibition of transcription with thiolutin, inhibition of transcription of ribosomal protein and RNA genes in a sly1-1 mutant at nonpermissive temperature, and inhibition of translation in a conditional prt1 mutant), Nmd3p remained associated with 60S subunits. In addition, Nmd3delta120, a truncated protein that lacked a nuclear localization signal, retained 60S binding. These results suggest that Nmd3p recruits nascent 60S subunits into the pool of free 60S subunits and exchanges on 60S subunits as they recycle during translation.

Base Sequence↗
PubMed2000

members only encodes a Drosophila nucleoporin required for rel protein import and immune response activation.

Many developmental and physiological responses rely on the selective translocation of transcriptional regulators in and out of the nucleus through the nuclear pores. Here we describe the Drosophila gene members only (mbo) encoding a nucleoporin homologous to the mammalian Nup88. The phenotypes of mbo mutants and mbo expression during development are cell specific, indicating that the nuclear import capacity of cells is differentially regulated. Using inducible assays for nucleocytoplasmic trafficking we show that mRNA export and classic NLS-mediated protein import are unaffected in mbo mutants. Instead, mbo is selectively required for the nuclear import of the yeast transcription factor GAL4 in a subset of the larval tissues. We have identified the first endogenous targets of the mbo nuclear import pathway in the Rel proteins Dorsal and Dif. In mbo mutants the upstream signaling events leading to the degradation of the IkappaB homolog Cactus are functional, but Dorsal and Dif remain cytoplasmic and the larval immune response is not activated in response to infection. Our results demonstrate that distinct nuclear import events require different nucleoporins in vivo and suggest a regulatory role for mbo in signal transduction.

Amino Acid Sequence↗
PubMed2000

Glyceraldehyde-3-phosphate dehydrogenase in neurodegeneration and apoptosis signaling.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a well-studied glycolytic enzyme that plays a key role in energy metabolism. GAPDH catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in the glycolytic pathway. As part of the conversion, GAPDH converts NAD+ to the high-energy electron carrier NADH. GAPDH has been referred to as a "housekeeping" protein and based on the view that GAPDH gene expression remains constant under changing cellular conditions, the levels of GAPDH mRNA have frequently been used to normalize northern blots. In recent years, that view has changed since GAPDH is now known to contribute to a number of diverse cellular functions unrelated to glycolysis. Normative functions of GAPDH now include nuclear RNA export, DNA replication, DNA repair, exocytotic membrane fusion, cytoskeletal organization and phosphotransferase activity. Pathologically, GAPDH has been implicated in apoptosis, neurodegenerative disease, prostate cancer and viral pathogenesis (see Sirover (1999) for a recent review of GAPDH functions). Most recently, it has been shown that GAPDH is a target for deprenyl related compounds (Carlile et al., 2000; Kragten et al., 1998) and may contribute to the neuroprotection offered by those compounds.

Animals↗