Multiple Nuclear Localization Signals Research Articles

Fusarium graminearum Effector FgNls1 Targets Plant Nuclei to Induce Wheat Head Blight.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most devastating diseases of wheat and barley worldwide. Effectors suppress host immunity and promote disease development. The genome of F. graminearum contains hundreds of effectors with unknown function. Therefore, investigations of the functions of these effectors will facilitate developing novel strategies to enhance wheat resistance to FHB. We characterized a F. graminearum effector, FgNls1, containing a signal peptide and multiple eukaryotic nuclear localization signals. A fusion protein of green fluorescent protein and FgNls1 accumulated in plant cell nuclei when transiently expressed in Nicotiana benthamiana. FgNls1 suppressed Bax-induced cell death when co-expressed in N. benthamiana. We revealed that the expression of FgNLS1 was induced in wheat spikes infected with F. graminearum. The Fgnls1 mutants significantly reduced initial infection and FHB spread within a spike. The function of FgNLS1 was restored in the Fgnls1-complemented strains. Wheat histone 2B was identified as an interacting protein by FgNls1-affinity chromatography. Furthermore, transgenic wheat plants that silence FgNLS1 expression had significantly lower FHB severity than control plants. This study demonstrates a critical role of FgNls1 in F. graminearum pathogenesis and indicates that host-induced gene silencing targeting F. graminearum effectors is a promising approach to enhance FHB resistance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Systematic Investigation of the Effects of Multiple SV40 Nuclear Localization Signal Fusion on the Genome Editing Activity of Purified SpCas9.

The emergence of CRISPR-Cas9 technology has revolutionized both basic and translational biomedical research. For Cas9 nuclease to exert genome editing activity, nuclear localization signal (NLS) derived from simian virus 40 (SV40) T antigen is commonly installed as genetic fusion to direct the intracellular Cas9 proteins to the nucleus of cells. Notably, previous studies have shown that multiple SV40 NLS fusion can improve the targeting activity of Cas9-derived genome-editing and base-editing tools. In addition, the multi-NLS fusion can increase the intracellular activity of Cas9 in the forms of both constitutive expression and directly delivered Cas9-guide RNA ribonucleoprotein (RNP) complex. However, the relationship between NLS fusion and intracellular Cas9 activity has not been fully understood, including the dependency of activity on the number or organization of NLS fusion. In the present study, we constructed and purified a set of Streptococcus pyogenes Cas9 (SpCas9) variants containing one to four NLS repeats at the N- or C-terminus of the proteins and systematically analyzed the effects of multi-NLS fusion on the activity of SpCas9 RNPs. It was found that multi-NLS fusion could improve the intracellular activity as lipofected or nucleofected Cas9 RNPs. Importantly, multi-NLS fusion could enhance the genome-editing activity of SpCas9 RNPs in primary and stem/progenitor cells and mouse embryos.

Genetic Patterns Found in the Nuclear Localization Signals (NLSs) Associated with EBV-1 and EBV-2 Provide New Insights into Their Contribution to Different Cell-Type Specificities.

Simple SummaryThe Epstein–Barr virus (EBV) has been implicated in several human neoplastic diseases. The EBV-1 can transform B cells into LCL more efficiently than EBV-2, and EBV-2 preferentially infects T-cell lymphocytes. The EBNA3A oncoprotein has an essential role in B-cell transformation. The six peptide motifs called nuclear localization signals (NLSs) from EBNA3A ensure nucleocytoplasmic protein trafficking. Multiple NLSs have been suggested to enhance EBNA3 function or different specificities to different cell types; however, a comprehensive assessment of their genetic variability has not been addressed. Our objective was to study the NLSs’ variability and their relationship with EBV types. Based on a comprehensive analysis of over a thousand EBNA3A sequences from different clinical manifestations and geographic locations, we found that EBNA3A from EBV-2 has two of the six NLSs altered, and genetic patterns in the NLSs are associated with EBV-1 and EBV-2.The Epstein–Barr virus (EBV) is a globally dispersed pathogen involved in several human cancers of B-cell and non-B-cell origin. EBV has been classified into EBV-1 and EBV-2, which have differences in their transformative ability. EBV-1 can transform B-cells into LCL more efficiently than EBV-2, and EBV-2 preferentially infects T-cell lymphocytes. The EBNA3A oncoprotein is a transcriptional regulator of virus and host cell genes, and is required in order to transform B-cells. EBNA3A has six peptide motifs called nuclear localization signals (NLSs) that ensure nucleocytoplasmic protein trafficking. The presence of multiple NLSs has been suggested to enhance EBNA3 function or different specificities in different cell types. However, studies about the NLS variability associated with EBV types are scarce. Based on a systematic sequence analysis considering more than a thousand EBNA3A sequences of EBV from different human clinical manifestations and geographic locations, we found differences in NLSs’ nucleotide structures among EBV types. Compared with the EBNA3A EBV-1, EBNA3A EBV-2 has two of the six NLSs altered, and these mutations were possibly acquired by recombination. These genetic patterns in the NLSs associated with EBV-1 and EBV-2 provide new information about the traits of EBNA3A in EBV biology.

Getting There: Thyroid Hormone Receptor Intracellular Trafficking

Trafficking of regulatory proteins into and out of the nucleus is fundamental to the function of all living cells. A long-standing interest in my lab is exploring the diversity of signals regulating traffic control of the thyroid hormone receptors, TRα1 and TRβ1, transcription factors that either activate or repress the expression of target genes in response to thyroid hormone. Although primarily residing in the nucleus, TRα1 and TRβ1 shuttle rapidly between the nucleus and cytoplasm. Set against a backdrop of my path as a woman in STEM, milestones in our understanding of TR's pathway to the nucleus will be reviewed, while at the same time the innovative contributions of the diverse community of talented scientists in my lab will be showcased. Along the way, we have identified multiple nuclear localization signals (NLSs) and nuclear export signals (NESs) within TRα1 and TRβ1 that interact with importins and exportins, respectively, to mediate translocation across the nuclear envelope. To begin to understand the molecular mechanisms underlying TR's journey, we have focused on the role of post-translational modifications and interactions with transcriptional coregulators in modulating intracellular localization. Using a combined approach in transfected human cells of fluorescence-based nucleocytoplasmic scoring, fluorescence recovery after photobleaching (FRAP), overexpression, and knockdown studies, we have revealed that acetylation promotes cytoplasmic localization of TR, while Mediator subunit MED1 and nuclear receptor corepressor 1 (NCoR1) are key regulators of its nuclear retention. Given the pivotal role of TRα1 in human health, development, and metabolism, we hypothesized that in some types of cancer and endocrine disorders, TRα1's highly regulated traffic control may be disrupted. To analyze key structural components of TRα1 and to further explore the correlation between TRα1 trafficking, misfolding, and disease, recently we have examined the impact of cancer-associated and Resistance to Thyroid Hormone (RTH) syndrome mutations on TRα1 localization patterns. Results show striking shifts towards a more cytoplasmic localization for many of the mutants and an increased tendency to form cytoplasmic and nuclear aggregates. Although the journey is far from over, what has emerged so far is that the fine balance between nuclear import, nuclear retention, and nuclear export of TR is a critical control point for modulating the cellular response to thyroid hormone.

Non-canonical role of wild-type SEC23B in the cellular stress response pathway

While germline recessive loss-of-function mutations in SEC23B in humans cause a rare form of anaemia, heterozygous change-of-function mutations result in increased predisposition to cancer. SEC23B encodes SEC23 homologue B, a component of coat protein complex II (COPII), which canonically transports proteins from the endoplasmic reticulum (ER) to the Golgi. Despite the association of SEC23B with anaemia and cancer, the precise pathophysiology of these phenotypic outcomes remains unknown. Recently, we reported that mutant SEC23B has non-canonical COPII-independent function, particularly within the ER stress and ribosome biogenesis pathways, and that may contribute to the pathobiology of cancer predisposition. In this study, we hypothesized that wild-type SEC23B has a baseline function within such cellular stress response pathways, with the mutant protein reflecting exaggerated effects. Here, we show that the wild-type SEC23B protein localizes to the nucleus in addition to classical distribution at the ER/Golgi interface and identify multiple putative nuclear localization and export signals regulating nuclear–cytoplasmic transport. Unexpectedly, we show that, independently of COPII, wild-type SEC23B can also localize to cell nucleoli under proteasome inhibition conditions, with distinct distribution patterns compared to mutant cells. Unbiased proteomic analyses through mass spectrometry further revealed that wild-type SEC23B interacts with a subset of nuclear proteins, in addition to central proteins in the ER stress, protein ubiquitination, and EIF2 signalling pathways. We validate the genotype-specific differential SEC23B–UBA52 (ribosomal protein RPL40) interaction. Finally, utilizing patient-derived lymphoblastoid cell lines harbouring either wild-type or mutant SEC23B, we show that SEC23B levels increase in response to ER stress, further corroborating its role as a cellular stress response sensor and/or effector. Overall, these observations suggest that SEC23B, irrespective of mutation status, has unexplored roles in the cellular stress response pathway, with implications relevant to cancer and beyond that, CDAII and normal cell biology.

Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V.

The L2 region of bovine adenovirus-3 (BAdV-3) encodes a Mastadenovirus genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81–120, 190–210, and 380–389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21–50 and 380–389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380–389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.

Structural basis for nuclear import selectivity of pioneer transcription factor SOX2

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.

Measurement of the nuclear concentration of α-ketoglutarate during adipocyte differentiation by using a fluorescence resonance energy transfer-based biosensor with nuclear localization signals.

α-Ketoglutarate (α-KG) also known as 2-oxoglutarate (2-OG) is an intermediate metabolite in the tricarboxylic acid (TCA) cycle and is also produced by the deamination of glutamate. It is an indispensable cofactor for a series of 2-oxoglutarate-dependent oxygenases including epigenetic modifiers such as ten-eleven translocation DNA demethylases (TETs) and JmjC domain-containing histone demethylases (JMJDs). Since these epigenetic enzymes target genomic DNA and histone in the nucleus, the nuclear concentration of α-KG would affect the levels of transcription by modulating the activity of the epigenetic enzymes. Thus, it is of great interest to measure the nuclear concentration of α-KG to elucidate the regulatory mechanism of these enzymes. Here, we report a novel fluorescence resonance energy transfer (FRET)-based biosensor with multiple nuclear localization signals (NLSs) to measure the nuclear concentration of α-KG. The probe contains the α-KG-binding GAF domain of NifA protein from Azotobacter vinelandii fused with EYFP and ECFP. Treatment of 3T3-L1 preadipocytes expressing this probe with either dimethyl-2-oxoglutarate (dimethyl-2-OG), a cell-permeable 2-OG derivative, or citrate elicited time- and dose-dependent changes in the FRET ratio, proving that this probe functions as an α-KG sensor. Measurement of the nuclear α-KG levels in the 3T3-L1 cells stably expressing the probe during adipocyte differentiation revealed that the nuclear concentration of α-KG increased in the early stage of differentiation and remained high thereafter. Thus, this nuclear-localized α-KG probe is a powerful tool for real-time monitoring of α-KG concentrations with subcellular resolution in living cells and is useful for elucidating the regulatory mechanisms of epigenetic enzymes.

Getting There: Thyroid Hormone Receptor Intracellular Trafficking

Trafficking of regulatory proteins into and out of the nucleus is fundamental to the function of all living cells. A long‐standing interest in my lab is exploring the diversity of signals regulating traffic control of the thyroid hormone receptors, TRα1 and TRβ1, transcription factors that either activate or repress the expression of target genes in response to thyroid hormone. Although primarily residing in the nucleus, TRα1 and TRβ1 shuttle rapidly between the nucleus and cytoplasm. Set against a backdrop of my path as a woman in STEM, milestones in our understanding of TR’s pathway to the nucleus will be reviewed, while at the same time the innovative contributions of the diverse community of talented scientists in my lab will be showcased. Along the way, we have identified multiple nuclear localization signals (NLSs) and nuclear export signals (NESs) within TRα1 and TRβ1 that interact with importins and exportins, respectively, to mediate translocation across the nuclear envelope. To begin to understand the molecular mechanisms underlying TR’s journey, we have focused on the role of post‐translational modifications and interactions with transcriptional coregulators in modulating intracellular localization. Using a combined approach in transfected human cells of fluorescence‐based nucleocytoplasmic scoring, fluorescence recovery after photobleaching (FRAP), overexpression, and knockdown studies, we have revealed that acetylation promotes cytoplasmic localization of TR, while Mediator subunit MED1 and nuclear receptor corepressor 1 (NCoR1) are key regulators of its nuclear retention. Given the pivotal role of TRα1 in human health, development, and metabolism, we hypothesized that in some types of cancer and endocrine disorders, TRα1’s highly regulated traffic control may be disrupted. To analyze key structural components of TRα1 and to further explore the correlation between TRα1 trafficking, misfolding, and disease, recently we have examined the impact of cancer‐associated and Resistance to Thyroid Hormone (RTH) syndrome mutations on TRα1 localization patterns. Results show striking shifts towards a more cytoplasmic localization for many of the mutants and an increased tendency to form cytoplasmic and nuclear aggregates. Although the journey is far from over, what has emerged so far is that the fine balance between nuclear import, nuclear retention, and nuclear export of TR is a critical control point for modulating the cellular response to thyroid hormone.Support or Funding InformationNIH 2R15DK058028

Multiple nuclear localization sequences in SRSF4 protein.

SRSF4 is one of the members of serine-/arginine (SR)-rich protein family involved in both constitutive and alternative splicing. SRSF4 is localized in the nucleus with speckled pattern, but its nuclear localization signal was not determined. Here, we have identified nuclear localization signals (NLSs) of SRSF4 by using a pyruvate kinase fusion system. As expected, arginine-/serine (RS)-rich domain of SRSF4 confers nuclear localization activity when it is fused to PK protein. We then further delineated the minimum sequences for nuclear localization in RS domain of SRSF4. Surprisingly, RS-rich region does not always have a nuclear localization activity. In addition, basic amino acid stretches that resemble to classical-type NLSs were identified. These results strongly suggest that SRSF4 protein uses two different nuclear import pathways with multiple NLSs in RS domain.

The subcellular localization of bHLH transcription factor TCF4 is mediated by multiple nuclear localization and nuclear export signals

Transcription factor 4 (TCF4) is a class I basic helix-loop-helix (bHLH) transcription factor which regulates the neurogenesis and specialization of cells. TCF4 also plays an important role in the development and functioning of the immune system. Additionally, TCF4 regulates the development of Sertoli cells and pontine nucleus neurons, myogenesis, melanogenesis and epithelial-mesenchymal transition. The ability of transcription factors to fulfil their function often depends on their intracellular trafficking between the nucleus and cytoplasm of the cell. The trafficking is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). We performed research on the TCF4 trafficking regulating sequences by mapping and detailed characterization of motifs potentially acting as the NLS or NES. We demonstrate that the bHLH domain of TCF4 contains an NLS that overlaps two NESs. The results of in silico analyses show high conservation of the sequences, especially in the area of the NLS and NESs. This high conservation is not only between mouse and human TCF4, but also between TCF4 and other mammalian E proteins, indicating the importance of these sequences for the functioning of bHLH class I transcription factors.

Nuclear Receptor Corepressor 1 Regulates Thyroid Hormone Receptor Nuclear Retention

The thyroid hormone receptor (TR) mediates gene transcription in response to thyroid hormone (T3) and significantly affects cell growth, development and metabolism. Although TR shuttles rapidly between the nucleus and cytoplasm, it is primarily nuclear localized at steady state. A shift in the balance between export and import leading to TR mislocalization may correlate with certain cancers or endocrine disorders. Previously, we have identified multiple nuclear localization signals and nuclear export signal within TR that interact with importins and exportins, respectively, to mediate shuttling. Given that TR contains multiple, competing import and export signals, the question arises: what factors are involved in nuclear retention of TR? In addition to DNA‐binding sites, the lab's prior research indicated that nuclear coactivator MED1 facilitates TR nuclear localization. Thus, we hypothesize that certain transcriptional coregulators, including Nuclear Receptor Corepressor 1 (NCoR1), contribute to TR nuclear retention. Using transient transfection assays and fluorescence microscopy, we measured the fluorescent intensity of GFP or mCherry‐tagged TR in the nucleus (N) and cytoplasm (C) of HeLa cells, and then calculated the N/C ratio as an indicator of intracellular localization patterns. We discovered that when NCoR1 is knocked down by shRNA, the amount of TR in the nucleus decreased significantly (lower N/C ratio). Inversely, when NCoR1 is over‐expressed, the amount of TR in the nucleus significantly increased (higher N/C ratio). An even more dramatic cytosolic shift was observed for a TR mutant that cannot bind to NCoR1 (AHT‐TR). Taken together, our results suggest that NCoR1 is a key regulator of TR nuclear retention. This finding will contribute to the development of a dynamic mathematical model that aims to predict TR's N/C ratio during the interaction with different complexes and TR localization's effect on transcriptional output of T3‐dependent genes.Support or Funding InformationNIH 2R15DK058028This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Thyroid hormone receptor localization in target tissues.

The thyroid hormone receptors, TRα1, TRβ1 and other subtypes, are members of the nuclear receptor superfamily that mediate the action of thyroid hormone signaling in numerous tissues to regulate important physiological and developmental processes. Their most well-characterized role is as ligand-dependent transcription factors; TRs bind thyroid hormone response elements in the presence or absence of thyroid hormone to facilitate the expression of target genes. Although primarily residing in the nucleus, TRα1 and TRβ1 shuttle rapidly between the nucleus and cytoplasm. We have identified multiple nuclear localization signals and nuclear export signals within TRα1 and TRβ1 that interact with importins and exportins, respectively, to mediate translocation across the nuclear envelope. More recently, enigmatic cytoplasmic functions have been ascribed to other TR subtypes, expanding the diversity of the cellular response to thyroid hormone. By integrating data on localization signal motifs, this review provides an overview of the complex interplay between TR's dynamic transport pathways and thyroid hormone signaling activities. We examine the variation in TR subtype response to thyroid hormone signaling, and what is currently known about regulation of the variety of tissue-specific localization patterns, including targeting to the nucleus, the mitochondria and the inner surface of the plasma membrane.

NLS copy-number variation governs efficiency of nuclear import--case study on dUTPases.

Nucleocytoplasmic trafficking of large macromolecules requires an active transport machinery. In many cases, this is initiated by binding of the nuclear localization signal (NLS) peptide of cargo proteins to importin-α molecules. Fine orchestration of nucleocytoplasmic trafficking is of particularly high importance for proteins involved in maintenance of genome integrity, such as dUTPases, which are responsible for prevention of uracil incorporation into the genome. In most eukaryotes, dUTPases have two homotrimeric isoforms: one of these contains three NLSs and is present in the cell nucleus, while the other is located in the cytoplasm or the mitochondria. Here we focus on the unusual occurrence of a pseudo-heterotrimeric dUTPase in Drosophila virilis that contains one NLS, and investigate its localization pattern compared to the homotrimeric dUTPase isoforms of Drosophila melanogaster. Although the interaction of individual NLSs with importin-α has been well characterized, the question of how multiple NLSs of oligomeric cargo proteins affect their trafficking has been less frequently addressed in adequate detail. Using the D. virilis dUTPase as a fully relevant physiologically occurring model protein, we show that NLS copy number influences the efficiency of nuclear import in both insect and mammalian cell lines, as well as in D. melanogaster and D. virilis tissues. Biophysical data indicate that NLS copy number determines the stoichiometry of complexation between importin-α and dUTPases. The main conclusion of our study is that, in D. virilis, a single dUTPase isoform efficiently reproduces the cellular dUTPase distribution pattern that requires two isoforms in D. melanogaster.

Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell.

Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin.

Characterization of Nuclear Localization Signals (NLSs) and Function of NLSs and Phosphorylation of Serine Residues in Subcellular and Subnuclear Localization of Transformer-2β (Tra2β)

The serine/arginine-rich (SR) proteins are one type of major actors in regulation of pre-mRNA splicing. Their functions are closely related to the intracellular spatial organization. The RS domain and phosphorylation status of SR proteins are two critical factors in determining the subcellular distribution. Mammalian Transformer-2β (Tra2β) protein, a member of SR proteins, is known to play multiple important roles in development and diseases. In the present study, we characterized the subcellular and subnuclear localization of Tra2β protein and its related mechanisms. The results demonstrated that in the brain the nuclear and cytoplasmic localization of Tra2β were correlated with its phosphorylation status. Using deletional mutation analysis, we showed that the nuclear localization of Tra2β was determined by multiple nuclear localization signals (NLSs) in the RS domains. The point-mutation analysis disclosed that phosphorylation of serine residues in the NLSs inhibited the function of NLS in directing Tra2β to the nucleus. In addition, we identified at least two nuclear speckle localization signals within the RS1 domain, but not in the RS2 domain. The nuclear speckle localization signals determined the localization of RS1 domain-contained proteins to the nuclear speckle. The function of the signals did not depend on the presence of serine residues. The results provide new insight into the mechanisms by which the subcellular and subnuclear localization of Tra2β proteins are regulated.

Vav1 regulates MHCII expression in murine resting and activated B cells

Vav1 is a guanine nucleotide exchange factor (GEF) for Rho GTPases, which is exclusively expressed in cells of the hematopoietic system. In addition to its well-documented GEF activity, it was suggested to have other functions due to the presence of multiple domains and nuclear localization signals in its protein structure. Although GEF-dependent and GEF-independent functions of vav have been implicated in T-cell development and T-cell receptor signaling, the role of vav1 in antigen-presenting cells is poorly understood. We found that vav1 is an important regulator of MHCII expression and transport. Microarray analysis of unstimulated bone marrow-derived macrophages revealed a novel role of vav1 in transcriptional regulation of the MHCII locus, possibly by indirect means. Primary immune cells from vav1-deficient mice had a significantly lower constitutive surface expression of MHCII with the strongest impact observed on splenic and peritoneal B cells. Impaired MHCII expression resulted in a diminished capacity for T-cell activation. Using 6-thio-GTP, a specific inhibitor of the GEF function of vav1, we were able to show that the GEF activity is required for MHCII upregulation in B cells after stimulation with LPS. Furthermore, our data show that vav1 not only affects transcription of the MHCII locus but also is an important regulator of MHCII protein transport to the cell surface.

Astrocyte Elevated Gene-1: Far More Than Just a Gene Regulated in Astrocytes

Since its original cloning by subtraction hybridization in 2002, it is now evident that Astrocyte elevated gene-1 (AEG-1) is a key contributor to the carcinogenic process in diverse organs. AEG-1 protein expression is elevated in advanced stages of many cancers, which correlates with poor survival. In specific cancers, such as breast and liver cancer, the AEG-1 gene itself is amplified, further supporting a seminal role in tumorigenesis. Overexpression and inhibition studies both in in vitro and in in vivo models reveal the importance of AEG-1 in regulating multiple physiologically and pathologically relevant processes including proliferation, invasion, metastasis, and gene expression. AEG-1 is a single-pass transmembrane protein with multiple nuclear localization signals and no known domains or motifs. Although pertinent roles of AEG-1 in the carcinogenic process are established, its potential function (promotion of metastasis only versus functioning as a bona fide oncogene) as well as localization (cell surface versus nucleus) remain areas requiring further clarification. The present review critically evaluates what is currently known about AEG-1 and provides new perspectives relative to this intriguing molecule that may provide a rational target for intervening in the cancer phenotype.

Controlling subcellular localization to alter function: Sending oncogenic Bcr– Abl to the nucleus causes apoptosis

Altering the subcellular localization of signal transducing proteins is a novel approach for therapeutic intervention. Mislocalization of tumor suppressors, oncogenes, or factors involved in apoptosis results in aberrant functioning of these proteins, leading to disease. In the case of chronic myelogenous leukemia (CML), cytoplasmic Bcr – Abl causes oncogenesis/proliferation. On the other hand, nuclear entrapment of endogenous Bcr – Abl (in K562 human leukemia cells) causes apoptosis. The goal of this study was to determine whether ectopically expressed Bcr–Abl could cause apoptosis of K562 cells when specifically directed to the nucleus via strong nuclear localization signals (NLSs). A single NLS from SV40 large T-antigen or four NLSs were subcloned to Bcr – Abl (1NLS– Bcr – Abl or 4NLS–Bcr–Abl). When transfected into K562 cells, only 4NLS– Bcr – Abl translocated to the nucleus. Bcr – Abl alone was found to localize in the cell cytoplasm, colocalizing with actin due to its actin binding domain. 1NLS– Bcr – Abl also localized with actin. Apoptosis induced by 4NLS– Bcr – Abl was evaluated 24 h post-transfection by morphologic determination, DNA staining, and caspase-3 assay. This is the first demonstration that altering the location of ectopically expressed Bcr – Abl can kill leukemia cells. Multiple NLSs are required to overcome Bcr – Abl binding to actin, thus driving it into the nucleus and causing apoptosis.