Rapid Nuclear Localization Research Articles

Scaffold protein RACK1 regulates BR signaling by modulating the nuclear localization of BZR1.

Brassinosteroids (BRs) are a group of plant-specific steroid hormones, which induces the rapid nuclear localization of the positive transcriptional factors BRASSINAZOLE RESISTANT1/2 (BZR1/2). However, the mechanisms underlying the regulation of nucleocytoplasmic shuttling of BZR1 remain to be fully elucidated. In this study, we show that the scaffold protein Receptor for Activated C Kinase 1 (RACK1) from Arabidopsis is involved in BR signaling cascades through mediating the nuclear localization of BZR1, which is tightly retained in the cytosol by the conserved scaffold protein 14-3-3s. RACK1 can interact with BZR1 and competitively decrease the 14-3-3 interaction with BZR1 in cytosol, which efficiently enhances the nuclear localization of BZR1. 14-3-3 also retains RACK1 in cytosol through their interaction. Conversely, BR treatment enhances the nuclear localization of BZR1 by disrupting the 14-3-3 interaction with RACK1 and BZR1. Our study uncovers a new mechanism that integrates two kinds of conserved scaffold proteins (RACK1 and 14-3-3) coordinating BR signaling event.

Abstract 514: Focal Adhesion Kinase Inhibition Prevents Prolonged Nuclear Factor-κB Activation and Reduces Atherosclerosis in ApoE-/- Mice

A key event in atherogenesis is the expression of cell adhesion molecules on endothelial cells (ECs) that recruit leukocytes to the vessel wall, where they accumulate over time and form atherosclerotic plaques filled with fatty lipids. Although lipid-lowering drugs have been beneficial in slowing down atherosclerosis, there is currently no treatment available to reduce the underlying triggers that promote EC activation. Focal adhesion kinase (FAK) is an integrin-associated protein tyrosine kinase that plays a key role in cytokine-induced pro-inflammatory molecule expression; however, the molecular mechanism has not been elucidated. We have revealed that a small molecule FAK inhibitor (PF-271) in human ECs reduces tumor necrosis factor-α (TNF-α)-induced sustained activation of nuclear factor-κB (NF-kB), a major inflammatory transcription factor. While NF-κB is normally bound to inhibitor of NF-κB α (IκBα) in the cytosol, TNF-α leads to rapid activation and nuclear localization of NF-κB after degradation of IκBα by activation of IκB kinase (IKK). Chronic exposure to cytokines leads to oscillations in NF-κB activation as IκBα is continually made by active NF-κB and degraded by active IKK. Surprisingly, we discovered that FAK inhibition blocks prolonged NF-κB activation through a time-dependent increase in IκBα protein stability, suggesting decreased IKK activity. By performing in vitro IKK kinase assays, we found that FAK inhibition did indeed reduce sustained IKK activity as seen by reduced serine phosphorylation of IκBα 3hr after TNF-α stimulation. Since both cell adhesion molecule expression and NF-κB in ECs is important for atherosclerotic lesion development, we tested the efficacy of FAK inhibition on atherogenesis in vivo . We found that oral delivery of PF-271 (twice daily, 8 weeks) significantly reduced atherosclerotic lesion area in ApoE-/- mice fed a western diet. Taken together, our data demonstrates that FAK inhibition potentially reduces atherogenesis by blocking sustained NF-κB signaling through decreased IKK activity.

Localization and interactions of Plasmodium falciparum SWIB/MDM2 homologues.

BackgroundMalaria remains a global health problem and the majority of deaths are caused by Plasmodium falciparum parasites. Due to the rapid emergence of drug-resistant strains, novel avenues of research on the biology of the parasite are needed. The massive proliferation of asexual, intra-erythrocytic parasites every 48 h could kill the human host prior to transmission of slow-developing gametocytes to the mosquito vector. A self-induced P. falciparum programmed cell death mechanism has been hypothesized to maintain this balance between the parasite and its two hosts, but molecular participants of the cell death pathway in P. falciparum have not been characterized. Proteins with SWIB/MDM2 domains play a key role in metazoan programmed cell death and this study provides the first evaluation of two parasite SWIB/MDM2 homologues, PF3D7_0518200 (PfMDM2) and PF3D7_0611400 (PfSWIB).MethodsThe function of these proteins was assessed by predicting their structural topology with the aid of bioinformatics and determining their location within live transgenic parasites, expressing green fluorescent protein-tagged PfMDM2 and PfSWIB under normal and elevated temperatures, which mimic fever and which are known to induce a programmed cell death response. Additionally, P. falciparum phage display library technology was used to identify binding partners for the two parasite SWIB/MDM2 domains.ResultsStructural features of the SWIB/MDM2 domains of PfMDM2 and PfSWIB, suggested that they are chromatin remodelling factors. The N-terminal signal sequence of PfMDM2 directed the protein to the mitochondrion under both normal and heat stress conditions. Plasmodium falciparum phage display library technology revealed that the C-terminal SWIB/MDM2 domain of PfMDM2 interacted with a conserved protein containing a LisH domain. PfSWIB localized to the cytoplasm under normal growth conditions, while approximately 10 % of the heat-stressed trophozoite-stage parasites presented a rapid but short-lived nuclear localization pattern. Two PfSWIB binding partners, a putative Aurora-related kinase and a member of the inner membrane complex, were identified.ConclusionThese novel data provide insight into the function of two parasite SWIB/MDM2 homologues and suggest that PfMDM2 plays a role within the mitochondrion and that PfSWIB is involved in a stage-specific, heat-stress, response pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1065-9) contains supplementary material, which is available to authorized users.

Entry of HIV in Primary Human Resting CD4+ T Cells Pretreated with the Chemokine CCL19

Resting CD4+ T cells latently infected with HIV are a major barrier to a cure and therefore antiretroviral therapy is required life-long. There are two major potential pathways leading to the establishment of latency. One pathway, which we refer to as postactivation latency, occurs with the reversion of an activated infected CD4+ T cell to the resting memory state with an integrated silenced provirus. The other pathway, preactivation latency, occurs via direct infection of resting CD4+ T cells. The relative contribution in vivo of preactivation and postactivation latency is unclear, but latency can be established in vitro via both pathways.1 We have previously demonstrated that although unstimulated resting CD4+ T cells are relatively resistant to HIV infection, incubation of resting CD4+ T cells with chemokines that bind the chemokine receptors, CCR7, CXCR3, and CCR6, which are all highly expressed on resting CD4+T cells, facilitates latent infection in vitro.2,3 Pretreatment with the chemokine CCL19, the ligand for CCR7, facilitated rapid nuclear localization and efficient integration. This was in part mediated via CCL19-induced changes in the actin cytoskeleton.3 Our aim here was to visualize the early events following HIV entry into a resting CD4+ T cell. We used fluorescent HIV to infect CCL19-treated resting CD4+ T cells to track the migration of virus to the nucleus. Resting CD4+ T cells were incubated with 30 nM CCL19 and infected at an MOI of 0.1 with HIVNL4.3 that was dual labeled with green fluorescent protein (GFP) fused to HIV Vpr and S15tomato fused to the viral membrane. GFPVpr associates with HIV cores and tracks HIV complexes in the cytoplasm of target cells. S15tomato is a fluorescent variant of S15mCherry that is no longer visible when virus particles fuse to target cells.4 Therefore, HIV that has fused and entered a cell can be identified as GFPVpr+ S15tomato−.4 Two hours after the addition of virus, cells were fixed and filamentous actin (F-actin) was stained using Alexa Fluor 350.phalloidin (Molecular Probes). The cells were placed in a Labtek II chamber slide and images were captured in a z series using a CoolSNAP camera (Photometrics) on a DeltaVision microscope (100×1.4 numerical aperture oil immersion lens, Applied Precision). Images were deconvolved using softWoRx deconvolution software (Applied Precision). A single z section of a deconvolved image is shown. Cells were also cultured for 4 days and quantification of integrated DNA was performed using Alu-LTR real time PCR.3 Reverse transcriptase (RT) was quantified in supernatant. The frequency of integrated HIV was 92,000 copies/106 CD4+ T cells and there was minimal RT production consistent with latent infection. In the image shown in Fig. 1, fused HIV complexes (GFPVpr+ S15tomato−) that have penetrated cortical F-actin at the cell periphery and are located in the resting CD4+ T cell cytoplasm can be seen. We are now examining how HIV interacts with other components of the cell cytoskeleton and defining key cellular proteins required for rapid transit of HIV to the nucleus in CCL19-treated resting CD4+ T cells. FIG. 1. Fused HIV in the cytoplasm of CCL19-treated resting CD4+ T cells. Resting CD4+ T cells pretreated with 30 nM CCL19 were infected for 2 h with HIVNL4.3 dual labeled with GFPVpr (yellow) and S15tomato (majenta). Fixed cells were stained ...

The Yeast Transcription Factor Crz1 Is Activated by Light in a Ca2+/Calcineurin-Dependent and PKA-Independent Manner

Light in the visible range can be stressful to non-photosynthetic organisms. The yeast Saccharomyces cerevisiae has earlier been reported to respond to blue light via activation of the stress-regulated transcription factor Msn2p. Environmental changes also induce activation of calcineurin, a Ca2+/calmodulin dependent phosphatase, which in turn controls gene transcription by dephosphorylating the transcription factor Crz1p. We investigated the connection between cellular stress caused by blue light and Ca2+ signalling in yeast by monitoring the nuclear localization dynamics of Crz1p, Msn2p and Msn4p. The three proteins exhibit distinctly different stress responses in relation to light exposure. Msn2p, and to a lesser degree Msn4p, oscillate rapidly between the nucleus and the cytoplasm in an apparently stochastic fashion. Crz1p, in contrast, displays a rapid and permanent nuclear localization induced by illumination, which triggers Crz1p-dependent transcription of its target gene CMK2. Moreover, increased extracellular Ca2+ levels stimulates the light-induced responses of all three transcription factors, e.g. Crz1p localizes much quicker to the nucleus and a larger fraction of cells exhibits permanent Msn2p nuclear localization at higher Ca2+ concentration. Studies in mutants lacking Ca2+ transporters indicate that influx of extracellular Ca2+ is crucial for the initial stages of light-induced Crz1p nuclear localization, while mobilization of intracellular Ca2+ stores appears necessary for a sustained response. Importantly, we found that Crz1p nuclear localization is dependent on calcineurin and the carrier protein Nmd5p, while not being affected by increased protein kinase A activity (PKA), which strongly inhibits light-induced nuclear localization of Msn2/4p. We conclude that the two central signalling pathways, cAMP-PKA-Msn2/4 and Ca2+-calcineurin-Crz1, are both activated by blue light illumination.

Interactions between MAP kinase and oestrogen receptor in human breast cancer

PurposeThe oestrogen receptor (ERα) may be activated in a ligand-dependent manner, via oestrogen, or in a ligand-independent manner, via signal transduction pathways. Mitogen Activated Protein Kinase (MAPK) is known to directly phosphorylate ERα at serine 118 in a ligand-independent manner. This study investigated the interaction between MAPK and ERα in breast cancer. Materials & methodsImmunohistochemical experiments were undertaken to determine the expression of MAPK, pMAPK and pER(ser118) in breast tumours to determine their clinical relevance. Immunofluorescent experiments were performed, on MCF-7 breast cancer cells, to monitor the phosphorylation and localisation of MAPK and ERα in response to oestrogen, heregulin and a MAPK inhibitor. ResultsOestrogen and Heregulin stimulated phosphorylation of ERα and its nuclear translocation, but heregulin induced this at levels much lower than those observed with oestrogen. Following stimulation with heregulin, but not oestrogen, treatment with MAPK inhibitor reduced the levels of nuclear pER(ser118). In cells treated with both oestrogen and heregulin, nuclear pER(ser118) was visible; but at levels comparable with heregulin treatment alone. ConclusionThis study confirms that ligand-mediated phosphorylation is associated with rapid nuclear localisation of ERα, due to oestrogen binding. ERα is phosphorylated at serine 118 in a ligand-independent manner. Preventing nuclear translocation of pMAPK reduced the levels of ligand-independent, but not ligand-dependent phosphorylation of ERα. Co-stimulation with both oestrogen and heregulin suggested that heregulin mediated signalling determines the subcellular localisation of ERα. Activation of ERα by direct phosphorylation may result in its rapid deactivation due to degradation or nuclear export.

Drosophila RecQ4 Is Directly Involved in Both DNA Replication and the Response to UV Damage in S2 Cells

The RecQ4 protein shows homology to both the S.cerevisiae DNA replication protein Sld2 and the DNA repair related RecQ helicases. Experimental data also suggest replication and repair functions for RecQ4, but the precise details of its involvement remain to be clarified.Here we show that depletion of DmRecQ4 by dsRNA interference in S2 cells causes defects consistent with a replication function for the protein. The cells show reduced proliferation associated with an S phase block, reduced BrdU incorporation, and an increase in cells with a subG1 DNA content. At the molecular level we observe reduced chromatin association of DNA polymerase-alpha and PCNA. We also observe increased chromatin association of phosphorylated H2AvD - consistent with the presence of DNA damage and increased apoptosis.Analysis of DmRecQ4 repair function suggests a direct role in NER, as the protein shows rapid but transient nuclear localisation after UV treatment. Re-localisation is not observed after etoposide or H2O2 treatment, indicating that the involvement of DmRecQ4 in repair is likely to be pathway specific.Deletion analysis of DmRecQ4 suggests that the SLD2 domain was essential, but not sufficient, for replication function. In addition a DmRecQ4 N-terminal deletion could efficiently re-localise on UV treatment, suggesting that the determinants for this response are contained in the C terminus of the protein. Finally several deletions show differential rescue of dsRNA generated replication and proliferation phenotypes. These will be useful for a molecular analysis of the specific role of DmRecQ4 in different cellular pathways.

Highly acetylated tubulin permits enhanced interactions with and trafficking of plasmids along microtubules

Microtubule-based transport is required for plasmid translocation to the nucleus during transfections, and having stable structures could enhance this movement. In previous studies in which the cytoskeleton was disrupted, we found that populations of microtubules remain that are stable and highly acetylated. By increasing the levels of acetylated tubulin through inhibition of the tubulin deacetylase HDAC6, we observe more rapid plasmid nuclear localization of transfected plasmids and greater levels of gene transfer. In this study, we sought to understand plasmid movement in cells with enhanced tubulin acetylation. Using variations of a microtubule spin down assay, we found that plasmids bound to hyper-acetylated microtubules to a greater degree than they did to unmodified microtubules. To determine if microtubule acetylation also affects cytoplasmic trafficking, plasmid movement was evaluated in real time by particle tracking in cells with varying levels of acetylated microtubules. We found that plasmids display greater net rates of movement, spend more time in productive motion and display longer runs of continuous motion in cells with highly acetylated microtubules compared to those with fewer modifications. These results all suggest that plasmid movement is enhanced along highly acetylated microtubules, reducing the time spent in the cytoplasm prior to nuclear import. Taken together, these findings provide a foundation for determining how modulation of microtubule acetylation can be used as a means to increase intracellular trafficking of plasmids and enhance gene therapy.

Gadolinium Compounds Signaling through TLR 4 and TLR 7 in Normal Human Macrophages: Establishment of a Proinflammatory Phenotype and Implications for the Pathogenesis of Nephrogenic Systemic Fibrosis

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity pathways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-κB, and increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-κB-inducible reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-1R-associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression. Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-6, TGF-β, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis.

Hypercapnia Induces Cleavage and Nuclear Localization of RelB Protein, Giving Insight into CO2 Sensing and Signaling

Carbon dioxide (CO(2)) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO(2) (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO(2) or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-κB pathway has been postulated to be important in signaling the cellular transcriptional response to CO(2). In this study, we demonstrate that in cells exposed to elevated CO(2), the NF-κB family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3β or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO(2)-sensitive NF-κB family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

The role of the protein kinase A pathway in the response to alkaline pH stress in yeast.

Exposure of Saccharomyces cerevisiae to alkaline pH provokes a stress condition that generates a compensatory reaction. In the present study we examined a possible role for the PKA (protein kinase A) pathway in this response. Phenotypic analysis revealed that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation enhances tolerance to this stress. We observed that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding with STRE (stress response element) sequences in many promoters. Consequently, a synthetic STRE–LacZ reporter shows a rapid induction in response to alkaline stress. A msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 min of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated genome expression remodelling. However, the relevance of attenuation of PKA in high pH tolerance is probably not restricted to regulation of Msn2 function.

High NaCl–induced activation of CDK5 increases phosphorylation of the osmoprotective transcription factor TonEBP/OREBP at threonine 135, which contributes to its rapid nuclear localization

When activated by high NaCl, tonicity-responsive enhancer-binding protein/osmotic response element-binding protein (TonEBP/OREBP) increases transcription of osmoprotective genes. High NaCl activates TonEBP/OREBP by increasing its phosphorylation, nuclear localization, and transactivating activity. In HEK293 cells, mass spectrometry shows phosphorylation of TonEBP/OREBP-S120, -S134, -T135, and -S155. When those residues are individually mutated to alanine, nuclear localization is greater for S155A, less for S134A and T135A, and unchanged for S120A. High osmolality increases phosphorylation at T135 in HEK293 cells and in rat renal inner medullas in vivo. In HEK293 cells, high NaCl activates cyclin-dependent kinase 5 (CDK5), which directly phosphorylates TonEBP/OREBP-T135. Inhibition of CDK5 activity reduces the rapid high NaCl-induced nuclear localization of TonEBP/OREBP but does not affect its transactivating activity. High NaCl induces nuclear localization of TonEBP/OREBP faster (≤2 h) than it increases its overall protein abundance (≥6 h). Inhibition of CDK5 reduces the increase in TonEBP/OREBP transcriptional activity that has occurred by 4 h after NaCl is raised, associated with less nuclear TonEBP/OREBP at that time, but does not reduce either activity or nuclear TonEBP/OREBP after 16 h. Thus high NaCl-induced increase of the overall abundance of TonEBP/OREBP, by itself, eventually raises its effective level in the nucleus, but its rapid CDK5-dependent nuclear localization accelerates the process, speeding transcription of osmoprotective target genes.

NFκB activation and stimulation of chemokine production in normal human macrophages by the gadolinium-based magnetic resonance contrast agent Omniscan: possible role in the pathogenesis of nephrogenic systemic fibrosis

ObjectiveNephrogenic systemic fibrosis (NSF) is a generalised fibrotic disorder occurring in certain individuals with renal insufficiency exposed to gadolinium-based contrast agents (GdBCA) for MRI. Histopathological examination of affected tissues shows...

A limited role for p53 in modulating the immediate phenotype of Apc loss in the intestine

Backgroundp53 is an important tumour suppressor with a known role in the later stages of colorectal cancer, but its relevance to the early stages of neoplastic initiation remains somewhat unclear. Although p53-dependent regulation of Wnt signalling activity is known to occur, the importance of these regulatory mechanisms during the early stages of intestinal neoplasia has not been demonstrated.MethodsWe have conditionally deleted the Adenomatous Polyposis coli gene (Apc) from the adult murine intestine in wild type and p53 deficient environments and subsequently compared the phenotype and transcriptome profiles in both genotypes.ResultsExpression of p53 was shown to be elevated following the conditional deletion of Apc in the adult small intestine. Furthermore, p53 status was shown to impact on the transcription profile observed following Apc loss. A number of key Wnt pathway components and targets were altered in the p53 deficient environment. However, the aberrant phenotype observed following loss of Apc (rapid nuclear localisation of β-catenin, increased levels of DNA damage, nuclear atypia, perturbed cell death, proliferation, differentiation and migration) was not significantly altered by the absence of p53.Conclusionp53 related feedback mechanisms regulating Wnt signalling activity are present in the intestine, and become activated following loss of Apc. However, the physiological Wnt pathway regulation by p53 appears to be overwhelmed by Apc loss and consequently the activity of these regulatory mechanisms is not sufficient to modulate the immediate phenotypes seen following Apc loss. Thus we are able to provide an explanation to the apparent contradiction that, despite having a Wnt regulatory capacity, p53 loss is not associated with early lesion development.

Nucleocytoplasmic Shuttling of BZR1 Mediated by Phosphorylation Is Essential inArabidopsisBrassinosteroid Signaling

Phytohormone brassinosteroids (BRs) play critical roles in plant growth and development. BR acts by modulating the phosphorylation status of two key transcriptional factors, BRI1 EMS SUPPRESSOR1 and BRASSINAZOLE RESISTANT1 (BZR1), through the action of BRASSINOSTEROID INSENSITIVE1/BRI1 ASSOCIATED RECEPTOR KINASE1 receptors and a GSK3 kinase, BRASSINOSTEROID INSENSITIVE2 (BIN2). It is still unknown how the perception of BR at the plasma membrane connects to the expression of BR target genes in the nucleus. We show here that BZR1 functions as a nucleocytoplasmic shuttling protein and GSK3-like kinases induce the nuclear export of BZR1 by modulating BZR1 interaction with the 14-3-3 proteins. BR-activated phosphatase mediates rapid nuclear localization of BZR1. Besides the phosphorylation domain for 14-3-3 binding, another phosphorylation domain in BZR1 is required for the BIN2-induced nuclear export of BZR1. Mutations of putative phosphorylation sites in two distinct domains enhance the nuclear retention of BZR1 and BR responses in transgenic plants. We propose that the spatial redistribution of BZR1 is critical for proper BR signaling in plant growth and development.

An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal Transduction in Arabidopsis

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

Targeting DNA Mismatches with Rhodium Intercalators Functionalized with a Cell-Penetrating Peptide

Cell-penetrating peptides are widely used to deliver cargo molecules into cells. Here we describe the synthesis, characterization, DNA binding, and cellular uptake studies of a series of metal-peptide conjugates containing oligoarginine as a cell-penetrating peptide. d-Octaarginine units are appended onto a rhodium intercalator containing the sterically expansive chrysenequinone diimine (chrysi) ligand to form Rh(chrysi)(phen)(bpy)(3+)-tethered oligoarginine conjugates, where the peptide is attached to the ancillary bpy ligand; some conjugates also include a fluorescein or thiazole orange tag. These complexes bind and with photoactivation selectively cleave DNA neighboring single-base mismatches. The presence of the oligoarginines is found to increase the nonspecific binding affinity of the complexes for both matched and mismatched DNA, but for these conjugates, photocleavage remains selective for the mismatched site, as assayed using both gel electrophoresis and mass spectrometry experiments. Significantly, the rhodium complex does not interfere with the delivery properties of the cell-penetrating peptide. Confocal microscopy experiments show rapid nuclear localization of the metal-peptide conjugates containing the tethered fluorescein. Mass spectrometry experiments confirm the association of the rhodium with the HeLa cells. These results provide a strategy for targeting mismatch-selective metal complexes inside cell nuclei.

Intracellular Delivery of Poly(ethylene glycol) Conjugated Antisense Oligonucleotide Using Cationic Lipids by Formation of Self-Assembled Polyelectrolyte Complex Micelles

A polyelectrolyte complex (PEC) micelle-based antisense oligodeoxynucleotide (ODN) delivery system was designed to overcome intrinsic limitations of cationic lipid-mediated gene transfer. Cationic lipid (Lipofectamine, LF) and ODN conjugated poly(ethylene glycol) (PEG) were ionically complexed to form self-assembled spherical PEC micelles. They have a distinctive structural feature: a charge-neutralized core surrounded by highly flexible PEG corona. The PEC micelles could be visualized as a nano-sized sphere by atomic force microscopy (AFM). The DNA/LF PEC micelles exhibited far improved transfection efficiency compared to those of conventional lipoplex formulations (ODN/LF) in the presence of serum. They showed enhanced cellular uptake followed by rapid nuclear localization of ODN in human epithelial carcinoma (KB) cells. In addition, anti-proliferative activity of c-raf gene-directed antisense ODN was almost fully maintained in KB cells in the presence of serum.

Ribonucleic Acid Polymerase II Binding Subunit 3 (Rpb3), a Potential Nuclear Target of Insulin-Like Growth Factor Binding Protein-3

IGF-binding protein (IGFBP)-3 has intrinsic antiproliferative and proapoptotic functions that are independent of IGF binding and may involve nuclear localization. We determined that exogenous IGFBP-3 rapidly translocates to myoblast nuclei and that a 22-residue peptide containing the metal binding domain (MBD) and nuclear localization sequence (NLS) can similarly direct chimeric GFP into myoblast nuclei. Furthermore, a non-IGF-binding IGFBP-3 mutant inhibited myoblast proliferation without stimulating apoptosis. These results suggest that IGFBP-3 inhibits muscle cell growth in an IGF-independent manner that may be influenced by its rapid nuclear localization. We therefore identified IGFBP-3 interacting proteins by screening a rat L6 myoblast cDNA library using the yeast two-hybrid assay and two N-terminal deletion mutants as bait: BP3/231 (231 residues, L61 to K291) and BP3/111 (K181-K291). Proteins previously known to interact with IGFBP-3 as well as several novel proteins were identified, including RNA polymerase II binding subunit 3 (Rpb3). The domain necessary for Rpb3 binding was subsequently identified using different IGFBP-3 deletion mutants and was localized to the MBD/NLS epitope. Rpb3/IGFBP-3 binding was confirmed by coimmunoprecipitation assays with specific antisera, whereas a NLS mutant IGFBP-3 did not associate with Rpb3, suggesting that a functional NLS is required. Rpb3 facilitates recruitment of the polymerase complex to specific transcription factors and is necessary for the transactivation of many genes. Its association with IGFBP-3 provides a functional role for IGFBP-3 in the direct modulation of gene transcription.

Mechanism of Uptake of C105Y, a Novel Cell-penetrating Peptide

C105Y, a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acid sequence corresponding to residues 359-374 of alpha1-antitrypsin, enhances gene expression from DNA nanoparticles. To investigate how this enhancement occurs, C105Y was fluorescently labeled to study its uptake and intracellular trafficking. When human hepatoma cells (HuH7) were incubated with fluorescently labeled C105Y for as little as 3 min, C105Y displayed nuclear and cytoplasmic staining with enrichment of fluorescent signal in the nucleus and nucleolus. Uptake and nucleolar localization were observed with the short sequence PFVYLI, but not with SIPPEVKFNK, and the D-isomer was readily taken up into cells but not into the nucleus. We found that the C105Y peptide is routed to the nucleolus very rapidly in an energy-dependent fashion, whereas membrane translocation and nuclear localization are energy-independent. When we tested the involvement of known endocytosis pathways in uptake and trafficking of this peptide, we demonstrated that C105Y peptide is internalized by a clathrin- and caveolin-independent pathway, although lipid raft-mediated endocytosis may play a role in peptide intracellular trafficking. Efficient energy-independent cell entry with rapid nuclear localization probably accounts for enhancement of gene expression from inclusion of C105Y into DNA nanoparticles.