Design of Conditionally Active STATs: Insights into STAT Activation and Gene Regulatory Function

ABSTRACT The STAT (signal transducer and activator of transcription) signaling pathway is activated by a large number of cytokines and growth factors. We sought to design a conditionally active STAT that could not only provide insight into basic questions about STAT function but also serve as a powerful tool to determine the precise biological role of STATs. To this end, we have developed a conditionally active STAT by fusing STATs with the ligand-binding domain of the estrogen receptor (ER). We have demonstrated that the resulting STAT-ER chimeras are estrogen-inducible transcription factors that retain the functional and biochemical characteristics of the cognate wild-type STATs. In addition, these tools have allowed us to evaluate separately the contribution of tyrosine phosphorylation and dimerization to STAT function. We have for the first time provided experimental data supporting the model that the only apparent role of STAT tyrosine phosphorylation is to drive dimerization, as dimerization alone is sufficient to unmask a latent STAT nuclear localization sequence and induce nuclear translocation, sequence-specific DNA binding, and transcriptional activity.

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Integrin signaling is critical for pathological angiogenesis

Journal of Experimental Medicine ◽

10.1084/jem.20060807 ◽

2006 ◽

Vol 203 (11) ◽

pp. 2495-2507 ◽

Cited By ~ 131

Author(s):

Ganapati H. Mahabeleshwar ◽

Weiyi Feng ◽

David R. Phillips ◽

Tatiana V. Byzova

Keyword(s):

Endothelial Cells ◽

Tyrosine Phosphorylation ◽

Ex Vivo ◽

Regulatory Function ◽

Vegf Receptor ◽

Wild Type ◽

Pathological Angiogenesis ◽

Β3 Integrin ◽

In Cells

The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in tissue remodeling upon injury. However, the molecular events underlying this complex process are not well understood and numerous issues remain controversial, including the regulatory function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in angiogenesis, we generated knock-in mice that express a mutant β3 integrin unable to undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed that neovascularization is impaired in mutant β3 knock-in mice. In an ex vivo angiogenesis assay, mutant β3 knock-in endothelial cells did not form complete capillaries in response to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine phosphorylation in mutant β3 knock-in cells resulted in impaired adhesion, spreading, and migration of endothelial cells. At the molecular level, VEGF stimulated complex formation between VEGF receptor-2 and β3 integrin in wild-type but not in mutant β3 knock-in endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in cells expressing mutant β3 compared to wild type, leading to impaired integrin activation in these cells. These findings provide novel mechanistic insights into the role of integrin–VEGF axis in pathological angiogenesis.

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Identification of a putative nuclear localization sequence within ANG II AT1A receptor associated with nuclear activation

AJP Cell Physiology ◽

10.1152/ajpcell.00337.2006 ◽

2007 ◽

Vol 292 (4) ◽

pp. C1398-C1408 ◽

Cited By ~ 40

Author(s):

Thomas A. Morinelli ◽

John R. Raymond ◽

Aleksander Baldys ◽

Qing Yang ◽

Mi-hye Lee ◽

...

Keyword(s):

Nuclear Localization ◽

Intracellular Signaling ◽

Nuclear Translocation ◽

Receptor Expression ◽

Nuclear Localization Sequence ◽

Ang Ii ◽

Wild Type ◽

Nuclear Activation ◽

Localization Sequence ◽

Carboxy Terminal

Angiotensin II (ANG II) type 1 (AT1) receptors, similar to other G protein-coupled receptors, undergo desensitization and internalization, and potentially nuclear localization, subsequent to agonist interaction. Evidence suggests that the carboxy-terminal tail may be involved in receptor nuclear localization. In the present study, we examined the carboxy-terminal tail of the receptor for specific regions responsible for the nuclear translocation phenomenon and resultant nuclear activation. Human embryonic kidney cells stably expressing either a wild-type AT1A receptor-green fluorescent protein (AT1AR/GFP) construct or a site-directed mutation of a putative nuclear localization sequence (NLS) [K307Q]AT1AR/GFP (KQ/AT1AR/GFP), were examined for differences in receptor nuclear trafficking and nuclear activation. Receptor expression, intracellular signaling, and ANG II-induced internalization of the wild-type/GFP construct and of the KQ/AT1AR/GFP mutant was similar. Laser scanning confocal microscopy showed that in cells expressing the AT1AR/GFP, trafficking of the receptor to the nuclear area and colocalization with lamin B occurred within 30 min of ANG II (100 nM) stimulation, whereas the KQ/AT1AR/GFP mutant failed to demonstrate nuclear localization. Immunoblotting of nuclear lysates with an anti-GFP antibody confirmed these observations. Nuclear localization of the wild-type receptor correlated with increase transcription for both EGR-1 and PTGS-2 genes while the nuclear-deficient KQ/AT1AR/GFP mutant demonstrated increases for only the EGR-1 gene. These results suggest that a NLS (KKFKKY; aa307–312) is located within the cytoplasmic tail of the AT1A receptor and that nuclear localization of the receptor corresponds with specific activation of transcription for the COX-2 gene PTGS-2.

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Analysis of myotilin turnover provides mechanistic insight into the role of myotilinopathy-causing mutations

Biochemical Journal ◽

10.1042/bj20101672 ◽

2011 ◽

Vol 436 (1) ◽

pp. 113-121 ◽

Cited By ~ 12

Author(s):

Pernilla von Nandelstadh ◽

Rabah Soliymani ◽

Marc Baumann ◽

Olli Carpen

Keyword(s):

Morphological Changes ◽

Protein Accumulation ◽

Cleavage Sites ◽

Wild Type ◽

Molecular Defects ◽

Mechanistic Insight ◽

First Time ◽

Insight Into ◽

Biological Differences

MFM (myofibrillar myopathies) are caused by mutations in several sarcomeric components, including the Z-disc protein myotilin. The morphological changes typical of MFM include Z-disc alterations and aggregation of dense filamentous sarcomeric material. The causes and mechanisms of protein aggregation in myotilinopathies and other forms of MFM remain unknown, although impaired degradation may explain, in part, the abnormal protein accumulation. In the present paper we have studied the mechanisms regulating myotilin turnover, analysed the consequences of defective myotilin degradation and tested whether disease-causing myotilin mutations result in altered protein turnover. The results indicate that myotilin is a substrate for the Ca2+-dependent protease calpain and identify two calpain cleavage sites in myotilin by MS. We further show that myotilin is degraded by the proteasome system in transfected COS7 cells and in myotubes, and that disease-causing myotilinopathy mutations result in reduced degradation. Finally, we show that proteolysis-inhibitor-induced reduction in myotilin turnover results in formation of intracellular myotilin and actin-containing aggregates, which resemble those seen in diseased muscle cells. These findings identify for the first time biological differences between wt (wild-type) and mutant myotilin. The present study provides novel information on the pathways controlling myotilin turnover and on the molecular defects associated with MFM.

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Salicylic Acid Accumulation Controlled by LSD1 Is Essential in Triggering Cell Death in Response to Abiotic Stress

Cells ◽

10.3390/cells10040962 ◽

2021 ◽

Vol 10 (4) ◽

pp. 962

Author(s):

Maciej Jerzy Bernacki ◽

Anna Rusaczonek ◽

Weronika Czarnocka ◽

Stanisław Karpiński

Keyword(s):

Cell Death ◽

Salicylic Acid ◽

Abiotic Stress ◽

Wild Type ◽

Pr Genes ◽

Genes Expression ◽

Salicylic Acid Accumulation ◽

Cell Death Phenotype ◽

Insight Into

Salicylic acid (SA) is well known hormonal molecule involved in cell death regulation. In response to a broad range of environmental factors (e.g., high light, UV, pathogens attack), plants accumulate SA, which participates in cell death induction and spread in some foliar cells. LESION SIMULATING DISEASE 1 (LSD1) is one of the best-known cell death regulators in Arabidopsis thaliana. The lsd1 mutant, lacking functional LSD1 protein, accumulates SA and is conditionally susceptible to many biotic and abiotic stresses. In order to get more insight into the role of LSD1-dependent regulation of SA accumulation during cell death, we crossed the lsd1 with the sid2 mutant, caring mutation in ISOCHORISMATE SYNTHASE 1(ICS1) gene and having deregulated SA synthesis, and with plants expressing the bacterial nahG gene and thus decomposing SA to catechol. In response to UV A+B irradiation, the lsd1 mutant exhibited clear cell death phenotype, which was reversed in lsd1/sid2 and lsd1/NahG plants. The expression of PR-genes and the H2O2 content in UV-treated lsd1 were significantly higher when compared with the wild type. In contrast, lsd1/sid2 and lsd1/NahG plants demonstrated comparability with the wild-type level of PR-genes expression and H2O2. Our results demonstrate that SA accumulation is crucial for triggering cell death in lsd1, while the reduction of excessive SA accumulation may lead to a greater tolerance toward abiotic stress.

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NMR and LC-MS-Based Metabolomics to Study Osmotic Stress in Lignan-Deficient Flax

Molecules ◽

10.3390/molecules26030767 ◽

2021 ◽

Vol 26 (3) ◽

pp. 767

Author(s):

Kamar Hamade ◽

Ophélie Fliniaux ◽

Jean-Xavier Fontaine ◽

Roland Molinié ◽

Elvis Otogo Nnang ◽

...

Keyword(s):

Stress Response ◽

Osmotic Stress ◽

Biosynthetic Pathway ◽

Potential Role ◽

Plant Secondary Metabolites ◽

Wild Type ◽

Osmotic Stress Response ◽

Transgenic Flax ◽

Insight Into

Lignans, phenolic plant secondary metabolites, are derived from the phenylpropanoid biosynthetic pathway. Although, being investigated for their health benefits in terms of antioxidant, antitumor, anti-inflammatory and antiviral properties, the role of these molecules in plants remains incompletely elucidated; a potential role in stress response mechanisms has been, however, proposed. In this study, a non-targeted metabolomic analysis of the roots, stems, and leaves of wild-type and PLR1-RNAi transgenic flax, devoid of (+) secoisolariciresinol diglucoside ((+) SDG)—the main flaxseed lignan, was performed using 1H-NMR and LC-MS, in order to obtain further insight into the involvement of lignan in the response of plant to osmotic stress. Results showed that wild-type and lignan-deficient flax plants have different metabolic responses after being exposed to osmotic stress conditions, but they both showed the capacity to induce an adaptive response to osmotic stress. These findings suggest the indirect involvement of lignans in osmotic stress response.

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Regulation of hair follicle development by the TNF signal ectodysplasin and its receptor Edar

Development ◽

10.1242/dev.129.10.2541 ◽

2002 ◽

Vol 129 (10) ◽

pp. 2541-2553 ◽

Cited By ~ 4

Author(s):

Johanna Laurikkala ◽

Johanna Pispa ◽

Han-Sung Jung ◽

Pekka Nieminen ◽

Marja Mikkola ◽

...

Keyword(s):

Signaling Pathway ◽

Hair Follicles ◽

Wild Type ◽

Mutant Mouse Embryos ◽

Anhidrotic Ectodermal Dysplasia ◽

Embryonic Morphogenesis ◽

Hair Follicle Development ◽

And Function ◽

Insight Into

X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for β-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.

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Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

Circulation Research ◽

10.1161/res.109.suppl_1.ap283 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Chengqun Huang ◽

Eric P Ratliff ◽

Genaro Hernandez ◽

Pamela Lee ◽

...

Keyword(s):

Ex Vivo ◽

Wild Type ◽

Simulated Ischemia ◽

Selective Targeting ◽

Cardioprotective Effects ◽

Mitochondrial Turnover ◽

First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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The role of phagocytosis-dependent activation of TFEB in the clearance of salmonella

10.32920/ryerson.14655972 ◽

2021 ◽

Author(s):

Erika Ospina Escobar

Keyword(s):

Transcription Factor ◽

Salmonella Typhimurium ◽

Nuclear Translocation ◽

Steady Increase ◽

Phagosome Maturation ◽

Salmonella Infection ◽

Complex Interplay ◽

Transcription Factor Eb ◽

Insight Into

During phagocytosis, macrophages engulf and sequester pathogens into phagosomes. Phagosomes then fuse with acidic and degradative lysosomes to degrade the internalized pathogen. We previously demonstrated that phagocytosis of IgG-opsonized particles and non-opsonized E.coli causes activation of the Transcription Factor EB (TFEB), which enhances the expression of lysosomal genes, increases the degradative capacity of lysosomes and boosts bactericidal activity. However, pathogens like Salmonella typhimurium have evolved mechanisms to evade and/or alter phagosome maturation to promote their own survival. We investigated: i) whether pathogens like Salmonella can alter TFEB activation and ii) whether phagocytosis-dependent activation of TFEB can counteract the pathogenicity of microorganisms. Here, we show that non-viable (heat-killed) S. typhimurium, pathogenic (EHEC and UPEC) and non-pathogenic E.coli (DH5α) all caused TFEB nuclear translocation in RAW macrophages, while strikingly live S. typhimurium maintained TFEB in the cytosol in the first hours post-infection. By contrast, Salmonella mutants for ΔsifA, ΔsopD2, ΔphoP all triggered TFEB activation in the first hour of infection. However, Salmonella infection eventually triggered a steady increase in nuclear TFEB after 4 h of infection, suggesting a more complex interplay between TFEB and Salmonella infection. We dissected the importance of TFEB activation towards Salmonella survivability by pre-activating TFEB before infection within WT macrophages and macrophages with a CRISPR-based deletion of TFEB. Our work suggests that Salmonella actively interferes with TFEB signaling in order to enhance its own survival. These results could provide insight into using TFEB as a target for the clearance of infections.

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The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

Journal of Cell Science ◽

10.1242/jcs.113.15.2771 ◽

2000 ◽

Vol 113 (15) ◽

pp. 2771-2781

Author(s):

P.S. Subramaniam ◽

J. Larkin ◽

M.G. Mujtaba ◽

M.R. Walter ◽

H.M. Johnson

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Nuclear Translocation ◽

Biological Activities ◽

Nuclear Localization Sequence ◽

Receptor Complex ◽

Ifn Gamma ◽

High Affinity ◽

Gamma Receptor ◽

Localization Sequence

We have recently shown that the nuclear localization of IFN gamma is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95–133) containing the NLS blocked the induction of STAT1 alpha nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1 alpha in IFN gamma treated cells. A deletion mutant of human IFN gamma, IFN gamma (1–123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K(d) similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1–123) to initiate the nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1 alpha nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFN gamma with a K(d) approx. 3 × 10(−8) M(−1) has been described by previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 alpha nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95–133), that contains the NLS motif, induced nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1 alpha.

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Complex Formation with Monomeric α-Tubulin and Importin 13 Fosters c-Jun Protein Stability and Is Required for c-Jun’s Nuclear Translocation and Activity

Cancers ◽

10.3390/cancers11111806 ◽

2019 ◽

Vol 11 (11) ◽

pp. 1806

Author(s):

Melanie Kappelmann-Fenzl ◽

Silke Kuphal ◽

Rosemarie Krupar ◽

Dirk Schadendorf ◽

Viktor Umansky ◽

...

Keyword(s):

Complex Formation ◽

Protein Stability ◽

Nuclear Translocation ◽

Nuclear Accumulation ◽

Nuclear Localization Sequence ◽

Microtubule Network ◽

Localization Sequence ◽

Dynamic Structures ◽

Main Regulator ◽

Factor C

Microtubules are highly dynamic structures, which consist of α- and β-tubulin heterodimers. They are essential for a number of cellular processes, including intracellular trafficking and mitosis. Tubulin-binding chemotherapeutics are used to treat different types of tumors, including malignant melanoma. The transcription factor c-Jun is a central driver of melanoma development and progression. Here, we identify the microtubule network as a main regulator of c-Jun activity. Monomeric α-tubulin fosters c-Jun protein stability by protein–protein interaction. In addition, this complex formation is necessary for c-Jun’s nuclear localization sequence binding to importin 13, and consequent nuclear import and activity of c-Jun. A reduction in monomeric α-tubulin levels by treatment with the chemotherapeutic paclitaxel resulted in a decline in the nuclear accumulation of c-Jun in melanoma cells in an experimental murine model and in patients’ tissues. These findings add important knowledge to the mechanism of the action of microtubule-targeting drugs and indicate the newly discovered regulation of c-Jun by the microtubule cytoskeleton as a novel therapeutic target for melanoma and potentially also other types of cancer.

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