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Sample records for 14-3-3 proteins regulate

  1. 14-3-3 Proteins are Regulators of Autophagy

    PubMed Central

    Pozuelo-Rubio, Mercedes

    2012-01-01

    14-3-3 proteins are implicated in the regulation of proteins involved in a variety of signaling pathways. 14-3-3-dependent protein regulation occurs through phosphorylation-dependent binding that results, in many cases, in the release of survival signals in cells. Autophagy is a cell digestion process that contributes to overcoming nutrient deprivation and is initiated under stress conditions. However, whether autophagy is a cell survival or cell death mechanism remains under discussion and may depend on context. Nevertheless, autophagy is a cellular process that determines cell fate and is tightly regulated by different signaling pathways, some of which, for example MAPK, PI3K and mTOR, are tightly regulated by 14-3-3 proteins. It is therefore important to understand the role of 14-3-3 protein in modulating the autophagic process. Within this context, direct binding of 14-3-3 to mTOR regulatory proteins, such as TSC2 and PRAS40, connects 14-3-3 with autophagy regulatory processes. In addition, 14-3-3 binding to human vacuolar protein sorting 34 (hVps34), a class III phosphatidylinositol-3-kinase (PI3KC3), indicates the involvement of 14-3-3 proteins in regulating autophagosome formation. hVps34 is involved in vesicle trafficking processes such as autophagy, and its activation is needed for initiation of autophagy. Chromatography and overlay techniques suggest that hVps34 directly interacts with 14-3-3 proteins under physiological conditions, thereby maintaining hVps34 in an inactive state. In contrast, nutrient starvation promotes dissociation of the 14-3-3–hVps34 complex, thereby enhancing hVps34 lipid kinase activity. Thus, 14-3-3 proteins are regulators of autophagy through regulating key components of the autophagic machinery. This review summarizes the role of 14-3-3 protein in the control of target proteins involved in regulating the master switches of autophagy. PMID:24710529

  2. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment.

  3. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment. PMID:25549848

  4. Involvement of 14-3-3 Proteins in Regulating Tumor Progression of Hepatocellular Carcinoma.

    PubMed

    Wu, Yi-Ju; Jan, Yee-Jee; Ko, Bor-Sheng; Liang, Shu-Man; Liou, Jun-Yang

    2015-01-01

    There are seven mammalian isoforms of the 14-3-3 protein, which regulate multiple cellular functions via interactions with phosphorylated partners. Increased expression of 14-3-3 proteins contributes to tumor progression of various malignancies. Several isoforms of 14-3-3 are overexpressed and associate with higher metastatic risks and poorer survival rates of hepatocellular carcinoma (HCC). 14-3-3β and 14-3-3ζ regulate HCC cell proliferation, tumor growth and chemosensitivity via modulating mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) and p38 signal pathways. Moreover, 14-3-3ε suppresses E-cadherin and induces focal adhesion kinase (FAK) expression, thereby enhancing epithelial-mesenchymal transition (EMT) and HCC cell migration. 14-3-3ζ forms complexes with αB-crystallin, which induces EMT and is the cause of sorafenib resistance in HCC. Finally, a recent study has indicated that 14-3-3σ induces heat shock protein 70 (HSP70) expression, which increases HCC cell migration. These results suggest that selective 14-3-3 isoforms contribute to cell proliferation, EMT and cell migration of HCC by regulating distinct targets and signal pathways. Targeting 14-3-3 proteins together with specific downstream effectors therefore has potential to be therapeutic and prognostic factors of HCC. In this article, we will overview 14-3-3's regulation of its downstream factors and contributions to HCC EMT, cell migration and proliferation. PMID:26083935

  5. 14-3-3 proteins regulate Tctp–Rheb interaction for organ growth in Drosophila

    PubMed Central

    Le, Thao Phuong; Vuong, Linh Thuong; Kim, Ah-Ram; Hsu, Ya-Chieh; Choi, Kwang-Wook

    2016-01-01

    14-3-3 family proteins regulate multiple signalling pathways. Understanding biological functions of 14-3-3 proteins has been limited by the functional redundancy of conserved isotypes. Here we provide evidence that 14-3-3 proteins regulate two interacting components of Tor signalling in Drosophila, translationally controlled tumour protein (Tctp) and Rheb GTPase. Single knockdown of 14-3-3ɛ or 14-3-3ζ isoform does not show obvious defects in organ development but causes synergistic genetic interaction with Tctp and Rheb to impair tissue growth. 14-3-3 proteins physically interact with Tctp and Rheb. Knockdown of both 14-3-3 isoforms abolishes the binding between Tctp and Rheb, disrupting organ development. Depletion of 14-3-3s also reduces the level of phosphorylated S6 kinase, phosphorylated Thor/4E-BP and cyclin E (CycE). Growth defects from knockdown of 14-3-3 and Tctp are suppressed by CycE overexpression. This study suggests a novel mechanism of Tor regulation mediated by 14-3-3 interaction with Tctp and Rheb. PMID:27151460

  6. Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity.

    PubMed

    Chi, Jen-Chih; Roeper, Juliane; Schwarz, Guenter; Fischer-Schrader, Katrin

    2015-03-01

    14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions. PMID:25578809

  7. 14-3-3 protein is a regulator of the mitochondrial and chloroplast ATP synthase

    PubMed Central

    Bunney, Tom D.; van Walraven, Hendrika S.; de Boer, Albertus H.

    2001-01-01

    Mitochondrial and chloroplast ATP synthases are key enzymes in plant metabolism, providing cells with ATP, the universal energy currency. ATP synthases use a transmembrane electrochemical proton gradient to drive synthesis of ATP. The enzyme complexes function as miniature rotary engines, ensuring energy coupling with very high efficiency. Although our understanding of the structure and functioning of the synthase has made enormous progress in recent years, our understanding of regulatory mechanisms is still rather preliminary. Here we report a role for 14-3-3 proteins in the regulation of ATP synthases. These 14-3-3 proteins are highly conserved phosphoserine/phosphothreonine-binding proteins that regulate a wide range of enzymes in plants, animals, and yeast. Recently, the presence of 14-3-3 proteins in chloroplasts was illustrated, and we show here that plant mitochondria harbor 14-3-3s within the inner mitochondrial-membrane compartment. There, the 14-3-3 proteins were found to be associated with the ATP synthases, in a phosphorylation-dependent manner, through direct interaction with the F1 β-subunit. The activity of the ATP synthases in both organelles is drastically reduced by recombinant 14-3-3. The rapid reduction in chloroplast ATPase activity during dark adaptation was prevented by a phosphopeptide containing the 14-3-3 interaction motif, demonstrating a role for endogenous 14-3-3 in the down-regulation of the CFoF1 activity. We conclude that regulation of the ATP synthases by 14-3-3 represents a mechanism for plant adaptation to environmental changes such as light/dark transitions, anoxia in roots, and fluctuations in nutrient supply. PMID:11274449

  8. Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.

    PubMed

    Liang, Xiubin; Da Paula, Ana Carina; Bozóky, Zoltán; Zhang, Hui; Bertrand, Carol A; Peters, Kathryn W; Forman-Kay, Julie D; Frizzell, Raymond A

    2012-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.

  9. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  10. 14-3-3 proteins: Macro-regulators with great potential for improving abiotic stress tolerance in plants.

    PubMed

    Liu, Qing; Zhang, Shaohong; Liu, Bin

    2016-08-12

    14-3-3 proteins (14-3-3s) are highly conserved regulatory proteins that are uniquely eukaryotic, and deeply involved in protein-protein interactions that mediate diverse signaling pathways. In plants, 14-3-3s have been validated to regulate many biological processes, such as metabolism, light and hormone signaling, cell-cycle control and protein trafficking. Recent years we have also witnessed an increasing number of reports describing the functions of 14-3-3s in plant stress responses through interactions with key proteins in both biotic and abiotic stresses. In this review, we highlight the advances that have been made in investigating the roles of 14-3-3s in plant abiotic stress tolerance. These advances provide a framework for our understanding of how signals are integrated to perceive and respond to the abiotic stresses in plants. PMID:27233603

  11. Cotton (Gossypium hirsutum) 14-3-3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling.

    PubMed

    Zhou, Ying; Zhang, Ze-Ting; Li, Mo; Wei, Xin-Zheng; Li, Xiao-Jie; Li, Bing-Ying; Li, Xue-Bao

    2015-02-01

    Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14-3-3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14-3-3L, Gh14-3-3e and Gh14-3-3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14-3-3 RNAi transgenic plants were significantly shorter than those of wild type. This 'short fibre' phenotype of the 14-3-3 RNAi cotton could be partially rescued by application of 2,4-epibrassinolide (BL). Expression levels of the BR-related and fibre-related genes were altered in the Gh14-3-3 transgenic fibres. Furthermore, we identified Gh14-3-3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14-3-3L/e/h were required for Gh14-3-3-GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14-3-3 proteins. Additionally, 14-3-3-regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14-3-3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.

  12. 14-3-3 proteins in plant-pathogen interactions.

    PubMed

    Lozano-Durán, Rosa; Robatzek, Silke

    2015-05-01

    14-3-3 proteins define a eukaryotic-specific protein family with a general role in signal transduction. Primarily, 14-3-3 proteins act as phosphosensors, binding phosphorylated client proteins and modulating their functions. Since phosphorylation regulates a plethora of different physiological responses in plants, 14-3-3 proteins play roles in multiple signaling pathways, including those controlling metabolism, hormone signaling, cell division, and responses to abiotic and biotic stimuli. Increasing evidence supports a prominent role of 14-3-3 proteins in regulating plant immunity against pathogens at various levels. In this review, potential links between 14-3-3 function and the regulation of plant-pathogen interactions are discussed, with a special focus on the regulation of 14-3-3 proteins in response to pathogen perception, interactions between 14-3-3 proteins and defense-related proteins, and 14-3-3 proteins as targets of pathogen effectors.

  13. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

    PubMed Central

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

  14. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis.

    PubMed

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis. PMID:26717241

  15. A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis.

    PubMed

    Sun, Xiaoli; Sun, Mingzhe; Jia, Bowei; Chen, Chao; Qin, Zhiwei; Yang, Kejun; Shen, Yang; Meiping, Zhang; Mingyang, Cong; Zhu, Yanming

    2015-01-01

    It is widely accepted that the 14-3-3 family proteins are key regulators of multiple stress signal transduction cascades. By conducting genome-wide analysis, researchers have identified the soybean 14-3-3 family proteins; however, until now, there is still no direct genetic evidence showing the involvement of soybean 14-3-3s in ABA responses. Hence, in this study, based on the latest Glycine max genome on Phytozome v10.3, we initially analyzed the evolutionary relationship, genome organization, gene structure and duplication, and three-dimensional structure of soybean 14-3-3 family proteins systematically. Our results suggested that soybean 14-3-3 family was highly evolutionary conserved and possessed segmental duplication in evolution. Then, based on our previous functional characterization of a Glycine soja 14-3-3 protein GsGF14o in drought stress responses, we further investigated the expression characteristics of GsGF14o in detail, and demonstrated its positive roles in ABA sensitivity. Quantitative real-time PCR analyses in Glycine soja seedlings and GUS activity assays in PGsGF14O:GUS transgenic Arabidopsis showed that GsGF14o expression was moderately and rapidly induced by ABA treatment. As expected, GsGF14o overexpression in Arabidopsis augmented the ABA inhibition of seed germination and seedling growth, promoted the ABA induced stomata closure, and up-regulated the expression levels of ABA induced genes. Moreover, through yeast two hybrid analyses, we further demonstrated that GsGF14o physically interacted with the AREB/ABF transcription factors in yeast cells. Taken together, results presented in this study strongly suggested that GsGF14o played an important role in regulation of ABA sensitivity in Arabidopsis.

  16. Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2016-09-01

    The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYK's interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYK's binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYK's interaction with Importin 7. PMID:27381982

  17. Posttranscriptional regulation of 14-3-3ζ by RNA-binding protein HuR modulating intestinal epithelial restitution after wounding.

    PubMed

    Hansraj, Natasha Z; Xiao, Lan; Wu, Jing; Chen, Gang; Turner, Douglas J; Wang, Jian-Ying; Rao, Jaladanki N

    2016-07-01

    The 14-3-3ζ is a member of the family of 14-3-3 proteins and participates in many aspects of cellular processes, but its regulation and involvement in gut mucosal homeostasis remain unknown. Here, we report that 14-3-3ζ expression is tightly regulated at the posttranscription level by RNA-binding protein HuR and plays an important role in early intestinal epithelial restitution after wounding. The 14-3-3ζ was highly expressed in the mucosa of gastrointestinal tract and in cultured intestinal epithelial cells (IECs). The 3' untranslated region (UTR) of the 14-3-3ζ mRNA was bound to HuR, and this association enhanced 14-3-3ζ translation without effect on its mRNA content. Conditional target deletion of HuR in IECs decreased the level of 14-3-3ζ protein in the intestinal mucosa. Silencing 14-3-3ζ by transfection with specific siRNA targeting the 14-3-3ζ mRNA suppressed intestinal epithelial restitution as indicated by a decrease in IEC migration after wounding, whereas ectopic overexpression of the wild-type 14-3-3ζ promoted cell migration. These results indicate that HuR induces 14-3-3ζ translation via interaction with its 3' UTR and that 14-3-3ζ is necessary for stimulation of IEC migration after wounding. PMID:27401462

  18. Posttranscriptional regulation of 14-3-3ζ by RNA-binding protein HuR modulating intestinal epithelial restitution after wounding.

    PubMed

    Hansraj, Natasha Z; Xiao, Lan; Wu, Jing; Chen, Gang; Turner, Douglas J; Wang, Jian-Ying; Rao, Jaladanki N

    2016-07-01

    The 14-3-3ζ is a member of the family of 14-3-3 proteins and participates in many aspects of cellular processes, but its regulation and involvement in gut mucosal homeostasis remain unknown. Here, we report that 14-3-3ζ expression is tightly regulated at the posttranscription level by RNA-binding protein HuR and plays an important role in early intestinal epithelial restitution after wounding. The 14-3-3ζ was highly expressed in the mucosa of gastrointestinal tract and in cultured intestinal epithelial cells (IECs). The 3' untranslated region (UTR) of the 14-3-3ζ mRNA was bound to HuR, and this association enhanced 14-3-3ζ translation without effect on its mRNA content. Conditional target deletion of HuR in IECs decreased the level of 14-3-3ζ protein in the intestinal mucosa. Silencing 14-3-3ζ by transfection with specific siRNA targeting the 14-3-3ζ mRNA suppressed intestinal epithelial restitution as indicated by a decrease in IEC migration after wounding, whereas ectopic overexpression of the wild-type 14-3-3ζ promoted cell migration. These results indicate that HuR induces 14-3-3ζ translation via interaction with its 3' UTR and that 14-3-3ζ is necessary for stimulation of IEC migration after wounding.

  19. 14-3-3 proteins: a historic overview.

    PubMed

    Aitken, Alastair

    2006-06-01

    This chapter includes a historic overview of 14-3-3 proteins with an emphasis on the differences between potentially cancer-relevant isoforms on the genomic, protein and functional level. The focus will therefore be on mammalian 14-3-3s although many important developments in the field have involved Drosophila 14-3-3 proteins for example and the cross-fertilisation from parallel studies on plant 14-3-3 should not be underestimated. In the major part of this review I will attempt to focus on some novel data and aspects of 14-3-3 structure and function, in particular regulation of 14-3-3 isoforms by oncogene-related protein kinase phosphorylation and aspects of 14-3-3 research with which newcomers to the field may be less familiar.

  20. Cofilin regulator 14-3-3zeta is an evolutionarily conserved protein required for phagocytosis and microbial resistance.

    PubMed

    Ulvila, Johanna; Vanha-aho, Leena-Maija; Kleino, Anni; Vähä-Mäkilä, Mari; Vuoksio, Milka; Eskelinen, Sinikka; Hultmark, Dan; Kocks, Christine; Hallman, Mikko; Parikka, Mataleena; Rämet, Mika

    2011-05-01

    Phagocytosis is an ancient cellular process that plays an important role in host defense. In Drosophila melanogaster phagocytic, macrophage-like hemocytes recognize and ingest microbes. We performed an RNAi-based in vitro screen in the Drosophila hemocyte cell line S2 and identified Abi, cpa, cofilin regulator 14-3-3ζ, tlk, CG2765, and CG15609 as mediators of bacterial phagocytosis. Of these identified genes, 14-3-3ζ had an evolutionarily conserved role in phagocytosis: bacterial phagocytosis was compromised when 14-3-3ζ was targeted with RNAi in primary Drosophila hemocytes and when the orthologous genes Ywhab and Ywhaz were silenced in zebrafish and mouse RAW 264.7 cells, respectively. In Drosophila and zebrafish infection models, 14-3-3ζ was required for resistance against Staphylococcus aureus. We conclude that 14-3-3ζ is essential for phagocytosis and microbial resistance in insects and vertebrates. PMID:21208897

  1. Structural Basis for the 14-3-3 Protein-dependent Inhibition of the Regulator of G Protein Signaling 3 (RGS3) Function*

    PubMed Central

    Rezabkova, Lenka; Man, Petr; Novak, Petr; Herman, Petr; Vecer, Jaroslav; Obsilova, Veronika; Obsil, Tomas

    2011-01-01

    Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins for the α-subunit of heterotrimeric G proteins. The function of certain RGS proteins is negatively regulated by 14-3-3 proteins, a family of highly conserved regulatory molecules expressed in all eukaryotes. In this study, we provide a structural mechanism for 14-3-3-dependent inhibition of RGS3-Gα interaction. We have used small angle x-ray scattering, hydrogen/deuterium exchange kinetics, and Förster resonance energy transfer measurements to determine the low-resolution solution structure of the 14-3-3ζ·RGS3 complex. The structure shows the RGS domain of RGS3 bound to the 14-3-3ζ dimer in an as-yet-unrecognized manner interacting with less conserved regions on the outer surface of the 14-3-3 dimer outside its central channel. Our results suggest that the 14-3-3 protein binding affects the structure of the Gα interaction portion of RGS3 as well as sterically blocks the interaction between the RGS domain and the Gα subunit of heterotrimeric G proteins. PMID:22027839

  2. 14-3-3 proteins and plant development.

    PubMed

    Fulgosi, Hrvoje; Soll, Jürgen; de Faria Maraschin, Simone; Korthout, Henrie A A J; Wang, Mei; Testerink, Christa

    2002-12-01

    The 14-3-3 proteins are a family of ubiquitous regulatory molecules which have been found in virtually every eukaryotic organism and tissue. Discovered 34 years ago, 14-3-3 proteins have first been studied in mammalian nervous tissues, but in the past decade their indispensable role in various plant regulatory and metabolic pathways has been increasingly established. We now know that 14-3-3 members regulate fundamental processes of nitrogen assimilation and carbon assimilation, play an auxiliary role in regulation of starch synthesis, ATP production, peroxide detoxification, and participate in modulation of several other important biochemical pathways. Plant development and seed germination appear also to be under control of factors whose interaction with 14-3-3 molecules is crucial for their activation. Located within the nucleus, 14-3-3 isoforms are constituents of transcription factor complexes and interact with components of abscisic acid (ABA)-induced gene expression machinery. In addition, in animal cells they participate in nucleo-cytoplasmic trafficking and molecular sequestration. Cytoplasmic 14-3-3 members form a guidance complex with chloroplast destined preproteins and facilitate their import into these photosynthetic organelles. Recently, several 14-3-3s have been identified within chloroplasts where they could be involved in targeting and insertion of thylakoid proteins. The identification of 14-3-3 isoform specificity, and in particular the elucidation of the signal transduction mechanisms connecting 14-3-3 members with physiological responses, are central and developing topics of current research in this field.

  3. Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.

    PubMed

    Jaumot, M; Hancock, J F

    2001-07-01

    Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

  4. 14-3-3 proteins as potential therapeutic targets

    PubMed Central

    Zhao, Jing; Meyerkord, Cheryl L.; Du, Yuhong; Khuri, Fadlo R.; Fu, Haian

    2011-01-01

    The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of client proteins in various signaling pathways that control diverse physiological and pathological processes. In response to environmental cues, 14-3-3 proteins orchestrate the highly regulated flow of signals through complex networks of molecular interactions to achieve well-controlled physiological outputs, such as cell proliferation or differentiation. Accumulating evidence now supports the concept that either an abnormal state of 14-3-3 protein expression, or dysregulation of 14-3-3/client protein interactions, contributes to the development of a large number of human diseases. In particular, clinical investigations in the field of oncology have demonstrated a correlation between upregulated 14-3-3 levels and poor survival of cancer patients. These studies highlight the rapid emergence of 14-3-3 proteins as a novel class of molecular target for potential therapeutic intervention. The current status of 14-3-3 modulator discovery is discussed. PMID:21983031

  5. Regulation of presynaptic anchoring of the scaffold protein Bassoon by phosphorylation-dependent interaction with 14-3-3 adaptor proteins.

    PubMed

    Schröder, Markus S; Stellmacher, Anne; Romorini, Stefano; Marini, Claudia; Montenegro-Venegas, Carolina; Altrock, Wilko D; Gundelfinger, Eckart D; Fejtova, Anna

    2013-01-01

    The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity.

  6. Molecular characterization of a novel 14-3-3 protein gene (Hb14-3-3c) from Hevea brasiliensis.

    PubMed

    Yang, Zi-Ping; Li, Hui-Liang; Guo, Dong; Tian, Wei-Min; Peng, Shi-Qing

    2012-04-01

    The cDNA encoding a 14-3-3 protein, designated as Hb14-3-3c, was isolated from Hevea brasiliensis. Hb14-3-3c was 1,269 bp long containing a 795 bp open reading frame encoding a putative protein of 264 amino acids, flanked by a 146 bp 5'UTR and a 328 bp 3' UTR. The predicted molecular mass of Hb14-3-3c is 29.67 kDa, with an isoelectric point of 4.52 and the deduced protein showed high similarity to the 14-3-3 protein from other plant species. Expression analysis revealed more significant accumulation of Hb14-3-3c transcripts in latex than in leaves, buds and flowers. The transcription of Hb14-3-3c in latex was induced by jasmonate and ethephon. Overproduction of recombinant Hb14-3-3c protein gave the Escherichia coli cells more tolerance on Co(2+), Cu(2+) and Zn(2+). Through yeast two-hybrid screening, 11 interaction partners of the Hb14-3-3c, which are involved in rubber biosynthesis, stress-related responses, defence etc., were identified in rubber tree latex. Taking these data together, it is proposed that the Hb14-3-3c may participate in regulation of rubber biosynthesis. Thus, the results of this study provide novel insights into the 14-3-3 signaling related to rubber biosynthesis, stress-related responses in rubber tree. PMID:21947841

  7. 14-3-3 Proteins in Guard Cell Signaling.

    PubMed

    Cotelle, Valérie; Leonhardt, Nathalie

    2015-01-01

    Guard cells are specialized cells located at the leaf surface delimiting pores which control gas exchanges between the plant and the atmosphere. To optimize the CO2 uptake necessary for photosynthesis while minimizing water loss, guard cells integrate environmental signals to adjust stomatal aperture. The size of the stomatal pore is regulated by movements of the guard cells driven by variations in their volume and turgor. As guard cells perceive and transduce a wide array of environmental cues, they provide an ideal system to elucidate early events of plant signaling. Reversible protein phosphorylation events are known to play a crucial role in the regulation of stomatal movements. However, in some cases, phosphorylation alone is not sufficient to achieve complete protein regulation, but is necessary to mediate the binding of interactors that modulate protein function. Among the phosphopeptide-binding proteins, the 14-3-3 proteins are the best characterized in plants. The 14-3-3s are found as multiple isoforms in eukaryotes and have been shown to be involved in the regulation of stomatal movements. In this review, we describe the current knowledge about 14-3-3 roles in the regulation of their binding partners in guard cells: receptors, ion pumps, channels, protein kinases, and some of their substrates. Regulation of these targets by 14-3-3 proteins is discussed and related to their function in guard cells during stomatal movements in response to abiotic or biotic stresses.

  8. Increased 14-3-3 phosphorylation observed in Parkinson's disease reduces neuroprotective potential of 14-3-3 proteins.

    PubMed

    Slone, Sunny Rae; Lavalley, Nicholas; McFerrin, Michael; Wang, Bing; Yacoubian, Talene Alene

    2015-07-01

    14-3-3 proteins are key regulators of cell survival. We have previously demonstrated that 14-3-3 levels are decreased in an alpha-synuclein (αsyn) mouse model of Parkinson's disease (PD), and that overexpression of certain 14-3-3 isoforms is protective in several PD models. Here we examine whether changes in 14-3-3 phosphorylation may contribute to the neurodegenerative process in PD. We examine three key 14-3-3 phosphorylation sites that normally regulate 14-3-3 function, including serine 58 (S58), serine 184 (S184), and serine/threonine 232 (S/T232), in several models of PD and in human PD brain. We observed that an increase in S232 phosphorylation is observed in rotenone-treated neuroblastoma cells, in cells overexpressing αsyn, and in human PD brains. Alterations in S58 phosphorylation were less consistent in these models, and we did not observe any phosphorylation changes at S184. Phosphorylation at S232 induced by rotenone is reduced by casein kinase inhibitors, and is not dependent on αsyn. Mutation of the S232 site affected 14-3-3θ's neuroprotective effects against rotenone and 1-methyl-4-phenylpyridinium (MPP(+)), with the S232D mutant lacking any protective effect compared to wildtype or S232A 14-3-3θ. The S232D mutant partially reduced the ability of 14-3-3θ to inhibit Bax activation in response to rotenone. Based on these findings, we propose that phosphorylation of 14-3-3s at serine 232 contributes to the neurodegenerative process in PD. PMID:25862939

  9. Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins.

    PubMed

    Oh, Chang-Sik; Martin, Gregory B

    2011-04-22

    Programmed cell death (PCD) associated with immunity is triggered when a plant disease resistance (R) protein recognizes a corresponding pathogen virulence protein. In tomato, detection by the host Pto kinase of the Pseudomonas syringae proteins AvrPto or AvrPtoB causes localized PCD. Previously, we reported that both MAPKKKα (mitogen-activated protein kinase kinase kinase) and the tomato 14-3-3 protein 7 (TFT7) positively regulate Pto-mediated PCD in tomato and Nicotiana benthamiana. In addition, in contrast to MAPKKKα, TFT7 is required for PCD mediated by four other R proteins. Here we investigate why TFT7 is required for PCD induced by diverse R proteins in plants. We discovered that a MAPKK, SlMKK2, which acts downstream of SlMAPKKKα, also interacts with TFT7 in plant cells. Gene silencing experiments revealed that the orthologous genes of both SlMKK2 and TFT7 in N. benthamiana are required for PCD mediated by the same set of R proteins. SlMKK2 and its orthologs contain a 14-3-3 binding site in their N terminus, and Thr(33) in this site is required for interaction with TFT7 in vivo. Like the structurally similar human 14-3-3ε protein, TFT7 forms a homodimer in vivo. Because TFT7 interacts with both SlMAPKKKα and SlMKK2 and also forms a homodimer, we propose that TFT7 may coordinately recruit these client proteins for efficient signal transfer, leading to PCD induction. PMID:21378171

  10. Eimeria tenella: 14-3-3 protein interacts with telomerase.

    PubMed

    Zhao, Na; Gong, Pengtao; Cheng, Baiqi; Li, Jianhua; Yang, Zhengtao; Li, He; Yang, Ju; Zhang, Guocai; Zhang, Xichen

    2014-10-01

    Telomerase, consisting of telomerase RNA and telomerase reverse transcriptase (TERT), is responsible for the maintenance of the end of linear chromosomes. TERT, as the catalytic subunit of telomerase, plays a critical role in telomerase activity. Researches indicate TERT-associated proteins participate in the regulation of telomerase assembly, posttranslational modification, localization, and enzymatic function. Here, the telomerase RNA-binding domain of Eimeria tenella TERT (EtTRBD) was cloned into pGBKT7 and performed as the bait. α-Galactosidase assay showed that the bait plasmid did not activate Gal4 reporter gene. Further, we isolated an EtTRBD-associated protein, 14-3-3, by yeast two-hybrid screening using the constructed bait plasmid. To confirm the interaction, EtTRBD and 14-3-3 were expressed by prokaryotic and eukaryotic expression systems. Pull-down assays by purified proteins demonstrated a direct bind between EtTRBD and 14-3-3. Co-immunoprecipitation techniques successfully validated that 14-3-3 interacted with EtTRBD in 293T cells. The protein-protein interaction provides a starting point for more in-depth studies on telomerase and telomere regulation in E. tenella.

  11. The role of the 14-3-3 protein family in health, disease, and drug development.

    PubMed

    Aghazadeh, Yasaman; Papadopoulos, Vassilios

    2016-02-01

    14-3-3 proteins regulate intracellular signaling pathways, such as signal transduction, protein trafficking, cell cycle, and apoptosis. In addition to the ubiquitous roles of 14-3-3 isoforms, unique tissue-specific functions are also described for each isoform. Owing to their role in regulating cell cycle, protein trafficking, and steroidogenesis, 14-3-3 proteins are prevalent in human diseases, such as cancer, neurodegeneration, and reproductive disorders, and, therefore, serve as valuable drug targets. In this review, we summarize the role of 14-3-3 proteins in normal and disease states, with a focus on 14-3-3γ and ɛ. We also discuss drug compounds targeting 14-3-3 proteins and their potential therapeutic uses. PMID:26456530

  12. Up-regulation and interaction of the plasma membrane H(+)-ATPase and the 14-3-3 protein are involved in the regulation of citrate exudation from the broad bean (Vicia faba L.) under Al stress.

    PubMed

    Chen, Qi; Guo, Chuan-Long; Wang, Ping; Chen, Xuan-Qin; Wu, Kong-Huan; Li, Kui-Zhi; Yu, Yong-Xiong; Chen, Li-Mei

    2013-09-01

    Our previous study showed that citrate excretion coupled with a concomitant release of protons was involved in aluminum (Al) resistance in the broad bean. Furthermore, genes encoding plasma membrane (PM) H(+)-ATPase (vha2) and the 14-3-3 protein (vf14-3-3b) were up-regulated by Al in Al-resistant (YD) broad bean roots. In this study, the roles of PM H(+)-ATPase (E.C. 3.6.3.6) and the 14-3-3 protein in the regulation of citrate secretion were further investigated in Al-resistant (YD) and Al-sensitive (AD) broad bean cultivars under Al stress. The results showed that greater citrate exudation was positively correlated with higher activities of PM H(+)-ATPase in roots of YD than AD. Real-time RT-PCR analysis revealed that vha2 was clearly up-regulated by Al in YD but not in AD roots, whereas the transcription levels of vf14-3-3b were elevated in a time-dependent manner in both YD and AD roots. Immunoprecipitation and Western analysis suggested that phosphorylation and interaction with the vf14-3-3b protein of the VHA2 were enhanced in YD roots but not in AD roots with increasing Al treatment time. Fusicoccin or adenosine 5'-monophosphate increased or decreased the interaction between the phosphorylated VHA2 and the vf14-3-3b protein, followed by an enhancement or reduction of the PM H(+)-ATPase activity and citrate exudation in both cultivars under Al stress conditions, respectively. Taken together, these results suggested that Al enhanced the expression and interaction of the PM H(+)-ATPase and the 14-3-3 protein, which thereby led to higher activity of the PM H(+)-ATPase and more citrate exudation from YD plants.

  13. Dynamic imaging of interaction between protein 14-3-3 and Bid in living cells

    NASA Astrophysics Data System (ADS)

    Chen, Tongsheng; Xing, Da; Wang, Jinjun

    2006-02-01

    The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3- interacting domain death agonist (Bid) may contribute to tumor necrosis factor α(TNF-α)-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid/tBid during TNF-α-induced cell death. The TNF-αtriggered Bid cleavage and tBid translocated to mitochondria. Human lung adenocarcinoma cells were co-transfected with both CFP-Bid and 14-3-3-YFP plasmids, and the dynamical interaction between the Bid/tBid and 14-3-3 were performed on laser confocal fluorescence microscope in single living cell during TNF-α-induced cell apoptosis. The Bid distribute equally only in the cytoplasm of healthy cells, and the 14-3-3 protein distribute not only in the cytoplasm but also in the nucleus of healthy cells. Our data showed that the tBid aggregate, but the 14-3-3 protein does not aggregate as the tBid, and the 14-3-3 protein separate from the aggregated tBid, implying that the 14-3-3 proteins do not interact with the aggregated tBid after TNF-αtreatment.

  14. 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing.

    PubMed

    Yam, Patricia T; Kent, Christopher B; Morin, Steves; Farmer, W Todd; Alchini, Ricardo; Lepelletier, Léa; Colman, David R; Tessier-Lavigne, Marc; Fournier, Alyson E; Charron, Frédéric

    2012-11-21

    Axons must switch responsiveness to guidance cues during development for correct pathfinding. Sonic Hedgehog (Shh) attracts spinal cord commissural axons ventrally toward the floorplate. We show that after crossing the floorplate, commissural axons switch their response to Shh from attraction to repulsion, so that they are repelled anteriorly by a posterior-high/anterior-low Shh gradient along the longitudinal axis. This switch is recapitulated in vitro with dissociated commissural neurons as they age, indicating that the switch is intrinsic and time dependent. 14-3-3 protein inhibition converted Shh-mediated repulsion of aged dissociated neurons to attraction and prevented the correct anterior turn of postcrossing commissural axons in vivo, an effect mediated through PKA. Conversely, overexpression of 14-3-3 proteins was sufficient to drive the switch from Shh-mediated attraction to repulsion both in vitro and in vivo. Therefore, we identify a 14-3-3 protein-dependent mechanism for a cell-intrinsic temporal switch in the polarity of axon turning responses.

  15. Hyperglycemia decreases expression of 14-3-3 proteins in an animal model of stroke.

    PubMed

    Jeon, Seong-Jun; Sung, Jin-Hee; Koh, Phil-Ok

    2016-07-28

    Diabetes is a severe metabolic disorder and a major risk factor for stroke. Stroke severity is worse in patients with diabetes compared to the non-diabetic population. The 14-3-3 proteins are a family of conserved acidic proteins that are ubiquitously expressed in cells and tissues. These proteins are involved in many cellular processes including metabolic pathways, signal transduction, protein trafficking, protein synthesis, and cell cycle control. This study investigated 14-3-3 proteins expression in the cerebral cortex of animals with diabetes, cerebral ischemic injury and a combination of both diabetes and cerebral ischemic injury. Diabetes was induced by intraperitoneal injection of streptozotocin (40mg/kg) in adult male rats. After 4 weeks of treatment, middle cerebral artery occlusion (MCAO) was performed for the induction of focal cerebral ischemia and cerebral cortex tissue was collected 24h after MCAO. We confirmed that diabetes increases infarct volume following MCAO compared to non-diabetic animals. In diabetic animals with MCAO injury, reduction of 14-3-3 β/α, 14-3-3 ζ/δ, 14-3-3 γ, and 14-3-3 ε isoforms was detected. The expression of these proteins was significantly decreased in diabetic animals with MCAO injury compared to diabetic-only and MCAO-only animals. Moreover, Western blot analysis ascertained the decreased expression of 14-3-3 family proteins in diabetic animals with MCAO injury, including β/α, ζ/δ, γ, ε, τ, and η isoforms. These results show the changes of 14-3-3 proteins expression in streptozotocin-induced diabetic animals with MCAO injury. Thus, these findings suggest that decreases in 14-3-3 proteins might be involved in the regulation of 14-3-3 proteins under the presence of diabetes following MCAO. PMID:27177727

  16. 14-3-3 Protein Masks the DNA Binding Interface of Forkhead Transcription Factor FOXO4*

    PubMed Central

    Silhan, Jan; Vacha, Petr; Strnadova, Pavla; Vecer, Jaroslav; Herman, Petr; Sulc, Miroslav; Teisinger, Jan; Obsilova, Veronika; Obsil, Tomas

    2009-01-01

    The role of 14-3-3 proteins in the regulation of FOXO forkhead transcription factors is at least 2-fold. First, the 14-3-3 binding inhibits the interaction between the FOXO and the target DNA. Second, the 14-3-3 proteins prevent nuclear reimport of FOXO factors by masking their nuclear localization signal. The exact mechanisms of these processes are still unclear, mainly due to the lack of structural data. In this work, we used fluorescence spectroscopy to investigate the mechanism of the 14-3-3 protein-dependent inhibition of FOXO4 DNA-binding properties. Time-resolved fluorescence measurements revealed that the 14-3-3 binding affects fluorescence properties of 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid moiety attached at four sites within the forkhead domain of FOXO4 that represent important parts of the DNA binding interface. Observed changes in 5-(((acetylamino)ethyl)amino) naphthalene-1-sulfonic acid fluorescence strongly suggest physical contacts between the 14-3-3 protein and labeled parts of the FOXO4 DNA binding interface. The 14-3-3 protein binding, however, does not cause any dramatic conformational change of FOXO4 as documented by the results of tryptophan fluorescence experiments. To build a realistic model of the FOXO4·14-3-3 complex, we measured six distances between 14-3-3 and FOXO4 using Förster resonance energy transfer time-resolved fluorescence experiments. The model of the complex suggests that the forkhead domain of FOXO4 is docked within the central channel of the 14-3-3 protein dimer, consistent with our hypothesis that 14-3-3 masks the DNA binding interface of FOXO4. PMID:19416966

  17. Suppression of death-associated protein kinase 2 by interaction with 14-3-3 proteins.

    PubMed

    Yuasa, Keizo; Ota, Reina; Matsuda, Shinya; Isshiki, Kinuka; Inoue, Masahiro; Tsuji, Akihiko

    2015-08-14

    Death-associated protein kinase 2 (DAPK2), a Ca(2+)/calmodulin-regulated serine/threonine kinase, induces apoptosis. However, the signaling mechanisms involved in this process are unknown. Using a proteomic approach, we identified 14-3-3 proteins as novel DAPK2-interacting proteins. The 14-3-3 family has the ability to bind to phosphorylated proteins via recognition of three conserved amino acid motifs (mode 1-3 motifs), and DAPK2 contains the mode 3 motif ((pS/pT)X1-2-COOH). The interaction of 14-3-3 proteins with DAPK2 was dependent on the phosphorylation of Thr(369), and effectively suppressed DAPK2 kinase activity and DAPK2-induced apoptosis. Furthermore, we revealed that the 14-3-3 binding site Thr(369) of DAPK2 was phosphorylated by the survival kinase Akt. Our findings suggest that DAPK2-induced apoptosis is negatively regulated by Akt and 14-3-3 proteins.

  18. Interaction network of the 14-3-3 protein in the ancient protozoan parasite Giardia duodenalis.

    PubMed

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Sayadi, Ahmed; Crescenzi, Marco; Pozio, Edoardo

    2012-05-01

    14-3-3s are phosphoserine/phosphotreonine binding proteins that play pivotal roles as regulators of multiple cellular processes in eukaryotes. The flagellated protozoan parasite Giardia duodenalis, the causing agent of giardiasis, is a valuable simplified eukaryotic model. A single 14-3-3 isoform (g14-3-3) is expressed in Giardia, and it is directly involved in the differentiation of the parasite into cyst. To define the overall functions of g14-3-3, the protein interactome has been investigated. A transgenic G. duodenalis strain was engineered to express a FLAG-tagged g14-3-3 under its own promoter. Affinity chromatography coupled with tandem mass spectrometry analysis have been used to purify and identify FLAG-g14-3-3-associated proteins from trophozoites and encysting parasites. A total of 314 putative g14-3-3 interaction partners were identified, including proteins involved in several pathways. Some interactions seemed to be peculiar of one specific stage, while others were shared among the different stages. Furthermore, the interaction of g14-3-3 with the giardial homologue of the CDC7 protein kinase (gCDC7) was characterized, leading to the identification of a multiprotein complex containing not only g14-3-3 and gCDC7 but also a newly identified and highly divergent homologue of DBF4, the putative regulatory subunit of gCDC7. The relevance of g14-3-3 interactions in G. duodenalis biology was discussed.

  19. Dual phosphorylation of Btk by Akt/protein kinase b provides docking for 14-3-3ζ, regulates shuttling, and attenuates both tonic and induced signaling in B cells.

    PubMed

    Mohammad, Dara K; Nore, Beston F; Hussain, Alamdar; Gustafsson, Manuela O; Mohamed, Abdalla J; Smith, C I Edvard

    2013-08-01

    Bruton's tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

  20. Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

    PubMed

    Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

    2009-06-01

    In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

  1. Structural Modulation of Phosducin by Phosphorylation and 14-3-3 Protein Binding

    PubMed Central

    Rezabkova, Lenka; Kacirova, Miroslava; Sulc, Miroslav; Herman, Petr; Vecer, Jaroslav; Stepanek, Miroslav; Obsilova, Veronika; Obsil, Tomas

    2012-01-01

    Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the Gtβγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function. PMID:23199924

  2. Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein.

    PubMed

    Llopis-Torregrosa, Vicent; Ferri-Blázquez, Alba; Adam-Artigues, Anna; Deffontaines, Emilie; van Heusden, G Paul H; Yenush, Lynne

    2016-07-15

    Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2. PMID:27261460

  3. 14-3-3ζ Interacts with Stat3 and Regulates Its Constitutive Activation in Multiple Myeloma Cells

    PubMed Central

    Li, Wenliang; Xiong, Qian; Yang, Mingkun; Zheng, Peng; Li, Chongyang; Pei, Jianfeng; Ge, Feng

    2012-01-01

    The 14-3-3 proteins are a family of regulatory signaling molecules that interact with other proteins in a phosphorylation-dependent manner and function as adapter or scaffold proteins in signal transduction pathways. One family member, 14-3-3ζ, is believed to function in cell signaling, cycle control, and apoptotic death. A systematic proteomic analysis done in our laboratory has identified signal transducers and activators of transcription 3 (Stat3) as a novel 14-3-3ζ interacting protein. Following our initial finding, in this study, we provide evidence that 14-3-3ζ interacts physically with Stat3. We further demonstrate that phosphorylation of Stat3 at Ser727 is vital for 14-3-3ζ interaction and mutation of Ser727 to Alanine abolished 14-3-3ζ/Stat3 association. Inhibition of 14-3-3ζ protein expression in U266 cells inhibited Stat3 Ser727 phosphorylation and nuclear translocation, and decreased both Stat3 DNA binding and transcriptional activity. Moreover, 14-3-3ζ is involved in the regulation of protein kinase C (PKC) activity and 14-3-3ζ binding to Stat3 protects Ser727 dephosphorylation from protein phosphatase 2A (PP2A). Taken together, our findings support the model that multiple signaling events impinge on Stat3 and that 14-3-3ζ serves as an essential coordinator for different pathways to regulate Stat3 activation and function in MM cells. PMID:22279540

  4. Structure-Function Analysis of PPP1R3D, a Protein Phosphatase 1 Targeting Subunit, Reveals a Binding Motif for 14-3-3 Proteins which Regulates its Glycogenic Properties.

    PubMed

    Rubio-Villena, Carla; Sanz, Pascual; Garcia-Gimeno, Maria Adelaida

    2015-01-01

    Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. It plays a key role in regulating glycogen synthesis, by dephosphorylating crucial enzymes involved in glycogen homeostasis such as glycogen synthase (GS) and glycogen phosphorylase (GP). To play this role, PP1 binds to specific glycogen targeting subunits that, on one hand recognize the substrates to be dephosphorylated and on the other hand recruit PP1 to glycogen particles. In this work we have analyzed the functionality of the different protein binding domains of one of these glycogen targeting subunits, namely PPP1R3D (R6) and studied how binding properties of different domains affect its glycogenic properties. We have found that the PP1 binding domain of R6 comprises a conserved RVXF motif (R102VRF) located at the N-terminus of the protein. We have also identified a region located at the C-terminus of R6 (W267DNND) that is involved in binding to the PP1 glycogenic substrates. Our results indicate that although binding to PP1 and glycogenic substrates are independent processes, impairment of any of them results in lack of glycogenic activity of R6. In addition, we have characterized a novel site of regulation in R6 that is involved in binding to 14-3-3 proteins (RARS74LP). We present evidence indicating that when binding of R6 to 14-3-3 proteins is prevented, R6 displays hyper-glycogenic activity although is rapidly degraded by the lysosomal pathway. These results define binding to 14-3-3 proteins as an additional pathway in the control of the glycogenic properties of R6.

  5. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    PubMed Central

    Sambandam, Sumitha A.T.; Kasetti, Ramesh Babu; Xue, Lei; Dean, Douglas C.; Lu, Qingxian; Li, Qiutang

    2015-01-01

    The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis. PMID:25668240

  6. Alternations of 14-3-3 θ and β protein levels in brain during experimental sepsis.

    PubMed

    Memos, Nikolaos; Kataki, Agapi; Chatziganni, Emmy; Nikolopoulou, Marilena; Skoulakis, Euthimios; Consoulas, Christos; Zografos, George; Konstadoulakis, Manousos

    2011-09-01

    The 14-3-3 family members play a crucial role in the determination of cell fate, exerting their antiapoptotic activity through directly interfering with the critical function of the mitochondrial core proapoptotic machinery. Dimerization of 14-3-3 is vital for the interaction with many of its client proteins and is regulated by phosphorylation. In a previous study, we observed time-dependent neuronal apoptosis during sepsis. Therefore, in the present study, we sought to evaluate the expression of 14-3-3 θ and β isoforms in septic brain and their association with apoptosis. Sepsis was induced by a CLP model in Wistar rats that were sacrificed at predefined time points. Flow cytometric analysis showed a sepsis-induced, time-dependent alteration of 14-3-3 θ and β isoforms in both Neun(+) and GFAP(+) cells. 14-3-3 θ was linearly correlated with apoptosis, and stratified analysis for alive and apoptotic neuronal cells demonstrated a gradual down-regulation of θ isoform in alive neurons and astrocytes. The phospho-P38 (pP38) MAP kinase levels were altered in a time-dependent manner during sepsis, presenting a peak at 6 hr post-CLP. A significant correlation between the two isoforms of 14-3-3 was observed in septic rats, with the θ isoform predominant at all time points. The hippocampus, Purkinje cells, and glia-like cells showed intense immunohistochemical reactivity for 14-3-3 θ isoform, whereas the choroid plexus showed constantly increased β isoform expression. Our results showed that sepsis alters the expression of both 14-3-3 θ and β isoforms in a time-, cell-, and topography-dependent manner. PMID:21618583

  7. Tomato 14-3-3 protein 7 (TFT7) positively regulates immunity-associated programmed cell death by enhancing accumulation and signaling ability of MAPKKKalpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Programmed cell death (PCD) is triggered when Pto, a serine-threonine protein kinase recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv. tomato. This PCD requires MAPKKKalpha as a positive regulator in tomato and Nicotiana benthamiana. To examine how PCD-eliciting activi...

  8. The Arabidopsis 14-3-3 Protein RARE COLD INDUCIBLE 1A Links Low-Temperature Response and Ethylene Biosynthesis to Regulate Freezing Tolerance and Cold Acclimation[C][W

    PubMed Central

    Catalá, Rafael; López-Cobollo, Rosa; Mar Castellano, M.; Angosto, Trinidad; Alonso, José M.; Ecker, Joseph R.; Salinas, Julio

    2014-01-01

    In plants, the expression of 14-3-3 genes reacts to various adverse environmental conditions, including cold, high salt, and drought. Although these results suggest that 14-3-3 proteins have the potential to regulate plant responses to abiotic stresses, their role in such responses remains poorly understood. Previously, we showed that the RARE COLD INDUCIBLE 1A (RCI1A) gene encodes the 14-3-3 psi isoform. Here, we present genetic and molecular evidence implicating RCI1A in the response to low temperature. Our results demonstrate that RCI1A functions as a negative regulator of constitutive freezing tolerance and cold acclimation in Arabidopsis thaliana by controlling cold-induced gene expression. Interestingly, this control is partially performed through an ethylene (ET)-dependent pathway involving physical interaction with different ACC SYNTHASE (ACS) isoforms and a decreased ACS stability. We show that, consequently, RCI1A restrains ET biosynthesis, contributing to establish adequate levels of this hormone in Arabidopsis under both standard and low-temperature conditions. We further show that these levels are required to promote proper cold-induced gene expression and freezing tolerance before and after cold acclimation. All these data indicate that RCI1A connects the low-temperature response with ET biosynthesis to modulate constitutive freezing tolerance and cold acclimation in Arabidopsis. PMID:25122152

  9. Expression of 14-3-3 transcript isoforms in response to ethanol exposure and their regulation by miRNAs.

    PubMed

    Mathew, Divya Elizabeth; Larsen, Kaitlyn; Janeczek, Paulina; Lewohl, Joanne M

    2016-09-01

    The 14-3-3 proteins are a family of highly conserved molecular chaperones involved in the regulation of a number of key cellular functions including metabolism, stress response, protein trafficking, cell-cycle control, signal transduction, transcription, apoptosis and neurotransmission. 14-3-3 proteins have also been implicated in the pathophysiology of neurodegenerative disorders including Alzheimer disease and Parkinson disease. Recent studies have also shown that 14-3-3s are differentially expressed in the frontal cortex of human alcoholics suggesting a potential role in the pathophysiology of alcohol use disorders. Here we measured the expression of 14-3-3 transcripts in HEK293T cells in response to chronic ethanol treatment. Five of the seven transcripts (14-3-3β, 14-3-3γ, 14-3-3ζ, 14-3-3ε and 14-3-3θ) were significantly down-regulated following chronic exposure to ethanol for a five day period with these changes persisting even after withdrawal from ethanol treatment. One transcript, 14-3-3σ, was significantly up-regulated following chronic ethanol exposure and 14-3-3η showed no differences in expression in the same treatment model. The pattern of expression changes is similar to those seen in the frontal cortex of human alcoholics. To investigate the role of miRNAs in mediating the expression changes we measured the expression of the 14-3-3 transcripts following transfection with miR-203, miR-144 and miR-7 mimics. Although these miRNAs had predicted target sites in the 3'untranslated region of each 14-3-3 isoform, only miR-203 resulted in a down-regulation of 14-3-3θ transcript. In addition, the expression of 14-3-3γ was upregulated following transfection with miR-7 and miR-144 mimics. MiRNA regulation of these isoforms following alcohol exposure may lead to alterations in neurotransmission, the balance between cell survival and cell death, as well as changing the rewarding effects of alcohol. PMID:27370936

  10. Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex.

    PubMed

    Pan, S; Sehnke, P C; Ferl, R J; Gurley, W B

    1999-08-01

    The 14-3-3 family of multifunctional proteins is highly conserved among animals, plants, and yeast. Several studies have shown that these proteins are associated with a G-box DNA binding complex and are present in the nucleus in several plant and animal species. In this study, 14-3-3 proteins are shown to bind the TATA box binding protein (TBP), transcription factor IIB (TFIIB), and the human TBP-associated factor hTAF(II)32 in vitro but not hTAF(II)55. The interactions with TBP and TFIIB were highly specific, requiring amino acid residues in the box 1 domain of the 14-3-3 protein. These interactions do not require formation of the 14-3-3 dimer and are not dependent on known 14-3-3 recognition motifs containing phosphoserine. The 14-3-3-TFIIB interaction appears to occur within the same domain of TFIIB that binds the human herpes simplex virus transcriptional activator VP16, because VP16 and 14-3-3 were able to compete for interaction with TFIIB in vitro. In a plant transient expression system, 14-3-3 was able to activate GAL4-dependent beta-glucuronidase reporter gene expression at low levels when translationally fused with the GAL4 DNA binding domain. The in vitro binding with general transcription factors TBP and TFIIB together with its nuclear location provide evidence supporting a role for 14-3-3 proteins as transcriptional activators or coactivators when part of a DNA binding complex. PMID:10449590

  11. Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

    PubMed Central

    Das, Maitreyi; Nuñez, Illyce; Rodriguez, Marbelys; Wiley, David J.; Rodriguez, Juan; Sarkeshik, Ali; Yates, John R.; Buchwald, Peter; Verde, Fulvia

    2015-01-01

    Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence. PMID:26246599

  12. An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel.

    PubMed

    Liang, Xiubin; Butterworth, Michael B; Peters, Kathryn W; Walker, William H; Frizzell, Raymond A

    2008-10-10

    Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport. PMID:18687683

  13. Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates

    PubMed Central

    Tinti, Michele; Johnson, Catherine; Toth, Rachel; Ferrier, David E. K.; MacKintosh, Carol

    2012-01-01

    14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a ‘lynchpin’, defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1–4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the –2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1–4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders. PMID:22870394

  14. Differential interaction and aggregation of 3-repeat and 4-repeat tau isoforms with 14-3-3{zeta} protein

    SciTech Connect

    Sadik, Golam; Tanaka, Toshihisa; Kato, Kiyoko; Yanagi, Kentaro; Kudo, Takashi; Takeda, Masatoshi

    2009-05-22

    Tau isoforms, 3-repeat (3R) and 4-repeat tau (4R), are differentially involved in neuronal development and in several tauopathies. 14-3-3 protein binds to tau and 14-3-3/tau association has been found both in the development and in tauopathies. To understand the role of 14-3-3 in the differential regulation of tau isoforms, we have performed studies on the interaction and aggregation of 3R-tau and 4R-tau, either phosphorylated or unphosphorylated, with 14-3-3{zeta}. We show by surface plasmon resonance studies that the interaction between unphosphorylated 3R-tau and 14-3-3{zeta} is {approx}3-folds higher than that between unphosphorylated 4R-tau and 14-3-3{zeta}. Phosphorylation of tau by protein kinase A (PKA) increases the affinity of both 3R- and 4R-tau for 14-3-3{zeta} to a similar level. An in vitro aggregation assay employing both transmission electron microscopy and fluorescence spectroscopy revealed the aggregation of unphosphorylated 4R-tau to be significantly higher than that of unphosphorylated 3R-tau following the induction of 14-3-3{zeta}. The filaments formed from 3R- and 4R-tau were almost similar in morphology. In contrast, the aggregation of both 3R- and 4R-tau was reduced to a similar low level after phosphorylation with PKA. Taken together, these results suggest that 14-3-3{zeta} exhibits a similar role for tau isoforms after PKA-phosphorylation, but a differential role for unphosphorylated tau. The significant aggregation of 4R-tau by 14-3-3{zeta} suggests that 14-3-3 may act as an inducer in the generation of 4R-tau-predominant neurofibrillary tangles in tauopathies.

  15. 14-3-3ζ regulates the mitochondrial respiratory reserve linked to platelet phosphatidylserine exposure and procoagulant function

    PubMed Central

    Schoenwaelder, Simone M.; Darbousset, Roxane; Cranmer, Susan L.; Ramshaw, Hayley S.; Orive, Stephanie L.; Sturgeon, Sharelle; Yuan, Yuping; Yao, Yu; Krycer, James R.; Woodcock, Joanna; Maclean, Jessica; Pitson, Stuart; Zheng, Zhaohua; Henstridge, Darren C.; van der Wal, Dianne; Gardiner, Elizabeth E.; Berndt, Michael C.; Andrews, Robert K.; James, David E.; Lopez, Angel F.; Jackson, Shaun P.

    2016-01-01

    The 14-3-3 family of adaptor proteins regulate diverse cellular functions including cell proliferation, metabolism, adhesion and apoptosis. Platelets express numerous 14-3-3 isoforms, including 14-3-3ζ, which has previously been implicated in regulating GPIbα function. Here we show an important role for 14-3-3ζ in regulating arterial thrombosis. Interestingly, this thrombosis defect is not related to alterations in von Willebrand factor (VWF)–GPIb adhesive function or platelet activation, but instead associated with reduced platelet phosphatidylserine (PS) exposure and procoagulant function. Decreased PS exposure in 14-3-3ζ-deficient platelets is associated with more sustained levels of metabolic ATP and increased mitochondrial respiratory reserve, independent of alterations in cytosolic calcium flux. Reduced platelet PS exposure in 14-3-3ζ-deficient mice does not increase bleeding risk, but results in decreased thrombin generation and protection from pulmonary embolism, leading to prolonged survival. Our studies define an important role for 14-3-3ζ in regulating platelet bioenergetics, leading to decreased platelet PS exposure and procoagulant function. PMID:27670677

  16. Binding of 14-3-3 reader proteins to phosphorylated DNMT1 facilitates aberrant DNA methylation and gene expression

    PubMed Central

    Estève, Pierre-Olivier; Zhang, Guoqiang; Ponnaluri, V.K. Chaithanya; Deepti, Kanneganti; Chin, Hang Gyeong; Dai, Nan; Sagum, Cari; Black, Karynne; Corrêa, Ivan R.; Bedford, Mark T.; Cheng, Xiaodong; Pradhan, Sriharsa

    2016-01-01

    Mammalian DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for maintenance methylation. Phosphorylation of Ser143 (pSer143) stabilizes DNMT1 during DNA replication. Here, we show 14-3-3 is a reader protein of DNMT1pSer143. In mammalian cells 14-3-3 colocalizes and binds DNMT1pSer143 post-DNA replication. The level of DNMT1pSer143 increased with overexpression of 14-3-3 and decreased by its depletion. Binding of 14-3-3 proteins with DNMT1pSer143 resulted in inhibition of DNA methylation activity in vitro. In addition, overexpression of 14-3-3 in NIH3T3 cells led to decrease in DNMT1 specific activity resulting in hypomethylation of the genome that was rescued by transfection of DNMT1. Genes representing cell migration, mobility, proliferation and focal adhesion pathway were hypomethylated and overexpressed. Furthermore, overexpression of 14-3-3 also resulted in enhanced cell invasion. Analysis of TCGA breast cancer patient data showed significant correlation for DNA hypomethylation and reduced patient survival with increased 14-3-3 expressions. Therefore, we suggest that 14-3-3 is a crucial reader of DNMT1pSer143 that regulates DNA methylation and altered gene expression that contributes to cell invasion. PMID:26553800

  17. Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein.

    PubMed

    Jeon, Yeong Ha; Ko, Kwan Young; Lee, Jea Hwang; Park, Ki Jun; Jang, Jun Ki; Kim, Ick Young

    2016-01-01

    Selenoprotein W (SelW) contains a selenocysteine (Sec, U) in a conserved CXXU motif corresponding to the CXXC redox motif of thioredoxin, suggesting a putative redox function of SelW. We have previously reported that the binding of 14-3-3 protein to its target proteins, including CDC25B, Rictor and TAZ, is inhibited by the interaction of 14-3-3 protein with SelW. However, the binding mechanism is unclear. In this study, we sought to determine the binding site of SelW to understand the regulatory mechanism of the interaction between SelW and 14-3-3 and its biological effects. Phosphorylated Ser(pS) or Thr(pT) residues in RSXpSXP or RXXXp(S/T)XP motifs are well-known common 14-3-3-binding sites, but Thr41, Ser59, and T69 of SelW, which are computationally predicted to serve are phosphorylation sites, were neither phosphorylation sites nor sites involved in the interaction. A mutant SelW in which Sec13 is changed to Ser (U13S) was unable to interact with 14-3-3 protein and thus did not inhibit the interaction of 14-3-3 to other target proteins. However, other Cys mutants of SelW(C10S, C33S and C37S) normally interacted with 14-3-3 protein. The interaction of SelW to 14-3-3 protein was enhanced by diamide or H2O2 and decreased by dithiothreitol (DTT). Taken together, these findings demonstrate that the Sec of SelW is involved in its interaction with 14-3-3 protein and that this interaction is increased under oxidative stress conditions. Thus, SelW may have a regulatory function in redox cell signaling by interacting with 14-3-3 protein. PMID:26474786

  18. 14-3-3ε and ζ Regulate Neurogenesis and Differentiation of Neuronal Progenitor Cells in the Developing Brain

    PubMed Central

    Wachi, Tomoka; Hunt, Robert F.; Baraban, Scott C.; Taya, Shinichiro; Ramshaw, Hayley; Kaibuchi, Kozo; Schwarz, Quenten P.; Lopez, Angel F.

    2014-01-01

    During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of β-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades. PMID:25186760

  19. 14-3-3β protein expression in eosinophilic meningitis caused by Angiostrongylus cantonensis infection

    PubMed Central

    2014-01-01

    Background Angiostrongylus cantonensis is a parasite endemic in the Southeast Asian and Pacific regions. Humans are incidentally infected either by eating uncooked intermediate hosts or by consuming vegetables containing the living third-stage larvae. The 14-3-3β protein is a cerebrospinal fluid (CSF) marker of neuronal damage during the development of Creutzfeldt-Jakob disease. In addition, increased 14-3-3β protein is also found in CSF from patients with a variety of neurological disorders. The goal of this study is to determine the roles of serum/CSF14-3-3β protein in patients with eosinophilic meningitis. Methods In a cohort study among nine Thai laborers with eosinophilic meningitis due to eating raw snails (Pomacea canaliculata), we examined the CSF weekly while patients were still hospitalized and followed up the serum for 6 months. The levels of 14-3-3β protein in CSF were analyzed by western blot and an in-house 14-3-3β enzyme-linked immunosorbent assay (ELISA) measurement was established and tested in an animal model of eosinophilic meningitis. Results The elevated 14-3-3β level was detected in the CSF from eight out of nine (81%) patients After 2 weeks of treatment, all patients showed a declined level or cleared of 14-3-3β protein in the CSF. By developing an in-house ELISA for measurement of 14-3-3β protein, it was found that the serum 14-3-3β level was significantly increased in patients during initial visit. . This finding was consistent to the animal experiment result in which there was severe blood brain barrier damage three weeks after infection and increased 14-3-3β protein expression in the CSF and serum by western blot and in house ELISA. After treatment, the serum 14-3-3β level in meningitis patients was rapidly returned to normal threshold. There was a correlation between initial CSF 14-3-3β level with severity of headache (r = 0.692, p = 0.039), CSF pleocytosis (r = 0.807, p = 0.009) and eosinophilia (r = 0

  20. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming.

    PubMed

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumorigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programmes by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anticancer metabolism therapy development in future. PMID:26179207

  1. The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming

    PubMed Central

    Phan, Liem; Chou, Ping-Chieh; Velazquez-Torres, Guermarie; Samudio, Ismael; Parreno, Kenneth; Huang, Yaling; Tseng, Chieh; Vu, Thuy; Gully, Chris; Su, Chun-Hui; Wang, Edward; Chen, Jian; Choi, Hyun-Ho; Fuentes-Mattei, Enrique; Shin, Ji-Hyun; Shiang, Christine; Grabiner, Brian; Blonska, Marzenna; Skerl, Stephen; Shao, Yiping; Cody, Dianna; Delacerda, Jorge; Kingsley, Charles; Webb, Douglas; Carlock, Colin; Zhou, Zhongguo; Hsieh, Yun-Chih; Lee, Jaehyuk; Elliott, Andrew; Ramirez, Marc; Bankson, Jim; Hazle, John; Wang, Yongxing; Li, Lei; Weng, Shaofan; Rizk, Nibal; Wen, Yu Ye; Lin, Xin; Wang, Hua; Wang, Huamin; Zhang, Aijun; Xia, Xuefeng; Wu, Yun; Habra, Mouhammed; Yang, Wei; Pusztai, Lajos; Yeung, Sai-Ching; Lee, Mong-Hong

    2015-01-01

    Summary Extensive reprogramming of cellular energy metabolism is a hallmark of cancer. Despite its importance, the molecular mechanism controlling this tumour metabolic shift remains not fully understood. Here we show that 14-3-3σ regulates cancer metabolic reprogramming and protects cells from tumourigenic transformation. 14-3-3σ opposes tumour-promoting metabolic programs by enhancing c-Myc poly-ubiquitination and subsequent degradation. 14-3-3σ demonstrates the suppressive impact on cancer glycolysis, glutaminolysis, mitochondrial biogenesis and other major metabolic processes of tumours. Importantly, 14-3-3σ expression levels predict overall and recurrence-free survival rates, tumour glucose uptake and metabolic gene expression in breast cancer patients. Thus, these results highlight that 14-3-3σ is an important regulator of tumour metabolism, and loss of 14-3-3σ expression is critical for cancer metabolic reprogramming. We anticipate that pharmacologically elevating the function of 14-3-3σ in tumours could be a promising direction for targeted anti-cancer metabolism therapy development in future. PMID:26179207

  2. Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

    PubMed

    Chen, Qi; Kan, Qi; Wang, Ping; Yu, Wenqian; Yu, Yuzhen; Zhao, Yan; Yu, Yongxiong; Li, Kunzhi; Chen, Limei

    2015-06-01

    Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

  3. Transcriptional Regulation of YWHAZ, the Gene Encoding 14-3-3ζ

    PubMed Central

    Kasinski, Andrea; Dong, Xueyuan; Khuri, Fadlo R.; Boss, Jeremy; Fu, Haian

    2014-01-01

    Aberrant expression of oncogenic 14-3-3 proteins is correlated with poor survival of cancer patients. While the underlying mechanism of the abnormal expression in tumors remains elusive for the six oncogenic 14-3-3 isoforms; the potential involvement of a transcriptional component has been suggested. Unfortunately, little experimental data has been reported to support this hypothesis. In this study we describe the genetic structure of YWHAZ, the gene encoding 14-3-3ζ, including the identification of previously unreported transcript variants. In total, five transcript variants were revealed and their expressions confirmed in a panel of cell lines. Expressed sequence tag (EST) database mining and in vitro rapid-amplification of cDNA ends (RACE) confirmed that one variant, 1c, represents >80% of the expressed transcripts, which is also the most efficiently translated. An analysis of the proximal promoter of this variant revealed a functional Cyclic-AMP Response Element (CRE). Factors that bound to the CRE element were recognized through fractionation and subsequent EMSAs. This analysis identified CREB and ATF-1 as the trans-interacting factors. Cell-based assays confirm that ATF-1, and to a lesser extent CREB, bind the endogenous YWHAZ promoter especially under TNF-α stimulating conditions. In support of a role of ATF-1 in the regulation of YWHAZ, silencing of ATF-1 resulted in a marked reduction in two of the five YWHAZ transcripts. These data suggest a novel mechanism for the transcriptional regulation of a major pro-survival gene, YWHAZ, by ATF-1. PMID:24690670

  4. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach.

    PubMed

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N-terminal sublibrary, and 0.77 and 269.13 for C-terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  5. Molecular tweezers modulate 14-3-3 protein-protein interactions.

    PubMed

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions. PMID:23422566

  6. Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice

    PubMed Central

    Bai, Ming-Yi; Zhang, Li-Ying; Gampala, Srinivas S.; Zhu, Sheng-Wei; Song, Wen-Yuan; Chong, Kang; Wang, Zhi-Yong

    2007-01-01

    Brassinosteroids (BR) are essential growth hormones found throughout the plant kingdom. BR bind to the receptor kinase BRI1 on the cell surface to activate a signal transduction pathway that regulates nuclear gene expression and plant growth. To understand the downstream BR signaling mechanism in rice, we studied the function of OsBZR1 using reverse genetic approaches and identified OsBZR1-interacting proteins. Suppressing OsBZR1 expression by RNAi resulted in dwarfism, erect leaves, reduced BR sensitivity, and altered BR-responsive gene expression in transgenic rice plants, demonstrating an essential role of OsBZR1 in BR responses in rice. Moreover, a yeast two-hybrid screen identified 14-3-3 proteins as OsBZR1-interacting proteins. Mutation of a putative 14-3-3-binding site of OsBZR1 abolished its interaction with the 14-3-3 proteins in yeast and in vivo. Such mutant OsBZR1 proteins suppressed the phenotypes of the Arabidopsis bri1–5 mutant and showed an increased nuclear distribution compared with the wild-type protein, suggesting that 14-3-3 proteins directly inhibit OsBZR1 function at least in part by reducing its nuclear localization. These results demonstrate a conserved function of OsBZR1 and an important role of 14-3-3 proteins in brassinosteroid signal transduction in rice. PMID:17699623

  7. Diagnosing Sporadic Creutzfeldt-Jakob Disease: Accuracy of CSF 14-3-3 Protein Test of the Spinal Fluid

    MedlinePlus

    ... JAKOB DISEASE: ACCURACY OF THE 14-3-3 PROTEIN TEST OF THE SPINAL FLUID This information sheet ... help you understand how the 14-3-3 protein test helps in diagnosing sporadic Creutzfeldt-Jakob disease ( ...

  8. 14-3-3 protein binds to the low molecular weight neurofilament (NFL) mRNA 3' UTR.

    PubMed

    Ge, Wei-Wen; Volkening, Kathryn; Leystra-Lantz, Cheryl; Jaffe, Howard; Strong, Michael J

    2007-01-01

    We have previously reported that altered stability of low molecular weight neurofilament (NFL) mRNA in lumbar spinal cord homogenates in amyotrophic lateral sclerosis (ALS) is associated with altered expression of trans-acting 3' UTR mRNA binding proteins. We have identified two hexanucleotide motifs as the main cis elements and, using LC/MS/MS of peptide digests of NFL 3' UTR interacting proteins from human spinal cord, observed that 14-3-3 proteins interact with these motifs. 14-3-3 beta, zeta, tau, gamma, and eta isoforms were found to be expressed in human spinal cord. Each isoform was expressed in vitro and shown to interact with NFL 3' UTR mRNA. Mutation of one or both motifs resulted in decreased 14-3-3 interaction, changes in predicted mRNA structure or alteration in stability of the mRNA. These data show a novel interaction for 14-3-3 with NFL mRNA, and suggests that 14-3-3 may play a role in regulating NFL mRNA stability.

  9. Modulation of GluK2a subunit-containing kainate receptors by 14-3-3 proteins.

    PubMed

    Sun, Changcheng; Qiao, Haifa; Zhou, Qin; Wang, Yan; Wu, Yuying; Zhou, Yi; Li, Yong

    2013-08-23

    Kainate receptors (KARs) are one of the ionotropic glutamate receptors that mediate excitatory postsynaptic currents (EPSCs) with characteristically slow kinetics. Although mechanisms for the slow kinetics of KAR-EPSCs are not totally understood, recent evidence has implicated a regulatory role of KAR-associated proteins. Here, we report that decay kinetics of GluK2a-containing receptors is modulated by closely associated 14-3-3 proteins. 14-3-3 binding requires PKC-dependent phosphorylation of serine residues localized in the carboxyl tail of the GluK2a subunit. In transfected cells, 14-3-3 binding to GluK2a slows desensitization kinetics of both homomeric GluK2a and heteromeric GluK2a/GluK5 receptors. Moreover, KAR-EPSCs at mossy fiber-CA3 synapses decay significantly faster in the 14-3-3 functional knock-out mice. Collectively, these results demonstrate that 14-3-3 proteins are an important regulator of GluK2a-containing KARs and may contribute to the slow decay kinetics of native KAR-EPSCs. PMID:23861400

  10. Dexamethasone downregulated the expression of CSF 14-3-3β protein in mice with eosinophilic meningitis caused by Angiostrongylus cantonensis infection.

    PubMed

    Tsai, Hung-Chin; Lee, Bi-Yao; Yen, Chuan-Min; Wann, Shue-Ren; Lee, Susan Shin-Jung; Chen, Yao-Shen; Tai, Ming-Hong

    2014-03-01

    Angiostrongylus cantonensis is the main causative agent of human eosinophilic meningitis in Southeast Asia and the Pacific Islands. A previous study demonstrated that the 14-3-3β protein is a neuropathological marker in monitoring neuronal damage in meningitis. Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infection. However, the mechanism by which steroids act in eosinophilic meningitis is unknown. We hypothesized that the beneficial effect of steroids on eosinophilic meningitis is partially mediated by the down-regulation of 14-3-3β protein expression in the cerebrospinal fluid (CSF). In this animal study, we determined the dynamic changes of 14-3-3β protein in mice with eosinophilic meningitis. The 14-3-3β protein in serum and CSF was increased in week 2 and 3 after infections. Dexamethasone administration significantly decreased the amounts of CSF 14-3-3β protein. By developing an in-house ELISA to measure 14-3-3β protein, it was found that the amounts of 14-3-3β protein in the CSF and serum increased over a three-week period after infection. There was a remarkable reduction of 14-3-3β protein in the CSF after 2 weeks of dexamethasone treatment. In conclusion, the administration of corticosteroids in mice with eosinophilic meningitis decreased the expression of 14-3-3β protein in the CSF.

  11. 14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels.

    PubMed

    Liang, Xiubin; Peters, Kathryn W; Butterworth, Michael B; Frizzell, Raymond A

    2006-06-16

    Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3beta. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of alpha-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3beta without altering total Nedd4-2. Aldosterone decreased the interaction of alpha-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3beta with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3beta blunted the aldosterone-induced increase in alpha-ENaC expression, returned alpha-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3beta with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3beta binding. Our studies show that aldosterone increases the expression of 14-3-3beta, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density. PMID:16613846

  12. Polycations Globally Enhance Binding of 14-3-3 omega to Target Proteins in Spinach Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The binding of 14-3-3' to phosphorylated NR (pNR) is stimulated by cations such as Mg2+ or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, Far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinac...

  13. Revealing the binding modes and the unbinding of 14-3-3σ proteins and inhibitors by computational methods.

    PubMed

    Hu, Guodong; Cao, Zanxia; Xu, Shicai; Wang, Wei; Wang, Jihua

    2015-01-01

    The 14-3-3σ proteins are a family of ubiquitous conserved eukaryotic regulatory molecules involved in the regulation of mitogenic signal transduction, apoptotic cell death, and cell cycle control. A lot of small-molecule inhibitors have been identified for 14-3-3 protein-protein interactions (PPIs). In this work, we carried out molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) method to study the binding mechanism between a 14-3-3σ protein and its eight inhibitors. The ranking order of our calculated binding free energies is in agreement with the experimental results. We found that the binding free energies are mainly from interactions between the phosphate group of the inhibitors and the hydrophilic residues. To improve the binding free energy of Rx group, we designed the inhibitor R9 with group R9 = 4-hydroxypheny. However, we also found that the binding free energy of inhibitor R9 is smaller than that of inhibitor R1. By further using the steer molecular dynamics (SMD) simulations, we identified a new hydrogen bond between the inhibitor R8 and residue Arg64 in the pulling paths. The information obtained from this study may be valuable for future rational design of novel inhibitors, and provide better structural understanding of inhibitor binding to 14-3-3σ proteins. PMID:26568041

  14. Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins*

    PubMed Central

    Yasuda, Shigetaka; Sato, Takeo; Maekawa, Shugo; Aoyama, Shoki; Fukao, Yoichiro; Yamaguchi, Junji

    2014-01-01

    Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr209, Ser247, Ser270, and Ser303 as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr209 and Ser247 on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr209 peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response. PMID:24722992

  15. Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest

    PubMed Central

    Kino, Tomoshige; Gragerov, Alexander; Valentin, Antonio; Tsopanomihalou, Maria; Ilyina-Gragerova, Galina; Erwin-Cohen, Rebecca; Chrousos, George P.; Pavlakis, George N.

    2005-01-01

    Vpr and selected mutants were used in a Saccharomyces cerevisiae two-hybrid screen to identify cellular interactors. We found Vpr interacted with 14-3-3 proteins, a family regulating a multitude of proteins in the cell. Vpr mutant R80A, which is inactive in cell cycle arrest, did not interact with 14-3-3. 14-3-3 proteins regulate the G2/M transition by inactivating Cdc25C phosphatase via binding to the phosphorylated serine residue at position 216 of Cdc25C. 14-3-3 overexpression in human cells synergized with Vpr in the arrest of cell cycle. Vpr did not arrest efficiently cells not expressing 14-3-3σ. This indicated that a full complement of 14-3-3 proteins is necessary for optimal Vpr function on the cell cycle. Mutational analysis showed that the C-terminal portion of Vpr, known to harbor its cell cycle-arresting activity, bound directly to the C-terminal part of 14-3-3, outside of its phosphopeptide-binding pocket. Vpr expression shifted localization of the mutant Cdc25C S216A to the cytoplasm, indicating that Vpr promotes the association of 14-3-3 and Cdc25C, independently of the presence of serine 216. Immunoprecipitations of cell extracts indicated the presence of triple complexes (Vpr/14-3-3/Cdc25C). These results indicate that Vpr promotes cell cycle arrest at the G2/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status. PMID:15708996

  16. Discovery and structural characterization of a small molecule 14-3-3 protein-protein interaction inhibitor

    PubMed Central

    Zhao, Jing; Du, Yuhong; Horton, John R.; Upadhyay, Anup K.; Lou, Bin; Bai, Yan; Zhang, Xing; Du, Lupei; Li, Minyong; Wang, Binghe; Zhang, Lixin; Barbieri, Joseph T.; Khuri, Fadlo R.; Cheng, Xiaodong; Fu, Haian

    2011-01-01

    The 14-3-3 family of phosphoserine/threonine-recognition proteins engage multiple nodes in signaling networks that control diverse physiological and pathophysiological functions and have emerged as promising therapeutic targets for such diseases as cancer and neurodegenerative disorders. Thus, small molecule modulators of 14-3-3 are much needed agents for chemical biology investigations and therapeutic development. To analyze 14-3-3 function and modulate its activity, we conducted a chemical screen and identified 4-[(2Z)-2-[4-formyl-6-methyl-5-oxo-3-(phosphonatooxymethyl)pyridin-2-ylidene]hydrazinyl]benzoate as a 14-3-3 inhibitor, which we termed FOBISIN (FOurteen-three-three BInding Small molecule INhibitor) 101. FOBISIN101 effectively blocked the binding of 14-3-3 with Raf-1 and proline-rich AKT substrate, 40 kDa and neutralized the ability of 14-3-3 to activate exoenzyme S ADP-ribosyltransferase. To provide a mechanistic basis for 14-3-3 inhibition, the crystal structure of 14-3-3ζ in complex with FOBISIN101 was solved. Unexpectedly, the double bond linking the pyridoxal-phosphate and benzoate moieties was reduced by X-rays to create a covalent linkage of the pyridoxal-phosphate moiety to lysine 120 in the binding groove of 14-3-3, leading to persistent 14-3-3 inactivation. We suggest that FOBISIN101-like molecules could be developed as an entirely unique class of 14-3-3 inhibitors, which may serve as radiation-triggered therapeutic agents for the treatment of 14-3-3-mediated diseases, such as cancer. PMID:21908710

  17. Unraveling 14-3-3 proteins in C4 panicoids with emphasis on model plant Setaria italica reveals phosphorylation-dependent subcellular localization of RS splicing factor.

    PubMed

    Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

    2015-01-01

    14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

  18. Unraveling 14-3-3 Proteins in C4 Panicoids with Emphasis on Model Plant Setaria italica Reveals Phosphorylation-Dependent Subcellular Localization of RS Splicing Factor

    PubMed Central

    Kumar, Karunesh; Muthamilarasan, Mehanathan; Bonthala, Venkata Suresh; Roy, Riti; Prasad, Manoj

    2015-01-01

    14-3-3 proteins are a large multigenic family of regulatory proteins ubiquitously found in eukaryotes. In plants, 14-3-3 proteins are reported to play significant role in both development and response to stress stimuli. Therefore, considering their importance, genome-wide analyses have been performed in many plants including Arabidopsis, rice and soybean. But, till date, no comprehensive investigation has been conducted in any C4 panicoid crops. In view of this, the present study was performed to identify 8, 5 and 26 potential 14-3-3 gene family members in foxtail millet (Si14-3-3), sorghum (Sb14-3-3) and maize (Zm14-3-3), respectively. In silico characterization revealed large variations in their gene structures; segmental and tandem duplications have played a major role in expansion of these genes in foxtail millet and maize. Gene ontology annotation showed the participation of 14-3-3 proteins in diverse biological processes and molecular functions, and in silico expression profiling indicated their higher expression in all the investigated tissues. Comparative mapping was performed to derive the orthologous relationships between 14-3-3 genes of foxtail millet and other Poaceae members, which showed a higher, as well as similar percentage of orthology among these crops. Expression profiling of Si14-3-3 genes during different time-points of abiotic stress and hormonal treatments showed a differential expression pattern of these genes, and sub-cellular localization studies revealed the site of action of Si14-3-3 proteins within the cells. Further downstream characterization indicated the interaction of Si14-3-3 with a nucleocytoplasmic shuttling phosphoprotein (SiRSZ21A) in a phosphorylation-dependent manner, and this demonstrates that Si14-3-3 might regulate the splicing events by binding with phosphorylated SiRSZ21A. Taken together, the present study is a comprehensive analysis of 14-3-3 gene family members in foxtail millet, sorghum and maize, which provides

  19. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H(+)-ATPase.

    PubMed Central

    Jahn, T; Fuglsang, A T; Olsson, A; Brüntrup, I M; Collinge, D B; Volkmann, D; Sommarin, M; Palmgren, M G; Larsson, C

    1997-01-01

    Accumulating evidence suggests that 14-3-3 proteins are involved in the regulation of plant plasma membrane H(+)-ATPase activity. However, it is not known whether the 14-3-3 protein interacts directly or indirectly with the H(+)-ATPase. In this study, detergent-solubilized plasma membrane H(+)-ATPase isolated from fusicoccin-treated maize shoots was copurified with the 14-3-3 protein (as determined by protein gel blotting), and the H(+)-ATPase was recovered in an activated state. In the absence of fusicoccin treatment, H(+)-ATPase and the 14-3-3 protein were well separated, and the H(+)-ATPase was recovered in a nonactivated form. Trypsin treatment removed the 10-kD C-terminal region from the H(+)-ATPase as well as the 14-3-3 protein. Using the yeast two-hybrid system, we could show a direct interaction between Arabidopsis 14-3-3 GF14-phi and the last 98 C-terminal amino acids of the Arabidopsis AHA2 plasma membrane H(+)-ATPase. We propose that the 14-3-3 protein is a natural ligand of the plasma membrane H(+)-ATPase, regulating proton pumping by displacing the C-terminal autoinhibitory domain of the H(+)-ATPase. PMID:9368417

  20. 14-3-3 zeta down-regulates p53 in mammary epithelial cells and confers luminal filling.

    PubMed

    Danes, Christopher G; Wyszomierski, Shannon L; Lu, Jing; Neal, Christopher L; Yang, Wentao; Yu, Dihua

    2008-03-15

    Recent progress in diagnostic tools allows many breast cancers to be detected at an early preinvasive stage. Thus, a better understanding of the molecular basis of early breast cancer progression is essential. Previously, we discovered that 14-3-3 zeta is overexpressed in >40% of advanced breast cancers, and this overexpression predicts poor patient survival. Here, we examined at what stage of breast disease 14-3-3 zeta overexpression occurs, and we found that increased expression of 14-3-3 zeta begins at atypical ductal hyperplasia, an early stage of breast disease. To determine whether 14-3-3 zeta overexpression is a decisive early event in breast cancer, we overexpressed 14-3-3 zeta in MCF10A cells and examined its effect in a three-dimensional culture model. We discovered that 14-3-3 zeta overexpression severely disrupted the acini architecture resulting in luminal filling. Proper lumen formation is a result of anoikis, apoptosis due to detachment from the basement membrane. We found that 14-3-3 zeta overexpression conferred resistance to anoikis. Additionally, 14-3-3 zeta overexpression in MCF10A cells and in mammary epithelial cells (MEC) from 14-3-3 zeta transgenic mice reduced expression of p53, which is known to mediate anoikis. Mechanistically, 14-3-3 zeta induced hyperactivation of the phosphoinositide 3-kinase/Akt pathway which led to phosphorylation and translocation of the MDM2 E3 ligase resulting in increased p53 degradation. Ectopic expression of p53 restored luminal apoptosis in 14-3-3 zeta-overexpressing MCF10A acini in three-dimensional cultures. These data suggest that 14-3-3 zeta overexpression is a critical event in early breast disease, and down-regulation of p53 is one of the mechanisms by which 14-3-3 zeta alters MEC acini structure and increases the risk of breast cancer.

  1. Ablation of the 14-3-3gamma Protein Results in Neuronal Migration Delay and Morphological Defects in the Developing Cerebral Cortex.

    PubMed

    Wachi, Tomoka; Cornell, Brett; Marshall, Courtney; Zhukarev, Vladimir; Baas, Peter W; Toyo-oka, Kazuhito

    2016-06-01

    14-3-3 proteins are ubiquitously-expressed and multifunctional proteins. There are seven isoforms in mammals with a high level of homology, suggesting potential functional redundancy. We previously found that two of seven isoforms, 14-3-3epsilon and 14-3-3zeta, are important for brain development, in particular, radial migration of pyramidal neurons in the developing cerebral cortex. In this work, we analyzed the function of another isoform, the protein 14-3-3gamma, with respect to neuronal migration in the developing cortex. We found that in utero 14-3-3gamma-deficiency resulted in delays in neuronal migration as well as morphological defects. Migrating neurons deficient in 14-3-3gamma displayed a thicker leading process stem, and the basal ends of neurons were not able to reach the boundary between the cortical plate and the marginal zone. Consistent with the results obtained from in utero electroporation, time-lapse live imaging of brain slices revealed that the ablation of the 14-3-3gamma proteins in pyramidal neurons slowed down their migration. In addition, the 14-3-3gamma deficient neurons showed morphological abnormalities, including increased multipolar neurons with a thicker leading processes stem during migration. These results indicate that the 14-3-3gamma proteins play an important role in radial migration by regulating the morphology of migrating neurons in the cerebral cortex. The findings underscore the pathological phenotypes of brain development associated with the disruption of different 14-3-3 proteins and will advance the preclinical data regarding disorders caused by neuronal migration defects.

  2. Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic.

    PubMed

    Schmitz, Matthias; Ebert, Elisabeth; Stoeck, Katharina; Karch, André; Collins, Steven; Calero, Miguel; Sklaviadis, Theodor; Laplanche, Jean-Louis; Golanska, Ewa; Baldeiras, Ines; Satoh, Katsuya; Sanchez-Valle, Raquel; Ladogana, Anna; Skinningsrud, Anders; Hammarin, Anna-Lena; Mitrova, Eva; Llorens, Franc; Kim, Yong Sun; Green, Alison; Zerr, Inga

    2016-05-01

    At present, the testing of 14-3-3 protein in cerebrospinal fluid (CSF) is a standard biomarker test in suspected sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis. Increasing 14-3-3 test referrals in CJD reference laboratories in the last years have led to an urgent need to improve established 14-3-3 test methods. The main result of our study was the validation of a commercially available 14-3-3 ELISA next to the commonly used Western blot method as a high-throughput screening test. Hereby, 14-3-3 protein expression was quantitatively analyzed in CSF of 231 sCJD and 2035 control patients. We obtained excellent sensitivity/specificity values of 88 and 96% that are comparable to the established Western blot method. Since standard protocols and preanalytical sample handling have become more important in routine diagnostic, we investigated in a further step the reproducibility and stability of 14-3-3 as a biomarker for human prion diseases. Ring trial data from 2009 to 2013 revealed an increase of Fleiss' kappa from 0.51 to 0.68 indicating an improving reliability of 14-3-3 protein detection. The stability of 14-3-3 protein under short-term and long-term storage conditions at various temperatures and after repeated freezing/thawing cycles was confirmed. Contamination of CSF samples with blood appears likely to be an important factor at a concentration of more than 2500 erythrocytes/μL. Hemolysis of erythrocytes with significant release of 14-3-3 protein started after 2 days at room temperature. We first define clear standards for the sample handling, short- and long-term storage of CSF samples as well as the handling of blood- contaminated samples which may result in artificially elevated CSF levels of 14-3-3.

  3. Site-specific regulatory interaction between spinach leaf sucrose-phosphate synthase and 14-3-3 proteins

    NASA Technical Reports Server (NTRS)

    Toroser, D.; Athwal, G. S.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    We report an Mg2+-dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.

  4. Transcription variants of the prostate-specific PrLZ gene and their interaction with 14-3-3 proteins

    SciTech Connect

    Wang, Ruoxiang; He, Hui; Sun, Xiaojuan; Xu, Jianchun; Marshall, Fray F.; Zhau, Haiyen; Chung, Leland W.K.; Fu, Haian; He, Dalin

    2009-11-20

    We have reported isolation and characterization of the prostate-specific and androgen-regulated PrLZ gene abnormally expressed in prostate cancer. PrLZ is a potential biomarker for prostate cancer and a candidate oncogene promoting cell proliferation and survival in prostate cancer cells. A full delineation of the PrLZ gene and its gene products may provide clues to the mechanisms regulating its expression and function. In this report, we identified three additional exons in the PrLZ gene and recognized five transcript variants from alternative splicing that could be detected by RT-PCR and Western blotting. Structural comparison demonstrated that the PrLZ proteins are highly conserved among species. PrLZ contains multiple potential sites for interaction with other proteins. We used mammalian two-hybrid assays to demonstrate that PrLZ isoforms interact with 14-3-3 proteins, and multiple sites in the PrLZ may be involved in the interaction. Alternative splicing may contribute to abnormally enhanced PrLZ levels in prostate cancer, and interaction with 14-3-3 proteins may be a mechanism by which PrLZ promotes cell proliferation and survival during prostate cancer development and progression. This information is a valuable addition to the investigation of the oncogenic properties of the PrLZ gene.

  5. Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

    PubMed Central

    Tutor, Antonio S.; Delpón, Eva; Caballero, Ricardo; Gómez, Ricardo; Núñez, Lucía; Vaquero, Miguel; Tamargo, Juan; Penela, Petronila

    2006-01-01

    We identify a new mechanism for the β1-adrenergic receptor (β1AR)-mediated regulation of human ether-a-go-go–related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3ε proteins is competed by wild type β1AR by means of a novel interaction between this receptor and 14-3-3ε. The association between β1AR and 14-3-3ε is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The β1AR/14-3-3ε association is direct, since it can be recapitulated using purified 14-3-3ε and β1AR fusion proteins and is abolished in cells expressing β1AR phosphorylation–deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that β1AR phosphorylation–deficient mutants do not recruit 14-3-3ε away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type β1AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both β1AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness. PMID:16914520

  6. Proteomic analysis of media from lung cancer cells reveals role of 14-3-3 proteins in cachexia

    PubMed Central

    McLean, Julie B.; Moylan, Jennifer S.; Horrell, Erin M. W.; Andrade, Francisco H.

    2015-01-01

    Aims: At the time of diagnosis, 60% of lung cancer patients present with cachexia, a severe wasting syndrome that increases morbidity and mortality. Tumors secrete multiple factors that contribute to cachectic muscle wasting, and not all of these factors have been identified. We used Orbitrap electrospray ionization mass spectrometry to identify novel cachexia-inducing candidates in media conditioned with Lewis lung carcinoma cells (LCM). Results: One-hundred and 58 proteins were confirmed in three biological replicates. Thirty-three were identified as secreted proteins, including 14-3-3 proteins, which are highly conserved adaptor proteins known to have over 200 binding partners. We confirmed the presence of extracellular 14-3-3 proteins in LCM via western blot and discovered that LCM contained less 14-3-3 content than media conditioned with C2C12 myotubes. Using a neutralizing antibody, we depleted extracellular 14-3-3 proteins in myotube culture medium, which resulted in diminished myosin content. We identified the proposed receptor for 14-3-3 proteins, CD13, in differentiated C2C12 myotubes and found that inhibiting CD13 via Bestatin also resulted in diminished myosin content. Conclusions: Our novel findings show that extracellular 14-3-3 proteins may act as previously unidentified myokines and may signal via CD13 to help maintain muscle mass. PMID:25972815

  7. Evolution of the 14-3-3 protein family: does the large number of isoforms in multicellular organisms reflect functional specificity?

    PubMed

    Rosenquist, M; Sehnke, P; Ferl, R J; Sommarin, M; Larsson, C

    2000-11-01

    14-3-3 proteins constitute a family of eukaryotic proteins that are key regulators of a large number of processes ranging from mitosis to apoptosis. 14-3-3s function as dimers and bind to particular motifs in their target proteins. To date, 14-3-3s have been implicated in regulation or stabilization of more than 35 different proteins. This number is probably only a fraction of the number of proteins that 14-3-3s bind to, as reports of new target proteins have become more frequent. An examination of 14-3-3 entries in the public databases reveals 153 isoforms, including alleloforms, reported in 48 different species. The number of isoforms range from 2, in the unicellular organism Saccharomyces cerevisiae, to 12 in the multicellular organism Arabidopsis thaliana. A phylogenetic analysis reveals that there are four major evolutionary lineages: Viridiplantae (plants), Fungi, Alveolata, and Metazoa (animals). A close examination of the aligned amino acid sequences identifies conserved amino acid residues and regions of importance for monomer stabilization, dimer formation, target protein binding, and the nuclear export function. Given the fact that 53% of the protein is conserved, including all amino acid residues in the target binding groove of the 14-3-3 monomer, one might expect little to no isoform specificity for target protein binding. However, using surface plasmon resonance we show that there are large differences in affinity between nine 14-3-3 isoforms of A. thaliana and a target peptide representing a novel binding motif present in the C terminus of the plant plasma membrane H(+)ATPase. Thus, our data suggest that one reason for the large number of isoforms found in multicellular organisms is isoform-specific functions. PMID:11080367

  8. 14-3-3 regulates the nuclear import of class IIa histone deacetylases

    SciTech Connect

    Nishino, Tomonori G.; Miyazaki, Masaya; Hoshino, Hideto; Miwa, Yoshihiro; Horinouchi, Sueharu; Yoshida, Minoru

    2008-12-19

    Class IIa histone deacetylases (HDACs) form complexes with a class of transcriptional repressors in the nucleus. While screening for compounds that could block the association of HDAC4 with the BTB domain-containing transcriptional repressor Bach2, we discovered that phorbol 12-myristate 13-acetate (PMA) induced the cytoplasmic retention of HDAC4 mutants lacking a nuclear export signal (NES). Although PMA treatment and PKD overexpression has been proposed to facilitate the nuclear export of class IIa HDACs by creating 14-3-3 binding sites containing phosphoserines, our experiments using HDAC mutants demonstrated that PMA greatly reduces nuclear import. PMA treatment repressed the NLS activity in a manner dependent on 14-3-3 binding. These results suggest that nuclear HDAC4 is not tethered in the nucleus, but instead shuttles between the nucleus and the cytoplasm. Phosphorylation-induced 14-3-3 binding biases the balance of nucleo-cytoplasmic shuttling toward the cytoplasm by inhibiting nuclear import.

  9. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein.

    PubMed Central

    Svennelid, F; Olsson, A; Piotrowski, M; Rosenquist, M; Ottman, C; Larsson, C; Oecking, C; Sommarin, M

    1999-01-01

    The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth. PMID:10590165

  10. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases.

  11. CSF Tau proteins reduce misdiagnosis of sporadic Creutzfeldt-Jakob disease suspected cases with inconclusive 14-3-3 result.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-09-01

    Cerebrospinal fluid (CSF) 14-3-3 protein supports sporadic Creutzfeldt-Jakob (sCJD) diagnosis, but often leads to weak-positive results and lacks standardization. In this study, we explored the added diagnostic value of Total Tau (t-Tau) and phosphorylated Tau (p-Tau) in sCJD diagnosis, particularly in the cases with inconclusive 14-3-3 result. 95 definite sCJD and 287 patients without prion disease (non-CJD) were included in this study. CSF samples were collected in routine clinical diagnosis and analysed for 14-3-3 detection by Western blot (WB). CSF t-Tau and p-Tau were quantified by commercial ELISA kits and PRNP and APOE genotyping assessed by PCR-RFLP. In a regression analysis of the whole cohort, 14-3-3 protein revealed an overall accuracy of 82 % (sensitivity = 96.7 %; specificity = 75.6 %) for sCJD. Regarding 14-3-3 clear positive results, we observed no added value either of t-Tau alone or p-Tau/t-Tau ratio in the model. On the other hand, considering 14-3-3 weak-positive cases, t-Tau protein increased the overall accuracy of 14-3-3 alone from 91 to 94 % and specificity from 74 to 93 % (p < 0.05), with no sensitivity improvement. However, inclusion of p-Tau/t-Tau ratio did not significantly improve the first model (p = 0.0595). Globally, t-Tau protein allowed a further discrimination of 65 % within 14-3-3 inconclusive results. Furthermore, PRNP MV genotype showed a trend to decrease 14-3-3 sensitivity (p = 0.051), but such effect was not seen on t-Tau protein. In light of these results, we suggest that t-Tau protein assay is of significant importance as a second marker in identifying 14-3-3 false-positive results among sCJD probable cases. PMID:27357003

  12. 14-3-3 isoforms bind directly exon B of the 5′-UTR of human surfactant protein A2 mRNA

    PubMed Central

    Noutsios, Georgios T.; Ghattas, Paul; Bennett, Stephanie

    2015-01-01

    Human surfactant protein (SP) A (SP-A), an innate immunity molecule, is encoded by two genes, SFTPA1 and SFTPA2. The 5′-untranslated splice variant of SP-A2 (ABD), but not SP-A1 (AD), contains exon B (eB). eB is an enhancer for transcription and translation and contains cis-regulatory elements. Specific trans-acting factors, including 14-3-3, bind eB. The 14-3-3 protein family contains seven isoforms that have been found by mass spectrometry in eB electromobility shift assays (Noutsios et al. Am J Physiol Lung Cell Mol Physiol 304: L722–L735, 2013). We used four different approaches to investigate whether 14-3-3 isoforms bind directly to eB. 1) eB RNA pulldown assays showed that 14-3-3 isoforms specifically bind eB. 2) RNA electromobility shift assay complexes were formed using purified 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, with wild-type eB RNA. 3 and 4) RNA affinity chromatography assays and surface plasmon resonance analysis showed that 14-3-3 isoforms β, γ, ε, η, σ, and τ, but not isoform ζ, specifically and directly bind eB. Inhibition of 14-3-3 isoforms γ, ε, η, and τ/θ with shRNAs in NCI-H441 cells resulted in downregulation of SP-A2 levels but did not affect SP-A1 levels. However, inhibition of 14-3-3 isoform σ was correlated with lower levels of SP-A1 and SP-A2. Inhibition of 14-3-3 isoform ζ/δ, which does not bind eB, had no effect on expression levels of SP-A1 and SP-A2. In conclusion, the 14-3-3 protein family affects differential regulation of SP-A1 and SP-A2 by binding directly to SP-A2 5′-UTR mRNA. PMID:26001776

  13. Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

    PubMed

    Aghazadeh, Yasaman; Martinez-Arguelles, Daniel B; Fan, Jinjiang; Culty, Martine; Papadopoulos, Vassilios

    2014-10-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

  14. Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

    PubMed

    Hyeon, Jae Wook; Kim, Su Yeon; Lee, Jeongmin; Park, Jun Sun; Hwang, Kyu Jam; Lee, Sol Moe; An, SeongSoo A; Lee, Myung Koo; Ju, Young Ran

    2015-01-01

    The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD. PMID:26507666

  15. Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory.

    PubMed

    Hyeon, Jae Wook; Kim, Su Yeon; Lee, Jeongmin; Park, Jun Sun; Hwang, Kyu Jam; Lee, Sol Moe; An, SeongSoo A; Lee, Myung Koo; Ju, Young Ran

    2015-10-28

    The 14-3-3 protein has been used as a biomarker for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). However, weakly positive 14-3-3 leads to false positive results and an incorrect diagnosis. We attempted to use quantitative data for tau protein to provide an accurate diagnosis based on weak 14-3-3 protein. Sixty-two patients with sCJD, including pathologically confirmed, clinically definite, and probable cases, and 89 non-CJD patients were investigated based on a Korean population. Among them, 20 sCJD and 14 non-CJD showed weakly positive 14-3-3. The total tau (t-tau) and phosphorylated tau (p-tau) protein levels were measured by ELISA, and the p-tau to t-tau ratio (p/t ratio) was calculated. The combined use of the 14-3-3 protein assay, t-tau levels, and p/t ratio improved the specificity of diagnosis compared with the use of the 14-3-3 protein assay alone (47% for 14-3-3 alone; 85.94% for 14-3-3 combined with t-tau; 90.62% for 14-3-3 combined with the p/t ratio). In addition, 18 of 20 sCJD and 12 of 14 non-CJD who were weakly positive for 14-3-3 were positive for the p/t ratio and negative for the p/t ratio, respectively. When used in combination with the 14-3-3 protein, the tau protein is useful as a biomarker for the precise diagnosis of sCJD.

  16. Brainstem deficiency of the 14-3-3 regulator of serotonin synthesis: a proteomics analysis in the sudden infant death syndrome.

    PubMed

    Broadbelt, Kevin G; Rivera, Keith D; Paterson, David S; Duncan, Jhodie R; Trachtenberg, Felicia L; Paulo, Joao A; Stapels, Martha D; Borenstein, Natalia S; Belliveau, Richard A; Haas, Elisabeth A; Stanley, Christina; Krous, Henry F; Steen, Hanno; Kinney, Hannah C

    2012-01-01

    Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42-75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MS(E)-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT(1A) receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process. PMID:21976671

  17. Brainstem Deficiency of the 14-3-3 Regulator of Serotonin Synthesis: A Proteomics Analysis in the Sudden Infant Death Syndrome*

    PubMed Central

    Broadbelt, Kevin G.; Rivera, Keith D.; Paterson, David S.; Duncan, Jhodie R.; Trachtenberg, Felicia L.; Paulo, Joao A.; Stapels, Martha D.; Borenstein, Natalia S.; Belliveau, Richard A.; Haas, Elisabeth A.; Stanley, Christina; Krous, Henry F.; Steen, Hanno; Kinney, Hannah C.

    2012-01-01

    Impaired brainstem responses to homeostatic challenges during sleep may result in the sudden infant death syndrome (SIDS). Previously we reported a deficiency of serotonin (5-HT) and its key biosynthetic enzyme, tryptophan hydroxylase (TPH2), in SIDS infants in the medullary 5-HT system that modulates homeostatic responses during sleep. Yet, the underlying basis of the TPH2 and 5-HT deficiency is unknown. In this study, we tested the hypothesis that proteomics would uncover previously unrecognized abnormal levels of proteins related to TPH2 and 5-HT regulation in SIDS cases compared with controls, which could provide novel insight into the basis of their deficiency. We first performed a discovery proteomic analysis of the gigantocellularis of the medullary 5-HT system in the same data set with deficiencies of TPH2 and 5-HT levels. Analysis in 6 SIDS cases and 4 controls revealed a 42–75% reduction in abundance in 5 of the 6 isoforms identified of the 14-3-3 signal transduction family, which is known to influence TPH2 activity (p < 0.07). These findings were corroborated in an additional SIDS and control sample using an orthogonal MSE-based quantitative proteomic strategy. To confirm these proteomics results in a larger data set (38 SIDS, 11 controls), we applied Western blot analysis in the gigantocellularis and found that 4/7 14-3-3 isoforms identified were significantly reduced in SIDS cases (p ≤ 0.02), with a 43% reduction in all 14-3-3 isoforms combined (p < 0.001). Abnormalities in 5-HT and TPH2 levels and 5-HT1A receptor binding were associated with the 14-3-3 deficits in the same SIDS cases. These data suggest a potential molecular defect in SIDS related to TPH2 regulation, as 14-3-3 is critical in this process. PMID:21976671

  18. The epsilon isoform of 14-3-3 protein is a component of the prion protein amyloid deposits of Gerstmann-Sträussler-Scheinker disease.

    PubMed

    Di Fede, Giuseppe; Giaccone, Giorgio; Limido, Lucia; Mangieri, Michela; Suardi, Silvia; Puoti, Gianfranco; Morbin, Michela; Mazzoleni, Giulia; Ghetti, Bernardino; Tagliavini, Fabrizio

    2007-02-01

    The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic events, such as transduction pathway modulation, cell cycle control, and apoptosis. Seven isoforms have been identified that are abundant in the brain, preferentially localized in neurons. Remarkable increases in 14-3-3 are seen in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease (CJD), and it has been found in pathologic inclusions of several neurodegenerative diseases. Moreover, the zeta isoform has been detected in prion protein (PrP) amyloid deposits of CJD patients. To further investigate the cerebral distribution of 14-3-3 in prion-related encephalopathies, we carried out an immunohistochemical and biochemical analysis of brain tissue from patients with Gerstmann-Sträussler-Scheinker disease (GSS) and sporadic, familial and acquired forms of CJD, using specific antibodies against the seven 14-3-3 isoforms. The study showed a strong immunoreactivity of PrP amyloid plaques of GSS patients for the 14-3-3 epsilon isoform, but not for the other isoforms. The epsilon isoform of 14-3-3 was not found in PrP deposits of CJD. These results indicate that the epsilon isoform of 14-3-3 is a component of PrP amyloid deposits of GSS and suggest that this is the sole 14-3-3 isoform specifically involved in the neuropathologic changes associated with this disorder.

  19. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization

    PubMed Central

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement. PMID:26791570

  20. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    PubMed

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-21

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

  1. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    SciTech Connect

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.; E-mail: andy.blakely@vanderbilt.edu

    2005-08-05

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH{sub 2}-terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking.

  2. 14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

    PubMed Central

    Liu, Lixin; Lin, Ye; Liu, Lili; Bian, Yanjie; Zhang, Li; Gao, Xuejun; Li, Qingzhang

    2015-01-01

    As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPAR

  3. 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides

    PubMed Central

    Madeira, Fábio; Tinti, Michele; Murugesan, Gavuthami; Berrett, Emily; Stafford, Margaret; Toth, Rachel; Cole, Christian; MacKintosh, Carol; Barton, Geoffrey J.

    2015-01-01

    Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulates many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets and to prioritize the downstream analysis of >2000 potential interactors identified in high-throughput experiments. Results: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix, support vector machines (SVM) and artificial neural network (ANN) classification methods were trained to discriminate experimentally determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from −6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful. Availability and implementation: A standalone prediction web server is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database. Contact: cmackintosh@dundee.ac.uk or gjbarton@dundee.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25735772

  4. The EFF-1A Cytoplasmic Domain Influences Hypodermal Cell Fusions in C. elegans But Is Not Dependent on 14-3-3 Proteins

    PubMed Central

    Shinn-Thomas, Jessica H.; del Campo, Jacob J.; Wang, Jianjun; Mohler, William A.

    2016-01-01

    Background Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1. Methodology/Principal Findings Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis. Conclusions/Significance Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions in vivo. However

  5. A Glycine soja 14-3-3 protein GsGF14o participates in stomatal and root hair development and drought tolerance in Arabidopsis thaliana.

    PubMed

    Sun, Xiaoli; Luo, Xiao; Sun, Mingzhe; Chen, Chao; Ding, Xiaodong; Wang, Xuedong; Yang, Shanshan; Yu, Qingyue; Jia, Bowei; Ji, Wei; Cai, Hua; Zhu, Yanming

    2014-01-01

    It is well established that 14-3-3 proteins are key regulators of multiple stress signal transduction cascades. However, the biological functions of soybean 14-3-3 proteins, especially in plant drought response, are not yet known. In this study, we characterized a Glycine soja 14-3-3 gene, GsGF14o, which is involved in plant development and drought response. GsGF14o expression was greatly induced by drought stress, as evidenced by the quantitative real-time PCR and β-glucuronidase (GUS) activity analysis. GsGF14o overexpression in Arabidopsis thaliana resulted in decreased drought tolerance during seed germination and seedling growth. Furthermore, silencing of AtGF14µ, the most homologous 14-3-3 gene of GsGF14o, led to enhanced drought tolerance at both the seed germination and seedling stage. Unexpectedly, GsGF14o transgenic lines showed reduced water loss and transpiration rates compared with wild-type plants, which was demonstrated to be the consequence of the decreased stomatal size. At the same time, the smaller stomata due to GsGF14o overexpression led to a relatively slow net photosynthesis rate, which led to a growth penalty under drought stress. We further demonstrated that GsGF14o overexpression caused deficits in root hair formation and development, and thereby reduced the water intake capacity of the transgenic root system. In addition, GsGF14o overexpression down-regulated the transcript levels of drought-responsive marker genes. Finally, we also investigated the tissue-specific accumulation of GsGF14o by using a GUS activity assay. Collectively, the results presented here confirm that GsGF14o plays a dual role in drought stress responses through its involvement in the regulation of stomatal size and root hair development.

  6. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene.

  7. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene. PMID:27261462

  8. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family

    PubMed Central

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M.

    2016-01-01

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems. PMID:27195976

  9. Controllability of protein-protein interaction phosphorylation-based networks: Participation of the hub 14-3-3 protein family.

    PubMed

    Uhart, Marina; Flores, Gabriel; Bustos, Diego M

    2016-05-19

    Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems.

  10. Spinach 14-3-3 protein interacts with the plasma membrane H(+)-ATPase and nitrate reductase in response to excess nitrate stress.

    PubMed

    Xu, Huini; Zhao, Xiuling; Guo, Chuanlong; Chen, Limei; Li, Kunzhi

    2016-09-01

    To investigate the function of 14-3-3 protein in response to excess nitrate stress, a 14-3-3 protein, designated as So14-3-3, was isolated from spinach. Phylogenetic analysis demonstrated that So14-3-3 belongs to non-ε group of 14-3-3 superfamily. Real time-quantitative RT-PCR and western blot analysis showed that So14-3-3 was induced by excess nitrate stress in spinach roots and leaves. After nitrate treatment, the phosphorylated H(+)-ATPase and nitrate reductase (NR) increased and decreased respectively. Co-Immunoprecipitation (Co-IP) suggested that the interaction of So14-3-3 with the phosphorylated H(+)-ATPase enhanced, but reduced with phosphorylated NR in spinach roots after nitrate treatment. Besides, 5 proteins interacted with So14-3-3 were found by Co-IP and LC-MS/MS analysis. So14-3-3 overexpressing transgenic tobacco plants showed enhanced tolerance to nitrate treatment at the germination and young seedlings stage. The transgenic plants showed longer root length, lower malondialdehyde (MDA), H2O2, protein carbonyl contents, relatively higher soluble sugar and protein contents, than the WT plants after nitrate treatment. The phosphorylation levels of H(+)-ATPase in transgenic plants were higher than the WT plants after nitrate treatment, whereas NR were lower. Additionally, in transgenic plants, the interaction of So14-3-3 with phosphorylated H(+)-ATPase and NR increased and decreased more than the WT plants under nitrate stress, leading to higher H(+)-ATPase and NR activities in transgenic plants. These data suggested that So14-3-3 might be involved in nitrate stress response by interacting with H(+)-ATPase and NR. PMID:27161584

  11. The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference

    PubMed Central

    Fiorillo, Annarita; di Marino, Daniele; Bertuccini, Lucia; Via, Allegra; Pozio, Edoardo; Camerini, Serena; Ilari, Andrea; Lalle, Marco

    2014-01-01

    The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3. PMID:24658679

  12. Down-regulation of 14-3-3β exerts anti-cancer effects through inducing ER stress in human glioma U87 cells: Involvement of CHOP–Wnt pathway

    SciTech Connect

    Cao, Lei; Lei, Hui; Chang, Ming-Ze; Liu, Zhi-Qin; Bie, Xiao-Hua

    2015-07-10

    We previously identified 14-3-3β as a tumor-specific isoform of 14-3-3 protein in astrocytoma, but its functional role in glioma cells and underlying mechanisms are poorly understood. In the present study, we investigated the effects of 14-3-3β inhibition in human glioma U87 cells using specific targeted small interfering RNA (siRNA). The results showed that 14-3-3β is highly expressed in U87 cells but not in normal astrocyte SVGp12 cells. Knockdown of 14-3-3β by Si-14-3-3β transfection significantly decreased the cell viability but increased the LDH release in a time-dependent fashion in U87 cells, and these effects were accompanied with G0/G1 cell cycle arrest and apoptosis. In addition, 14-3-3β knockdown induced ER stress in U87 cells, as evidenced by ER calcium release, increased expression of XBP1S mRNA and induction of ER related pro-apoptotic factors. Down-regulation of 14-3-3β significantly decreased the nuclear localization of β-catenin and inhibited Topflash activity, which was shown to be reversely correlated with CHOP. Furthermore, Si-CHOP and sFRP were used to inhibit CHOP and Wnt, respectively. The results showed that the anti-cancer effects of 14-3-3β knockdown in U87 cells were mediated by increased expression of CHOP and followed inhibition of Wnt/β-catenin pathway. In summary, the remarkable efficiency of 14-3-3β knockdown to induce apoptotic cell death in U87 cells may find therapeutic application for the treatment of glioma patients. - Highlights: • Knockdown of 14-3-3β leads to cytotoxicity in human glioma U87 cells. • Knockdown of 14-3-3β induces cell cycle arrest and apoptosis in U87 cells. • Knockdown of 14-3-3β results in ER stress in U87 cells. • Knockdown of 14-3-3β inhibits Wnt/β-catenin pathway via CHOP activation.

  13. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress.

    PubMed

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-04-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein-protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress.

  14. Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ

    PubMed Central

    Bersani, C; Xu, L-D; Vilborg, A; Lui, W-O; Wiman, K G

    2014-01-01

    Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3′-UTR and decreases its stability through an ARE in the 3′-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels. PMID:24469038

  15. 14-3-3ζ up-regulates hypoxia-inducible factor-1α in hepatocellular carcinoma via activation of PI3K/Akt/NF-кB signal transduction pathway

    PubMed Central

    Tang, Yufu; Lv, Pengfei; Sun, Zhongyi; Han, Lei; Luo, Bichao; Zhou, Wenping

    2015-01-01

    14-3-3ζ protein, a member of 14-3-3 family, plays important roles in multiple cellular processes. Our previous study showed that 14-3-3ζ could bind to regulate the expression of hypoxia-inducible factor-1α (HIF-1α), which is induced by hypoxia and a crucial factor for induction of tumor metastasis. Moreover, we also have confirmed the response of 14-3-3ζ to hypoxia in our unpublished data as well. Thus, in the present study, we attempted to reveal that whether the regulation effect of 14-3-3ζ on HIF-1α functioned in a similar pattern as hypoxia. Stable regulation of 14-3-3ζ in human HCC cell line SMMC-772 and HCC-LM3 was achieved. The regulation of 14-3-3ζ on HIF-1α mRNA transcription was evaluated by luciferase activity assay and quantitative real-time PCR (qPCR). The effect of 14-3-3ζ on the production of HIF-1α and pathways determining HIF-1α’s response to hypoxia was assessed using western blotting assay. Our results showed that regulation of 14-3-3ζ expression influenced the activity of HIF-1α, phosphatidyl inositol 3-kinase (PI3K), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and nuclear factor kappa B (NF-кB). Blocking of these pathways using indicated inhibitors revealed that 14-3-3ζ enhanced the production of HIF-1α via the activation of PI3K/Akt/NF-кB pathway, which was identical to hypoxia induced HIF-1α expression. For the first time, our study described the key role of 14-3-3ζ in the HIF-1α production in HCC cells. And the molecule exerted its function on HIF-1α both by directly binding to it and via PI3K/Akt/NF-кB signal transduction pathway. PMID:26884855

  16. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.

  17. A Negative Regulatory Mechanism Involving 14-3-3ζ Limits Signaling Downstream of ROCK to Regulate Tissue Stiffness in Epidermal Homeostasis.

    PubMed

    Kular, Jasreen; Scheer, Kaitlin G; Pyne, Natasha T; Allam, Amr H; Pollard, Anthony N; Magenau, Astrid; Wright, Rebecca L; Kolesnikoff, Natasha; Moretti, Paul A; Wullkopf, Lena; Stomski, Frank C; Cowin, Allison J; Woodcock, Joanna M; Grimbaldeston, Michele A; Pitson, Stuart M; Timpson, Paul; Ramshaw, Hayley S; Lopez, Angel F; Samuel, Michael S

    2015-12-21

    ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities. PMID:26702834

  18. Involvement of 14-3-3 protein GRF9 in root growth and response under polyethylene glycol-induced water stress

    PubMed Central

    He, Yuchi; Wu, Jingjing; Lv, Bing; Li, Jia; Gao, Zhiping; Xu, Weifeng; Baluška, František; Shi, Weiming; Shaw, Pang Chui; Zhang, Jianhua

    2015-01-01

    Plant 14-3-3 proteins are phosphoserine-binding proteins that regulate a wide array of targets via direct protein–protein interactions. In this study, the role of a 14-3-3 protein, GRF9, in plant response to water stress was investigated. Arabidopsis wild-type, GRF9-deficient mutant (grf9), and GRF9-overexpressing (OE) plants were treated with polyethylene glycol (PEG) to induce mild water stress. OE plant showed better whole-plant growth and root growth than the wild type under normal or water stress conditions while the grf9 mutant showed worse growth. In OE plants, GRF9 favours the allocation of shoot carbon to roots. In addition, GRF9 enhanced proton extrusion, mainly in the root elongation zone and root hair zone, and maintained root growth under mild water stress. Grafting among the wild type, OE, and grf9 plants showed that when OE plants were used as the scion and GRF9 was overexpressed in the shoot, it enhanced sucrose transport into the root, and when OE plants were used as rootstock and GRF9 was overexpressed in the root, it caused more release of protons into the root surface under water stress. Taken together, the results suggest that under PEG-induced water stress, GRF9 is involved in allocating more carbon from the shoot to the root and enhancing proton secretion in the root growing zone, and this process is important for root response to mild water stress. PMID:25873671

  19. 14-3-3γ regulates cell viability and milk fat synthesis in lipopolysaccharide-induced dairy cow mammary epithelial cells

    PubMed Central

    LIU, LIXIN; ZHANG, LI; LIN, YE; BIAN, YANJIE; GAO, XUEJUN; QU, BO; LI, QINGZHANG

    2016-01-01

    Our previous study demonstrated that 14-3-3γ overexpression was able to inhibit the production of lipopolysaccharide (LPS)-induced cytokines in dairy cow mammary epithelial cells (DCMECs) by inhibiting the activation of nuclear factor-κB (NF-κB) signaling pathways. However, the association between 14-3-3γ overexpression and milk fat synthesis in LPS-induced DCMECs remains unclear. Therefore, the present study investigated the effect of 14-3-3γ on cell viability and milk fat synthesis in LPS-induced DCMECs. The results of the MTT assay and lactate dehydrogenase activity assay demonstrated that 14-3-3γ overexpression was able to attenuate LPS-induced cytotoxicity in DCMECs, and increase the viability of the cells. In addition, the results of reverse transcription-quantitative polymerase chain reaction suggested that mRNA expression levels of genes associated with milk fat synthesis, including sterol regulatory element binding protein (SREBP1), peroxisome proliferator-activated receptor-γ (PPARG), cluster of differentiation 36, acetyl-coA carboxylase (ACC), fatty acid synthase (FAS) and fatty acid binding protein-3, were significantly upregulated in cells overexpressing the 14-3-3γ protein. In addition, as compared with the LPS-treated group, the activities of FAS and ACC were significantly increased. Furthermore, western blotting demonstrated that 14-3-3γ overexpression enhanced the protein expression levels of phosphorylated SREBP1 and PPARG. These results suggested that high levels of 14-3-3γ protein were able to attenuate LPS-induced cell damage and promote milk fat synthesis in LPS-induced DCMECs by increasing the cell viability and upregulating the expression levels of transcription factors associated with milk fat synthesis. PMID:27073437

  20. Dual role for 14-3-3 proteins and ABF transcription factors in gibberellic acid and abscisic acid signalling in barley (Hordeum vulgare) aleurone cells.

    PubMed

    Schoonheim, Peter J; Costa Pereira, Daniel D A; De Boer, Albertus H

    2009-05-01

    The balance of gibberellins [gibberellic acid (GA)] and abscisic acid (ABA) is a determining factor during transition of embryogenesis and seed germination. Recently, we showed that 14-3-3 proteins are important in ABA signalling in barley aleurone cells. Using 14-3-3 RNAi constructs in the barley aleurone transient expression system, we demonstrate here that silencing of each 14-3-3 isoform suppresses GA induction of the alpha-amylase gene. 14-3-3 Proteins interact with ABA-responsive element (ABRE) binding factors HvABF1, 2 and 3, and here we show that these transcription factors also interact with the ABA-responsive kinase PKABA1, a kinase that mediates cross-talk between the GA and ABA pathway. ABF1 and ABF2 have a function in both signalling pathways as: (1) ectopic expression of wild-type ABF1 and mutant ABF2, lacking the 14-3-3 interaction domain, transactivates the ABA inducible HVA1 gene; and (2) GA induction of the alpha-amylase gene is repressed by ectopic expression of wild-type ABF1 and 2. Mutant ABF1 and 2 were still effective repressors of GA signalling. In summary, our data provide evidence that 14-3-3 proteins and members of the ABF transcription factor family have a regulatory function in the GA pathway and suggest that PKABA1 and ABF transcription factors are cross-talk intermediates in ABA and GA signalling.

  1. 14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules.

    PubMed

    Lai, Kenneth K Y; Chan, Kin Tak; Choi, Mei Yuk; Wang, Hector K; Fung, Eva Y M; Lam, Ho Yu; Tan, Winnie; Tung, Lai Nar; Tong, Daniel K H; Sun, Raymond W Y; Lee, Nikki P; Law, Simon

    2016-02-01

    Esophageal squamous cell carcinoma (ESCC) is the predominant type of esophageal cancer in Asia. Cisplatin is commonly used in chemoradiation for unresectable ESCC patients. However, the treatment efficacy is diminished in patients with established cisplatin resistance. To understand the mechanism leading to the development of cisplatin resistance in ESCC, we compared the proteomes from a cisplatin-resistant HKESC-2R cell line with its parental-sensitive counterpart HKESC-2 to identify key molecule involved in this process. Mass spectrometry analysis detected 14-3-3σ as the most abundant molecule expressed exclusively in HKESC-2R cells, while western blot result further validated it to be highly expressed in HKESC-2R cells when compared to HKESC-2 cells. Ectopic expression of 14-3-3σ increased cisplatin resistance in HKESC-2 cells, while its suppression sensitized SLMT-1 cells to cisplatin. Among the molecules involved in drug detoxification, drug transportation, and DNA repair, the examined DNA repair molecules HMGB1 and XPA were found to be highly expressed in HKESC-2R cells with high 14-3-3σ expression. Subsequent manipulation of 14-3-3σ by both overexpression and knockdown approaches concurrently altered the expression of HMGB1 and XPA. 14-3-3σ, HMGB1, and XPA were preferentially expressed in cisplatin-resistant SLMT-1 cells when compared to those more sensitive to cisplatin. In ESCC patients with poor response to cisplatin-based chemoradiation, their pre-treatment tumors expressed higher expression of HMGB1 than those with response to such treatment. In summary, our results demonstrate that 14-3-3σ induces cisplatin resistance in ESCC cells and that 14-3-3σ-mediated cisplatin resistance involves DNA repair molecules HMGB1 and XPA. Results from this study provide evidences for further work in researching the potential use of 14-3-3σ and DNA repair molecules HMGB1 and XPA as biomarkers and therapeutic targets for ESCC.

  2. 14-3-3ζ coordinates adipogenesis of visceral fat

    PubMed Central

    Lim, Gareth E.; Albrecht, Tobias; Piske, Micah; Sarai, Karnjit; Lee, Jason T. C; Ramshaw, Hayley S.; Sinha, Sunita; Guthridge, Mark A.; Acker-Palmer, Amparo; Lopez, Angel F.; Clee, Susanne M.; Nislow, Corey; Johnson, James D.

    2015-01-01

    The proteins that coordinate complex adipogenic transcriptional networks are poorly understood. 14-3-3ζ is a molecular adaptor protein that regulates insulin signalling and transcription factor networks. Here we report that 14-3-3ζ-knockout mice are strikingly lean from birth with specific reductions in visceral fat depots. Conversely, transgenic 14-3-3ζ overexpression potentiates obesity, without exacerbating metabolic complications. Only the 14-3-3ζ isoform is essential for adipogenesis based on isoform-specific RNAi. Mechanistic studies show that 14-3-3ζ depletion promotes autophagy-dependent degradation of C/EBP-δ, preventing induction of the master adipogenic factors, Pparγ and C/EBP-α. Transcriptomic data indicate that 14-3-3ζ acts upstream of hedgehog signalling-dependent upregulation of Cdkn1b/p27Kip1. Indeed, concomitant knockdown of p27Kip1 or Gli3 rescues the early block in adipogenesis induced by 14-3-3ζ knockdown in vitro. Adipocyte precursors in 14-3-3ζKO embryos also appear to have greater Gli3 and p27Kip1 abundance. Together, our in vivo and in vitro findings demonstrate that 14-3-3ζ is a critical upstream driver of adipogenesis. PMID:26220403

  3. A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

    PubMed Central

    Andersen, Joshua L.; Thompson, J. Will; Lindblom, Kelly R.; Johnson, Erika S.; Yang, Chih-Sheng; Lilley, Lauren R.; Freel, Christopher D.; Moseley, M. Arthur; Kornbluth, Sally

    2011-01-01

    While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation. PMID:21884983

  4. Interaction of Ubinuclein-1, a nuclear and adhesion junction protein, with the 14-3-3 epsilon protein in epithelial cells: implication of the PKA pathway.

    PubMed

    Conti, Audrey; Sueur, Charlotte; Lupo, Julien; Brazzolotto, Xavier; Burmeister, Wim P; Manet, Evelyne; Gruffat, Henri; Morand, Patrice; Boyer, Véronique

    2013-03-01

    Ubinuclein-1 is a NACos (Nuclear and Adhesion junction Complex components) protein which shuttles between the nucleus and tight junctions, but its function in the latter is not understood. Here, by co-immunoprecipitation and confocal analysis, we show that Ubinuclein-1 interacts with the 14-3-3ɛ protein both in HT29 colon cells, and AGS gastric cells. This interaction is mediated by an Ubinuclein-1 phosphoserine motif. We show that the arginine residues (R56, R60 and R132) which form the 14-3-3ɛ ligand binding site are responsible for the binding of 14-3-3ɛ to phosphorylated Ubinuclein-1. Furthermore, we demonstrate that in vitro Ubinuclein-1 can be directly phosphorylated by cAMP-dependent protein kinase A. This in vitro phosphorylation allows binding of wildtype 14-3-3ɛ. Moreover, treatment of the cells with inhibitors of the cAMP-dependent protein kinase, KT5720 or H89, modifies the subcellular localization of Ubinuclein-1. Indeed, KT5720 and H89 greatly increase the staining of Ubinuclein-1 at the tight junctions in AGS gastric cells. In the presence of the kinase inhibitor KT5720, the amount of Ubinuclein-1 in the NP40 insoluble fraction is increased, together with actin. Moreover, treatment of the cells with KT5720 or H89 induces the concentration of Ubinuclein-1 at tricellular intersections of MDCK cells. Taken together, our findings demonstrate novel cell signaling trafficking by Ubinuclein-1 via association with 14-3-3ɛ following Ubinuclein-1 phosphorylation by the cAMP-dependent protein kinase-A.

  5. Nuclear localization and interaction of RolB with plant 14-3-3 proteins correlates with induction of adventitious roots by the oncogene rolB.

    PubMed

    Moriuchi, Hiroshi; Okamoto, Chiho; Nishihama, Ryuichi; Yamashita, Ichiro; Machida, Yasunori; Tanaka, Nobukazu

    2004-04-01

    The rooting-locus gene B (rolB) on the T-DNA of the root-inducing (Ri) plasmid in Agrobacterium rhizogenes is responsible for the induction of transformed adventitious roots, although the root induction mechanism is unknown. We report here that the RolB protein of pRi1724 (1724RolB) is associated with Nicotianatabacum14-3-3-like protein omegaII (Nt14-3-3 omegaII) in tobacco bright yellow (BY)-2 cells. Nt14-3-3 omegaII directly interacts with 1724RolB protein. Green fluorescent protein (GFP)-fused 1724RolB is localized to the nucleus. GFP-fused mutant 1724RolB proteins having a deletion or amino acid substitution are unable to interact with Nt14-3-3 omegaII and also show impaired nuclear localization. Moreover, these 1724RolB mutants show decreased capacity for adventitious root induction. These results suggest that adventitious root induction by 1724RolB protein correlates with its interaction with Nt14-3-3 omegaII and the nuclear localization of 1724RolB protein. PMID:15078329

  6. Genetic variations of 14-3-3E1 isoform in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly conserved family of 14-3-3 proteins functions in the regulation of a wide variety of cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. Several studies have observed diffe...

  7. Identification of a functional splice variant of 14-3-3E1 in rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The 14-3-3 proteins are a family of regulatory proteins involved in diverse cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. In this study, we report the identification and charact...

  8. 14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation.

    PubMed

    Dar, Ashraf; Wu, David; Lee, Nicholas; Shibata, Etsuko; Dutta, Anindya

    2014-11-01

    Cdt2 is the substrate recognition adaptor of CRL4(Cdt2) E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCF(FbxO11)-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4(Cdt2) substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression.

  9. 14-3-3 Proteins Play a Role in the Cell Cycle by Shielding Cdt2 from Ubiquitin-Mediated Degradation

    PubMed Central

    Dar, Ashraf; Wu, David; Lee, Nicholas; Shibata, Etsuko

    2014-01-01

    Cdt2 is the substrate recognition adaptor of CRL4Cdt2 E3 ubiquitin ligase complex and plays a pivotal role in the cell cycle by mediating the proteasomal degradation of Cdt1 (DNA replication licensing factor), p21 (cyclin-dependent kinase [CDK] inhibitor), and Set8 (histone methyltransferase) in S phase. Cdt2 itself is attenuated by SCFFbxO11-mediated proteasomal degradation. Here, we report that 14-3-3 adaptor proteins interact with Cdt2 phosphorylated at threonine 464 (T464) and shield it from polyubiquitination and consequent proteasomal degradation. Depletion of 14-3-3 proteins promotes the interaction of FbxO11 with Cdt2. Overexpressing 14-3-3 proteins shields Cdt2 that has a phospho-mimicking mutation (T464D [change of T to D at position 464]) but not Cdt2(T464A) from ubiquitination. Furthermore, the delay of the cell cycle in the G2/M phase and decrease in cell proliferation seen upon depletion of 14-3-3γ is partly due to the accumulation of the CRL4Cdt2 substrate, Set8 methyltransferase. Therefore, the stabilization of Cdt2 is an important function of 14-3-3 proteins in cell cycle progression. PMID:25154416

  10. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization. PMID:25748451

  11. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  12. Protein Kinase CK2 Interacts at the Neuromuscular Synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 Proteins and Phosphorylates the Latter Two*

    PubMed Central

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-01-01

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800–1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction. PMID:26198629

  13. Protein kinase CK2 interacts at the neuromuscular synapse with Rapsyn, Rac1, 14-3-3γ, and Dok-7 proteins and phosphorylates the latter two.

    PubMed

    Herrmann, Dustin; Straubinger, Marion; Hashemolhosseini, Said

    2015-09-11

    Previously, we demonstrated that the protein kinase CK2 associates with and phosphorylates the receptor tyrosine kinase MuSK (muscle specific receptor tyrosine kinase) at the neuromuscular junction (NMJ), thereby preventing fragmentation of the NMJs (Cheusova, T., Khan, M. A., Schubert, S. W., Gavin, A. C., Buchou, T., Jacob, G., Sticht, H., Allende, J., Boldyreff, B., Brenner, H. R., and Hashemolhosseini, S. (2006) Genes Dev. 20, 1800-1816). Here, we asked whether CK2 interacts with other proteins involved in processes at the NMJ, which would be consistent with the previous observation that CK2 appears enriched at the NMJ. We identified the following proteins to interact with protein kinase CK2: (a) the α and β subunits of the nicotinic acetylcholine receptors with weak interaction, (b) dishevelled (Dsh), and (c) another four proteins, Rapsyn, Rac1, 14-3-3γ, and Dok-7, with strong interaction. CK2 phosphorylated 14-3-3γ at serine residue 235 and Dok-7 at several serine residues but does not phosphorylate Rapsyn or Rac1. Furthermore, phosphomimetic Dok-7 mutants aggregated nicotinic acetylcholine receptors in C2C12 myotubes with significantly higher frequency than wild type Dok-7. Additionally, we mapped the interacting epitopes of all four binding partners to CK2 and thereby gained insights into the potential role of the CK2/Rapsyn interaction.

  14. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

    PubMed

    Silva, Julhiany de Fátima da; Vicentim, Juliana; Oliveira, Haroldo Cesar de; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; Silva, Juliana Leal Monteiro da; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-06-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  15. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

    PubMed Central

    da Silva, Julhiany de Fátima; Vicentim, Juliana; de Oliveira, Haroldo Cesar; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; da Silva, Juliana Leal Monteiro; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-01-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis. PMID:26038961

  16. 14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation1[W][OA

    PubMed Central

    Radwan, Osman; Wu, Xia; Govindarajulu, Manjula; Libault, Marc; Neece, David J.; Oh, Man-Ho; Berg, R. Howard; Stacey, Gary; Taylor, Christopher G.; Huber, Steven C.; Clough, Steven J.

    2012-01-01

    The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules. PMID:23060368

  17. A rare case of rapidly progressive dementia with elevated RT-QuIC and negative 14-3-3 and tau proteins.

    PubMed

    Trikamji, Bhavesh; Hamlin, Clive; Baldwin, Kelly J

    2016-05-01

    Creutzfeldt-Jakob disease (CJD) is characterized by rapidly progressing dementia with death usually occurring within 6 months. There is no verified disease-specific pre-mortem diagnostic test besides brain biopsy. We describe a 66 y old previously high functioning male who presented with a 5 month history of rapidly progressive dementia. Neurological examination revealed a score of 19/30 on MOCA testing. An extensive workup into various causes of dementia including electroencephalography and imaging studies was unremarkable. The cerebrospinal fluid was sent to National Prion Disease Center and it revealed elevated RT-QuIC levels with negative 14-3-3 and T tau proteins. Based on literature review, our case is one of few living subjects with elevated RT-QuIC levels and negative 14-3-3 and tau proteins. PMID:27249661

  18. An Analysis of CAF-1-interacting Proteins Reveals Dynamic and Direct Interactions with the KU Complex and 14-3-3 Proteins*

    PubMed Central

    Hoek, Maarten; Myers, Michael P.; Stillman, Bruce

    2011-01-01

    CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly. PMID:21209461

  19. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-01-01

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country. PMID:27622622

  20. Positive 14-3-3 and tau proteins in a sporadic Creutzfeldt-Jakob disease case and a brief perspective of prion diseases in Colombia.

    PubMed

    Escandón-Vargas, Kevin; Zorrilla-Vaca, Andrés; Corral-Prado, Raúl Heli

    2016-02-24

    Prion diseases are rare neurodegenerative disorders occurring worldwide and affecting both humans and animals. Herein, we present the case of a patient diagnosed with definite sporadic Creutzfeldt-Jakob disease in Cali, Colombia. Besides neurological examination, 14-3-3 and tau proteins were valuable tools supporting the diagnosis. We also present a brief perspective of the prion diseases reported in Colombia to date. Although the incidence of prion diseases is unknown in Colombia, our literature review revealed that one case of scrapie in 1981 and 29 human sporadic cases of Creutzfeldt-Jakob disease have been documented and published in our country.

  1. Echinococcus multilocularis laminated-layer components and the E14t 14-3-3 recombinant protein decrease NO production by activated rat macrophages in vitro.

    PubMed

    Andrade, M Amparo; Siles-Lucas, Mar; Espinoza, Elsa; Pérez Arellano, José Luis; Gottstein, Bruno; Muro, Antonio

    2004-05-01

    Echinococcus multilocularis and Echinococcus granulosus cause alveolar and cystic (unilocular) echinococcosis, respectively, in humans and animals. It is known that these parasites can affect, among other molecules, nitric oxide (NO) production by periparasitic host cells. Nevertheless, detailed dissection of parasite components specifically affecting cell NO production has not been done to date. We compare the effect of E. granulosus and E. multilocularis defined metacestode structural (laminated-layer associated) and metabolic (14-3-3 protein, potentially related with E. multilocularis metacestode tumor-like growth) components on the NO production by rat alveolar macrophages in vitro. Our results showed that none of these antigens could stimulate macrophage NO production in vitro. However, a reversed effect of some Echinococcus antigens on NO in vitro production was found when cells were previously exposed to LPS stimulation. This inhibitory effect was found when E. multilocularis laminated-layer (LL) or cyst wall (CW) soluble components from both species were used. Pre-stimulation of cells with LPS also resulted in a strong, dose-dependent reduction of NO and iNOS mRNA production after incubation of cells with the E14t protein. Thus, the E. multilocularis 14-3-3 protein appears to be one of the components accounting for the suppressive effect of the CW and LL metacestode extracts.

  2. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  3. Molecular Modeling of Differentially Phosphorylated Serine 10 and Acetylated lysine 9/14 of Histone H3 Regulates their Interactions with 14-3-3ζ, MSK1, and MKP1

    PubMed Central

    Sharma, Ajit K.; Mansukh, Abhilasha; Varma, Ashok; Gadewal, Nikhil; Gupta, Sanjay

    2013-01-01

    Histone modifications occur in precise patterns, with several modifications known to affect the binding of proteins. These interactions affect the chromatin structure, gene regulation, and cell cycle events. The dual modifications on the H3 tail, serine10 phosphorylation, and lysine14 acetylation (H3Ser10PLys14Ac) are reported to be crucial for interaction with 14-3-3ζ. However, the mechanism by which H3Ser10P along with neighboring site-specific acetylation(s) is targeted by its regulatory proteins, including kinase and phosphatase, is not fully understood. We carried out molecular modeling studies to understand the interaction of 14-3-3ζ, and its regulatory proteins, mitogen-activated protein kinase phosphatase-1 (MKP1), and mitogen- and stress-activated protein kinase-1 (MSK1) with phosphorylated H3Ser10 alone or in combination with acetylated H3Lys9 and Lys14. In silico molecular association studies suggested that acetylated Lys14 and phosphorylated Ser10 of H3 shows the highest binding affinity towards 14-3-3ζ. In addition, acetylation of H3Lys9 along with Ser10PLys14Ac favors the interaction of the phosphatase, MKP1, for dephosphorylation of H3Ser10P. Further, MAP kinase, MSK1 phosphorylates the unmodified H3Ser10 containing N-terminal tail with maximum affinity compared to the N-terminal tail with H3Lys9AcLys14Ac. The data clearly suggest that opposing enzymatic activity of MSK1 and MKP1 corroborates with non-acetylated and acetylated, H3Lys9Lys14, respectively. Our in silico data highlights that site-specific phosphorylation (H3Ser10P) and acetylation (H3Lys9 and H3Lys14) of H3 are essential for the interaction with their regulatory proteins (MKP1, MSK1, and 14-3-3ζ) and plays a major role in the regulation of chromatin structure. PMID:24027420

  4. The Tomato 14-3-3 protein TFT4 modulates H+ efflux, basipetal auxin transport, and the PKS5-J3 pathway in the root growth response to alkaline stress.

    PubMed

    Xu, Weifeng; Jia, Liguo; Shi, Weiming; Baluska, Frantisek; Kronzucker, Herbert J; Liang, Jiansheng; Zhang, Jianhua

    2013-12-01

    Alkaline stress is a common environmental stress, in particular in salinized soils. Plant roots respond to a variety of soil stresses by regulating their growth, but the nature of the regulatory pathways engaged in the alkaline stress response (ASR) is not yet understood. Previous studies show that PIN-FORMED2, an auxin (indole-3-acetic acid [IAA]) efflux transporter, PKS5, a protein kinase, and DNAJ HOMOLOG3 (J3), a chaperone, play key roles in root H(+) secretion by regulating plasma membrane (PM) H(+)-ATPases directly or by targeting 14-3-3 proteins. Here, we investigated the expression of all 14-3-3 gene family members (TOMATO 14-3-3 PROTEIN1 [TFT1]-TFT12) in tomato (Solanum lycopersicum) under ASR, showing the involvement of four of them, TFT1, TFT4, TFT6, and TFT7. When these genes were separately introduced into Arabidopsis (Arabidopsis thaliana) and overexpressed, only the growth of TFT4 overexpressors was significantly enhanced when compared with the wild type under stress. H(+) efflux and the activity of PM H(+)-ATPase were significantly enhanced in the root tips of TFT4 overexpressors. Microarray analysis and pharmacological examination of the overexpressor and mutant plants revealed that overexpression of TFT4 maintains primary root elongation by modulating PM H(+)-ATPase-mediated H(+) efflux and basipetal IAA transport in root tips under alkaline stress. TFT4 further plays important roles in the PKS5-J3 signaling pathway. Our study demonstrates that TFT4 acts as a regulator in the integration of H(+) efflux, basipetal IAA transport, and the PKS5-J3 pathway in the ASR of roots and coordinates root apex responses to alkaline stress for the maintenance of primary root elongation. PMID:24134886

  5. Exon B of human surfactant protein A2 mRNA, alone or within its surrounding sequences, interacts with 14-3-3; role of cis-elements and secondary structure

    PubMed Central

    Noutsios, Georgios T.; Silveyra, Patricia; Bhatti, Faizah

    2013-01-01

    Human surfactant protein A, an innate immunity molecule, is encoded by two genes: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5′ untranslated (5′UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis-regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441 proteins to wild-type eB, eB mutant, AD, and ABD 5′UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found that 1) proteins bind eB mRNA in a sequence-specific manner, with two cis-elements identified within eB to be important; 2) eB secondary structure is necessary for binding; 3) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; 4) other ribosomal and cytoskeletal proteins, and translation factors, are also present in the eB mRNA-protein complex; 5) knockdown of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis-elements within eB of hSP-A2 mRNA in a sequence- and secondary structure-specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA. PMID:23525782

  6. Spatial coordination of chloroplast and plasma membrane activities in Chara cells and its disruption through inactivation of 14-3-3 proteins.

    PubMed

    Bulychev, A A; van den Wijngaard, P W J; de Boer, A H

    2005-01-01

    In Chara corallina cells exposed to continuous light, external pH (pH(o)) and photosystem II (PSII) photochemical yield show correlated banding patterns. Photosynthetic activity is low in cell regions producing alkaline zones and high in the acid regions. We addressed the question whether (and how) photosynthetic activity and plasma membrane (PM) H+-pumping and H+-conductance are coupled in the different bands. First, PM H+-pump activity was stimulated with fusicoccin. This resulted in a more acidic pH in the acid bands without disturbing the correlation of photosynthetic electron transport and H+ fluxes across the PM. Next, H+-pump activity was reduced through microinjection of a phosphorylated peptide matching the canonical 14-3-3 binding motif RSTpSTP in the acid cell region. Microinjection induced a rapid (~5 min) rise in pH(o) by ca. 1.0 unit near the injection site, whereas the injection of the non-phosphorylated peptide had no effect. This pH rise confirms the supposed inhibition of the H+-pump upon the detachment of 14-3-3 proteins from the H+-ATPase. However, the PSII yield in the cell regions corresponding to the new alkaline peak remained high, which violated the normal inverse relations between the pH(o) and PSII photochemical yield. We conclude that the injection of the competitive inhibitor of the H+-ATPase disrupts the balanced operation of PM H+-transport and photosynthetic electron flow and promotes electron flow through alternative pathways.

  7. 14-3-3 in Thoracic Aortic Aneurysms

    PubMed Central

    Chakravarti, Ritu; Gupta, Karishma; Swain, Mamuni; Willard, Belinda; Scholtz, Jaclyn; Svensson, Lars G.; Roselli, Eric E.; Pettersson, Gosta; Johnston, Douglas R.; Soltesz, Edward G.; Yamashita, Michifumi; Stuehr, Dennis; Daly, Thomas M.; Hoffman, Gary S.

    2015-01-01

    Objective Large vessel vasculitides (LVV) are a group of autoimmune diseases characterized by injury to and anatomic modifications of large vessels, including the aorta and its branch vessels. Disease etiology is unknown. This study was undertaken to identify antigen targets within affected vessel walls in aortic root, ascending aorta, and aortic arch surgical specimens from patients with LVV, including giant cell arteritis, Takayasu arteritis, and isolated focal aortitis. Methods Thoracic aortic aneurysm specimens and autologous blood were acquired from consenting patients who underwent aorta reconstruction procedures. Aorta proteins were extracted from both patients with LVV and age-, race-, and sex-matched disease controls with noninflammatory aneurysms. A total of 108 serum samples from patients with LVV, matched controls, and controls with antinuclear antibodies, different forms of vasculitis, or sepsis were tested. Results Evaluation of 108 serum samples and 22 aortic tissue specimens showed that 78% of patients with LVV produced antibodies to 14-3-3 proteins in the aortic wall (93.7% specificity), whereas controls were less likely to do so (6.7% produced antibodies). LVV patient sera contained autoantibody sufficient to immunoprecipitate 14-3-3 protein(s) from aortic lysates. Three of 7 isoforms of 14-3-3 were found to be up-regulated in aorta specimens from patients with LVV, and 2 isoforms (ε and ζ) were found to be antigenic in LVV. Conclusion This is the first study to use sterile, snap-frozen thoracic aorta biopsy specimens to identify autoantigens in LVV. Our findings indicate that 78% of patients with LVV have antibody reactivity to 14-3-3 protein(s). The precise role of these antibodies and 14-3-3 proteins in LVV pathogenesis deserves further study. PMID:25917817

  8. Identification and expression analysis of four 14-3-3 genes during fruit ripening in banana (Musa acuminata L. AAA group, cv. Brazilian).

    PubMed

    Li, Mei-Ying; Xu, Bi-Yu; Liu, Ju-Hua; Yang, Xiao-Liang; Zhang, Jian-Bin; Jia, Cai-Hong; Ren, Li-Cheng; Jin, Zhi-Qiang

    2012-02-01

    To investigate the regulation of 14-3-3 proteins in banana (Musa acuminata L. AAA group, cv. Brazilian) fruit postharvest ripening, four cDNAs encoding 14-3-3 proteins were isolated from banana and designated as Ma-14-3-3a, Ma-14-3-3c, Ma-14-3-3e, and Ma-14-3-3i, respectively. Amino acid sequence alignment showed that the four 14-3-3 proteins shared a highly conserved core structure and variable C-terminal as well as N-terminal regions with 14-3-3 proteins from other plant species. Phylogenetic analysis revealed that the four 14-3-3 genes belong to the non-ε groups. They were differentially and specifically expressed in various tissues. Real-time RT-PCR analysis indicated that these four genes function differentially during banana fruit postharvest ripening. Three genes, Ma-14-3-3a, Ma-14-3-3c, and Ma-14-3-3e, were significantly induced by exogenous ethylene treatment. However, gene function differed in naturally ripened fruits. Ethylene could induce Ma-14-3-3c expression during postharvest ripening, but expression patterns of Ma-14-3-3a and Ma-14-3-3e suggest that these two genes appear to be involved in regulating ethylene biosynthesis during fruit ripening. No obvious relationship emerged between Ma-14-3-3i expression in naturally ripened and 1-MCP (1-methylcyclopropene)-treated fruit groups during fruit ripening. These results indicate that the 14-3-3 proteins might be involved in various regulatory processes of banana fruit ripening. Further studies will mainly focus on revealing the detailed biological mechanisms of these four 14-3-3 genes in regulating banana fruit postharvest ripening. PMID:22009053

  9. Interactome analysis of the six cotton 14-3-3s that are preferentially expressed in fibres and involved in cell elongation

    PubMed Central

    Zhang, Ze-Ting; Zhou, Ying; Li, Yang; Shao, Su-Qiang; Li, Bing-Ying; Shi, Hai-Yan; Li, Xue-Bao

    2010-01-01

    Proteins of the 14-3-3 family regulate a divergent set of signalling pathways in all eukaryotic organisms. In this study, several cDNAs encoding 14-3-3 proteins were isolated from a cotton fibre cDNA library. The Gh14-3-3 genes share high sequence homology at the nucleotide level in the coding region and at the amino acid level. Real-time quantitative RT-PCR analysis indicated that the expression of these Gh14-3-3 genes is developmentally regulated in fibres, and reached their peak at the stage of rapid cell elongation of fibre development. Furthermore, overexpression of Gh14-3-3a, Gh14-3-3e, and Gh14-3-3L in fission yeast promoted atypical longitudinal growth of the host cells. Yeast two-hybrid analysis revealed that the interaction between cotton 14-3-3 proteins is isoform selective. Through yeast two-hybrid screening, 38 novel interaction partners of the six 14-3-3 proteins (Gh14-3-3a, Gh14-3-3e, Gh14-3-3f, Gh14-3-3g, Gh14-3-3h, and Gh14-3-3L), which are involved in plant development, metabolism, signalling transduction, and other cellular processes, were identified in cotton fibres. Taking these data together, it is proposed that the Gh14-3-3 proteins may participate in regulation of fibre cell elongation. Thus, the results of this study provide novel insights into the 14-3-3 signalling related to fibre development of cotton. PMID:20519337

  10. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome

    PubMed Central

    Tinti, Michele; Madeira, Fábio; Murugesan, Gavuthami; Hoxhaj, Gerta; Toth, Rachel; MacKintosh, Carol

    2014-01-01

    The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate ‘lynchpins’, which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the ‘lynchpin’ site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology. Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py PMID:24501395

  11. ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome.

    PubMed

    Tinti, Michele; Madeira, Fábio; Murugesan, Gavuthami; Hoxhaj, Gerta; Toth, Rachel; Mackintosh, Carol

    2014-01-01

    The dimeric 14-3-3 proteins dock onto pairs of phosphorylated Ser and Thr residues on hundreds of proteins, and thereby regulate many events in mammalian cells. To facilitate global analyses of these interactions, we developed a web resource named ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome, which integrates multiple data sets on 14-3-3-binding phosphoproteins. ANIA also pinpoints candidate 14-3-3-binding phosphosites using predictor algorithms, assisted by our recent discovery that the human 14-3-3-interactome is highly enriched in 2R-ohnologues. 2R-ohnologues are proteins in families of two to four, generated by two rounds of whole genome duplication at the origin of the vertebrate animals. ANIA identifies candidate 'lynchpins', which are 14-3-3-binding phosphosites that are conserved across members of a given 2R-ohnologue protein family. Other features of ANIA include a link to the catalogue of somatic mutations in cancer database to find cancer polymorphisms that map to 14-3-3-binding phosphosites, which would be expected to interfere with 14-3-3 interactions. We used ANIA to map known and candidate 14-3-3-binding enzymes within the 2R-ohnologue complement of the human kinome. Our projections indicate that 14-3-3s dock onto many more human kinases than has been realized. Guided by ANIA, PAK4, 6 and 7 (p21-activated kinases 4, 6 and 7) were experimentally validated as a 2R-ohnologue family of 14-3-3-binding phosphoproteins. PAK4 binding to 14-3-3 is stimulated by phorbol ester, and involves the 'lynchpin' site phosphoSer99 and a major contribution from Ser181. In contrast, PAK6 and PAK7 display strong phorbol ester-independent binding to 14-3-3, with Ser113 critical for the interaction with PAK6. These data point to differential 14-3-3 regulation of PAKs in control of cell morphology. Database URL: https://ania-1433.lifesci.dundee.ac.uk/prediction/webserver/index.py.

  12. Genome-Wide Identification, Phylogeny, and Expression Analyses of the 14-3-3 Family Reveal Their Involvement in the Development, Ripening, and Abiotic Stress Response in Banana

    PubMed Central

    Li, Meiying; Ren, Licheng; Xu, Biyu; Yang, Xiaoliang; Xia, Qiyu; He, Pingping; Xiao, Susheng; Guo, Anping; Hu, Wei; Jin, Zhiqiang

    2016-01-01

    Plant 14-3-3 proteins act as critical components of various cellular signaling processes and play an important role in regulating multiple physiological processes. However, less information is known about the 14-3-3 gene family in banana. In this study, 25 14-3-3 genes were identified from the banana genome. Based on the evolutionary analysis, banana 14-3-3 proteins were clustered into ε and non-ε groups. Conserved motif analysis showed that all identified banana 14-3-3 genes had the typical 14-3-3 motif. The gene structure of banana 14-3-3 genes showed distinct class-specific divergence between the ε group and the non-ε group. Most banana 14-3-3 genes showed strong transcript accumulation changes during fruit development and postharvest ripening in two banana varieties, indicating that they might be involved in regulating fruit development and ripening. Moreover, some 14-3-3 genes also showed great changes after osmotic, cold, and salt treatments in two banana varieties, suggested their potential role in regulating banana response to abiotic stress. Taken together, this systemic analysis reveals the involvement of banana 14-3-3 genes in fruit development, postharvest ripening, and response to abiotic stress and provides useful information for understanding the functions of 14-3-3 genes in banana. PMID:27713761

  13. Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14-3-3 Proteins.

    PubMed

    Xiao, Yihong; Wu, Weining; Gao, Jiming; Smith, Nikki; Burkard, Christine; Xia, Dong; Zhang, Minxia; Wang, Chengbao; Archibald, Alan; Digard, Paul; Zhou, En-Min; Hiscox, Julian A

    2016-05-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins. PMID:26709850

  14. Dynamic interaction between 14-3-3zeta and bax during TNF-α-induced apoptosis in living cells

    NASA Astrophysics Data System (ADS)

    Gao, Xuejuan; Xing, Da; Chen, Tongsheng

    2006-09-01

    Bax, a proapoptotic member of the Bcl-2 family, localizes largely in the cytoplasm but redistributes to mitochondria and undergoes oligomerization to induce the release of apoptogenic factors such as cytochrome c in response to apoptotic stimuli. Cytoplasmic protein 14-3-3zeta binds to Bax and, upon apoptotic stimulation, releases Bax by a caspase-independent mechanism. However, the direct interaction of the cytoplasmic 14-3-3zeta and Bax in living cells has not been observed. In present study, to monitor the dynamic interaction between 14-3-3zeta and Bax in living cells in real time during apoptosis induced by tumor necrosis factor (TNF-α), DsRed-14-3-3zeta plasmid is constructed. By cotransfecting DsRed- 14-3-3zeta and GFP-Bax plasmids into human lung adenocarcinoma cells (ASTC-a-1), we observe the dynamic interaction between Bax and 14-3-3zeta using fluorescence resonance energy transfer (FRET) technique on laser scanning confocal microscope. The results show that 14-3-3zeta remains in the cytoplasm but GFP-Bax translocates to mitochondria completely after TNF-α stimulation. These results reveal that 14-3-3zeta binds directly to Bax in healthy cells, and that 14-3-3zeta negatively regulates Bax translocation to mitochondria during TNF-α-induced apoptosis.

  15. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program.

    PubMed

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD.

  16. Characteristics of Korean patients with suspected Creutzfeldt-Jakob disease with 14-3-3 protein in cerebrospinal fluid: Preliminary study of the Korean Creutzfeldt-Jakob disease active surveillance program

    PubMed Central

    Lim, Jae-Sung; Kwon, Hyung-Min; Jang, Jae-Won; Ju, Young-Ran; Kim, SuYeon; Park, Young Ho; Park, So Young; Kim, SangYun

    2015-01-01

    Abstract Although Korea had a national surveillance system for Creutzfeldt-Jakob disease (CJD), it was mainly dependent on attending physician's reports. Thus, little prospective data about the epidemiology, characteristics, and final diagnoses of suspected patients were available. We have established a nationwide network for the active surveillance of patients with suspected CJD. When the requested cerebrospinal fluid (CSF) samples tested positive for 14-3-3 protein, we investigated the clinical characteristics of the corresponding patients and followed them until their final diagnoses were confirmed. A total of 218 samples were requested for CSF assays from May 2010 to August 2012, and 106 (48.6%) were positive for 14-3-3 protein. In 89 patients with complete clinical data, 38 (42.7%) were diagnosed with probable CJD and the estimated annual occurrence of CJD was 16.3 persons-per-year. The most common diagnoses of the remainder were central nervous system infection and any-cause encephalopathy. Non-CJD subjects showed worse initial consciousness levels than CJD patients. This preliminary study showed that the number of reported cases of CJD and the true positivity rates of CSF 14-3-3 protein assays were both low in Korea. An active surveillance system is urgently needed to provide the latest nationwide epidemiological data of CJD. PMID:25996401

  17. Visualization and Biochemical Analyses of the Emerging Mammalian 14-3-3-Phosphoproteome*

    PubMed Central

    Johnson, Catherine; Tinti, Michele; Wood, Nicola T.; Campbell, David G.; Toth, Rachel; Dubois, Fanny; Geraghty, Kathryn M.; Wong, Barry H. C.; Brown, Laura J.; Tyler, Jennifer; Gernez, Aurélie; Chen, Shuai; Synowsky, Silvia; MacKintosh, Carol

    2011-01-01

    Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease. PMID:21725060

  18. 14-3-3 fusion oncogenes in high-grade endometrial stromal sarcoma

    PubMed Central

    Lee, Cheng-Han; Ou, Wen-Bin; Mariño-Enriquez, Adrian; Zhu, Meijun; Mayeda, Mark; Wang, Yuexiang; Guo, Xiangqian; Brunner, Alayne L.; Amant, Frédéric; French, Christopher A.; West, Robert B.; McAlpine, Jessica N.; Gilks, C. Blake; Yaffe, Michael B.; Prentice, Leah M.; McPherson, Andrew; Jones, Steven J. M.; Marra, Marco A.; Shah, Sohrab P.; van de Rijn, Matt; Huntsman, David G.; Dal Cin, Paola; Debiec-Rychter, Maria; Nucci, Marisa R.; Fletcher, Jonathan A.

    2012-01-01

    14-3-3 proteins are ubiquitously expressed regulators of various cellular functions, including proliferation, metabolism, and differentiation, and altered 14-3-3 expression is associated with development and progression of cancer. We report a transforming 14-3-3 oncoprotein, which we identified through conventional cytogenetics and whole-transcriptome sequencing analysis as a highly recurrent genetic mechanism in a clinically aggressive form of uterine sarcoma: high-grade endometrial stromal sarcoma (ESS). The 14-3-3 oncoprotein results from a t(10;17) genomic rearrangement, leading to fusion between 14-3-3ε (YWHAE) and either of two nearly identical FAM22 family members (FAM22A or FAM22B). Expression of YWHAE–FAM22 fusion oncoproteins was demonstrated by immunoblot in t(10;17)-bearing frozen tumor and cell line samples. YWHAE–FAM22 fusion gene knockdowns were performed with shRNAs and siRNAs targeting various FAM22A exons in an t(10;17)-bearing ESS cell line (ESS1): Fusion protein expression was inhibited, with corresponding reduction in cell growth and migration. YWHAE–FAM22 maintains a structurally and functionally intact 14-3-3ε (YWHAE) protein-binding domain, which is directed to the nucleus by a FAM22 nuclear localization sequence. In contrast to classic ESS, harboring JAZF1 genetic fusions, YWHAE–FAM22 ESS display high-grade histologic features, a distinct gene-expression profile, and a more aggressive clinical course. Fluorescence in situ hybridization analysis demonstrated absolute specificity of YWHAE–FAM22A/B genetic rearrangement for high-grade ESS, with no fusions detected in other uterine and nonuterine mesenchymal tumors (55 tumor types, n = 827). These discoveries reveal diagnostically and therapeutically relevant models for characterizing aberrant 14-3-3 oncogenic functions. PMID:22223660

  19. 14-3-3, an integrator of cell mechanics and cytokinesis.

    PubMed

    Robinson, Douglas N

    2010-11-01

    One of the goals of understanding cytokinesis is to uncover the molecular regulation of the cellular mechanical properties that drive cell shape change. Such regulatory pathways are likely to be used at multiple stages of a cell's life, but are highly featured during cell division. Recently, we demonstrated that 14-3-3 (encoded by a single gene in the social amoeba Dictyostelium discoideum) serves to integrate key cytoskeletal components-microtubules, Rac and myosin II-to control cell mechanics and cytokinesis. As 14-3-3 proteins are frequently altered in a variety of human tumors, we extend these observations to suggest possible additional roles for how 14-3-3 proteins may contribute to tumorigenesis. PMID:21686271

  20. Royal Jelly-Mediated Prolongevity and Stress Resistance in Caenorhabditis elegans Is Possibly Modulated by the Interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 Proteins.

    PubMed

    Wang, Xiaoxia; Cook, Lauren F; Grasso, Lindsay M; Cao, Min; Dong, Yuqing

    2015-07-01

    Recent studies suggest that royal jelly (RJ) and its related substances may have antiaging properties. However, the molecular mechanisms underlying the beneficial effects remain elusive. We report that the effects of RJ and enzyme-treated RJ (eRJ) on life span and health span in Caenorhabditis elegans (C elegans) are modulated by the sophisticated interplays of DAF-16, SIR-2.1, HCF-1, and 14-3-3 proteins. Dietary supplementation with RJ or eRJ increased C. elegans life span in a dose-dependent manner. The RJ and eRJ consumption increased the tolerance of C elegans to oxidative stress, ultraviolet irradiation, and heat shock stress. Our genetic analyses showed that RJ/eRJ-mediated life-span extension requires insulin/IGF-1 signaling and the activities of DAF-16, SIR-2.1, HCF-1, and FTT-2, a 14-3-3 protein. Earlier studies reported that DAF-16/FOXO, SIR-2.1/SIRT1, FTT-2, and HCF-1 have extensive interplays in worms and mammals. Our present findings suggest that RJ/eRJ-mediated promotion of longevity and stress resistance in C elegans is dependent on these conserved interplays. From an evolutionary point of view, this study not only provides new insights into the molecular mechanisms of RJ's action on health span promotion in C elegans, but also has imperative implications in using RJ/eRJ as nutraceuticals to delay aging and age-related disorders.

  1. Molecular and biochemical mining of heat-shock and 14-3-3 proteins in drug-induced protoscolices of Echinococcus granulosus and the detection of a candidate gene for anthelmintic resistance.

    PubMed

    Pan, D; Das, S; Bera, A K; Bandyopadhyay, S; Bandyopadhyay, S; De, S; Rana, T; Das, S K; Suryanaryana, V V; Deb, J; Bhattacharya, D

    2011-06-01

    Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus is a disease that affects both humans and animals. In humans the disease is treated by surgery with a supplementary option of chemotherapy with a benzimidazole compound. During the present study heat-shock protein 60 (HSP 60) was identified as one of the most frequently expressed biomolecules by E. granulosus after albendazole treatment. Data were correlated with 14-3-3 protein signature, and overexpression of this molecule after albendazole induction was an indicator of cell survival and signal transduction during in vitro maintenance of E. granulosus for up to 72 h. This observation was further correlated with a uniform expression pattern of a housekeeping gene (actin II). Out of three β-tubulin gene isoforms of E. granulosus, β-tubulin gene isoform 2 showed a conserved point mutation indicative of benzimidazole resistance.

  2. Akt Phosphorylates Connexin43 on Ser373, a “Mode-1” Binding Site for 14-3-3

    PubMed Central

    PARK, DARREN J.; WALLICK, CHRISTOPHER J.; MARTYN, KENDRA D.; LAU, ALAN F.; JIN, CHENGSHI; WARN-CRAMER, BONNIE J.

    2009-01-01

    Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques. PMID:18163231

  3. Genome-Wide Identification, Classification, and Expression Analysis of 14-3-3 Gene Family in Populus

    PubMed Central

    Tian, Fengxia; Wang, Tan; Xie, Yuli; Zhang, Jin; Hu, Jianjun

    2015-01-01

    Background In plants, 14-3-3 proteins are encoded by a large multigene family and are involved in signaling pathways to regulate plant development and protection from stress. Although twelve Populus 14-3-3s were identified based on the Populus trichocarpa genome V1.1 in a previous study, no systematic analysis including genome organization, gene structure, duplication relationship, evolutionary analysis and expression compendium has been conducted in Populus based on the latest P. trichocarpa genome V3.0. Principal Findings Here, a comprehensive analysis of Populus 14-3-3 family is presented. Two new 14-3-3 genes were identified based on the latest P. trichocarpa genome. In P. trichocarpa, fourteen 14-3-3 genes were grouped into ε and non-ε group. Exon-intron organizations of Populus 14-3-3s are highly conserved within the same group. Genomic organization analysis indicated that purifying selection plays a pivotal role in the retention and maintenance of Populus 14-3-3 family. Protein conformational analysis indicated that Populus 14-3-3 consists of a bundle of nine α-helices (α1-α9); the first four are essential for formation of the dimer, while α3, α5, α7, and α9 form a conserved peptide-binding groove. In addition, α1, α3, α5, α7, and α9 were evolving at a lower rate, while α2, α4, and α6 were evolving at a relatively faster rate. Microarray analyses showed that most Populus 14-3-3s are differentially expressed across tissues and upon exposure to various stresses. Conclusions The gene structures and their coding protein structures of Populus 14-3-3s are highly conserved among group members, suggesting that members of the same group might also have conserved functions. Microarray and qRT-PCR analyses showed that most Populus 14-3-3s were differentially expressed in various tissues and were induced by various stresses. Our investigation provided a better understanding of the complexity of the 14-3-3 gene family in poplars. PMID:25867623

  4. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    SciTech Connect

    Xin, Ying; Lu, Qingxian; Li, Qiutang

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  5. The role of 14-3-3{beta} in transcriptional activation of estrogen receptor {alpha} and its involvement in proliferation of breast cancer cells

    SciTech Connect

    Kim, Yoonseo; Kim, Hyungjin; Jang, Sung-Wuk; Ko, Jesang

    2011-10-14

    Highlights: {yields} 14-3-3{beta} interacts with ER{alpha} and the interaction is Akt-dependent. {yields} 14-3-3{beta} regulates the transcriptional activity of ER{alpha} in a ligand-dependent manner. {yields} 14-3-3{beta} increases expressions of ER{alpha} target genes. {yields} 14-3-3{beta} increases breast cancer cell proliferation. -- Abstract: The estrogen receptor (ER) functions as a transcription factor that mediates the effects of estrogen. ER{alpha}, which plays a crucial role in the development and progression of breast cancer, is activated by estrogen binding, leading to receptor phosphorylation, dimerization, and recruitment of co-activators and chaperons to the estrogen-bound receptor complex. The 14-3-3 proteins bind to target proteins via phosphorylation and influence many cellular events by altering their subcellular localization or acting as a chaperone. However, regulation of ER{alpha} expression and transactivation by the 14-3-3 proteins has not been reported. We demonstrate that 14-3-3{beta} functions as a positive regulator of ER{alpha} through a direct protein-protein interaction in an estrogen-dependent manner. Ectopic expression of 14-3-3{beta} stimulated ER{alpha}-mediated transcriptional activity in MCF-7 breast cancer cells. Enhanced ER{alpha} transcriptional activity due to 14-3-3{beta} increased the expressions of the endogenous ER{alpha} target genes, leading to proliferation of breast cancer cells. We suggest that 14-3-3{beta} has oncogenic potential in breast cancer via binding to ER{alpha} and activation of the transcriptional activity of ER{alpha}.

  6. 14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase

    SciTech Connect

    Mizuno, Emi; Kitamura, Naomi; Komada, Masayuki

    2007-10-01

    The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 {epsilon}, {gamma}, and {zeta} as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS{sup 680}SP, by phosphatase treatment, and by competition with the Ser{sup 680}-phosphorylated RSYS{sup 680}SP peptide. Metabolic labeling with [{sup 32}P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser{sup 680} is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser{sup 680} in a phosphorylation-dependent manner. The mutation at Ser{sup 680} led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3{epsilon} inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser{sup 680} and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.

  7. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  8. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics.

  9. 14-3-3 phosphoprotein interaction networks - does isoform diversity present functional interaction specification?

    PubMed

    Paul, Anna-Lisa; Denison, Fiona C; Schultz, Eric R; Zupanska, Agata K; Ferl, Robert J

    2012-01-01

    The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question - does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis. PMID:22934100

  10. Higher order Arabidopsis 14-3-3 mutants show 14-3-3 involvement in primary root growth both under control and abiotic stress conditions

    PubMed Central

    van Kleeff, P. J. M.; Jaspert, N.; Li, K. W.; Rauch, S.; Oecking, C.; de Boer, A. H.

    2014-01-01

    Arabidopsis 14-3-3 proteins are a family of conserved proteins that interact with numerous partner proteins in a phospho-specific manner, and can affect the target proteins in a number of ways; e.g. modification of enzymatic activity. We isolated T-DNA insertion lines in six 14-3-3 genes within the non-epsilon group that phylogenetically group in three closely related gene pairs. In total, 6 single, 3 double, 12 triple, and 3 quadruple mutants were generated. The mutants were phenotyped for primary root growth on control plates: single and double mutants were indistinguishable from WT, whereas six triples and all quadruples showed a shorter primary root. In addition, length of the first epidermal cell with a visible root hair bulge (LEH) was used to determine primary root elongation on medium containing mannitol and 1-aminocyclopropane-1-carboxylic acid (ACC). This analysis showed clear differences depending on the stress and 14-3-3 gene combinations. Next to the phenotypic growth analyses, a 14-3-3 pull-down assay on roots treated with and without mannitol showed that mannitol stress strongly affects the 14-3-3 interactome. In conclusion, we show gene specificity and functional redundancy among 14-3-3 proteins in primary root elongation under control and under abiotic stress conditions and changes in the 14-3-3 interactome during the onset of stress adaptation. PMID:25189593

  11. The Peripheral Binding of 14-3-3γ to Membranes Involves Isoform-Specific Histidine Residues

    PubMed Central

    Ying, Ming; Halskau, Øyvind; Baumann, Anne; Rodriguez-Larrea, David; Costas, Miguel; Underhaug, Jarl; Sanchez-Ruiz, Jose M.; Martinez, Aurora

    2012-01-01

    Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states. PMID:23189152

  12. Keratin 23, a novel DPC4/Smad4 target gene which binds 14-3-3ε

    PubMed Central

    2011-01-01

    Background Inactivating mutations of SMAD4 are frequent in metastatic colorectal carcinomas. In previous analyses, we were able to show that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas in vitro cell growth was not affected. Using this cellular model system, we searched for new Smad4 targets comparing nuclear subproteomes derived from Smad4 re-expressing and Smad4 negative SW480 cells. Methods High resolution two-dimensional (2D) gel electrophoresis was applied to identify novel Smad4 targets in the nuclear subproteome of Smad4 re-expressing SW480 cells. The identified candidate protein Keratin 23 was further characterized by tandem affinity purification. Immunoprecipitation, subfractionation and immunolocalization studies in combination with RNAi were used to validate the Keratin 23-14-3-3ε interaction. Results We identified keratins 8 and 18, heat shock proteins 60 and 70, plectin 1, as well as 14-3-3ε and γ as novel proteins present in the KRT23-interacting complex. Co-immunoprecipitation and subfractionation analyses as well as immunolocalization studies in our Smad4-SW480 model cells provided further evidence that KRT23 associates with 14-3-3ε and that Smad4 dependent KRT23 up-regulation induces a shift of the 14-3-3ε protein from a nuclear to a cytoplasmic localization. Conclusion Based on our findings we propose a new regulatory circuitry involving Smad4 dependent up-regulation of KRT23 (directly or indirectly) which in turn modulates the interaction between KRT23 and 14-3-3ε leading to a cytoplasmic sequestration of 14-3-3ε. This cytoplasmic KRT23-14-3-3 interaction may alter the functional status of the well described 14-3-3 scaffold protein, known to regulate key cellular processes, such as signal transduction, cell cycle control, and apoptosis and may thus be a previously unappreciated facet of the Smad4 tumor suppressive circuitry. PMID

  13. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa

    PubMed Central

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C.; Bisht, Naveen C.

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1–5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  14. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C; Bisht, Naveen C

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses.

  15. Class-Specific Evolution and Transcriptional Differentiation of 14-3-3 Family Members in Mesohexaploid Brassica rapa.

    PubMed

    Chandna, Ruby; Augustine, Rehna; Kanchupati, Praveena; Kumar, Roshan; Kumar, Pawan; Arya, Gulab C; Bisht, Naveen C

    2016-01-01

    14-3-3s are highly conserved, multigene family proteins that have been implicated in modulating various biological processes. The presence of inherent polyploidy and genome complexity has limited the identification and characterization of 14-3-3 proteins from globally important Brassica crops. Through data mining of Brassica rapa, the model Brassica genome, we identified 21 members encoding 14-3-3 proteins namely, BraA.GRF14.a to BraA.GRF14.u. Phylogenetic analysis indicated that B. rapa contains both ε (epsilon) and non-ε 14-3-3 isoforms, having distinct intron-exon structural organization patterns. The non-ε isoforms showed lower divergence rate (Ks < 0.45) compared to ε protein isoforms (Ks > 0.48), suggesting class-specific divergence pattern. Synteny analysis revealed that mesohexaploid B. rapa genome has retained 1-5 orthologs of each Arabidopsis 14-3-3 gene, interspersed across its three fragmented sub-genomes. qRT-PCR analysis showed that 14 of the 21 BraA.GRF14 were expressed, wherein a higher abundance of non-ε transcripts was observed compared to the ε genes, indicating class-specific transcriptional bias. The BraA.GRF14 genes showed distinct expression pattern during plant developmental stages and in response to abiotic stress, phytohormone treatments, and nutrient deprivation conditions. Together, the distinct expression pattern and differential regulation of BraA.GRF14 genes indicated the occurrence of functional divergence of B. rapa 14-3-3 proteins during plant development and stress responses. PMID:26858736

  16. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination.

    PubMed

    Lam, Tonika; Thomas, Lisa M; White, Clayton A; Li, Guideng; Pone, Egest J; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5'-AGCT-3' repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S-S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180-198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR.

  17. 14-3-3s are Potential Biomarkers for HIV-related Neurodegeneration

    PubMed Central

    Morales, Diana; Skoulakis, Efthimios M.; Acevedo, Summer F.

    2013-01-01

    Over the last decade, it has become evident that 14-3-3 proteins are essential for primary cell functions. These proteins are abundant throughout the body, including the central nervous system (CNS) and interact with other proteins in both cell cycle and apoptotic pathways. Examination of cerebral spinal fluid (CSF) in humans, suggest that 14-3-3s including 14-3-3ε (YWHAE), are upregulated in several neurological diseases and loss or duplication of the YWHAE gene leads to Miller-Dieker Syndrome (MDS). The goal of this review is to examine the utility of 14-3-3s as a marker of Human Immune deficiency virus (HIV)-dependent neurodegeneration, and also as a tool to track disease progression. To that end we describe mechanisms implicating 14-3-3s in neurological diseases and summarize evidence of its interactions with HIV accessory and co-receptor proteins. PMID:22811265

  18. Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205.

    PubMed

    Ganguly, Surajit; Weller, Joan L; Ho, Anthony; Chemineau, Philippe; Malpaux, Benoit; Klein, David C

    2005-01-25

    The nocturnal increase in circulating melatonin in vertebrates is regulated by the activity of arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the melatonin pathway (serotonin --> N-acetylserotonin --> melatonin). Large changes in activity are linked to cyclic AMP-dependent protein kinase-mediated phosphorylation of AANAT T31. Phosphorylation of T31 promotes binding of AANAT to the dimeric 14-3-3 protein, which activates AANAT by increasing arylalkylamine affinity. In the current study, a putative second AANAT cyclic AMP-dependent protein kinase phosphorylation site, S205, was found to be approximately 55% phosphorylated at night, when T31 is approximately 40% phosphorylated. These findings indicate that ovine AANAT is dual-phosphorylated. Moreover, light exposure at night decreases T31 and S205 phosphorylation, consistent with a regulatory role of both sites. AANAT peptides containing either T31 or S205 associate with 14-3-3zeta in a phosphorylation-dependent manner; binding through phosphorylated (p)T31 is stronger than that through pS205, consistent with the location of only pT31 in a mode I binding motif, one of two recognized high-affinity 14-3-3-binding motifs AANAT protein binds to 14-3-3zeta through pT31 or pS205. Two-site binding lowers the Km for arylalkylamine substrate to approximately 30 microM. In contrast, single-site pS205 binding increases the Km to approximately 1,200 microM. Accordingly, the switch from dual to single pS205 binding of AANAT to 14-3-3 changes the Km for substrates by approximately 40-fold. pS205 seems to be part of a previously unrecognized 14-3-3-binding motif-pS/pT (X1-2)-COOH, referred to here as mode III.

  19. Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

    SciTech Connect

    Han, Dingding; Ye, Guangming; Liu, Tingting; Chen, Cong; Yang, Xianmei; Wan, Bo; Pan, Yuanwang; Yu, Long

    2010-05-28

    14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1' insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal {alpha}-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.

  20. Clinical implication of 14-3-3 epsilon expression in gastric cancer

    PubMed Central

    Leal, Mariana Ferreira; Calcagno, Danielle Queiroz; Demachki, Sâmia; Assumpção, Paulo Pimentel; Chammas, Roger; Burbano, Rommel Rodríguez; Smith, Marília de Arruda Cardoso

    2012-01-01

    AIM: To evaluate for the first time the protein and mRNA expression of 14-3-3ε in gastric carcinogenesis. METHODS: 14-3-3ε protein expression was determined by western blotting, and mRNA expression was examined by real-time quantitative RT-PCR in gastric tumors and their matched non-neoplastic gastric tissue samples. RESULTS: Authors observed a significant reduction of 14-3-3ε protein expression in gastric cancer (GC) samples compared to their matched non-neoplastic tissue. Reduced levels of 14-3-3ε were also associated with diffuse-type GC and early-onset of this pathology. Our data suggest that reduced 14-3-3ε may have a role in gastric carcinogenesis process. CONCLUSION: Our results reveal that the reduced 14-3-3ε expression in GC and investigation of 14-3-3ε interaction partners may help to elucidate the carcinogenesis process. PMID:22509086

  1. 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression

    PubMed Central

    Mukhopadhyay, Amitabha; Sehgal, Lalit; Bose, Arunabha; Gulvady, Anushree; Senapati, Parijat; Thorat, Rahul; Basu, Srikanta; Bhatt, Khyati; Hosing, Amol S.; Balyan, Renu; Borde, Lalit; Kundu, Tapas K.; Dalal, Sorab N.

    2016-01-01

    More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. PMID:27253419

  2. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR

    PubMed Central

    Stevers, Loes M.; Lam, Chan V.; Leysen, Seppe F. R.; Meijer, Femke A.; van Scheppingen, Daphne S.; de Vries, Rens M. J. M.; Carlile, Graeme W.; Milroy, Lech G.; Thomas, David Y.; Brunsveld, Luc; Ottmann, Christian

    2016-01-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein–protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3–CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  3. Decreased expression of 14-3-3 in Paracoccidioides brasiliensis confirms its involvement in fungal pathogenesis.

    PubMed

    Marcos, Caroline Maria; Silva, Julhiany de Fátima ds; Oliveira, Haroldo Cesar de; Assato, Patrícia Akemi; Singulani, Junya de Lacorte; Lopez, Angela Maria; Tamayo, Diana Patricia; Hernandez-Ruiz, Orville; McEwen, Juan G; Mendes-Giannini, Maria José Soares; Fusco-Almeida, Ana Marisa

    2016-01-01

    The interaction between the fungal pathogen Paracoccidioides brasiliensis and host cells is usually mediated by specific binding events between adhesins on the fungal surface and receptors on the host extracellular matrix or cell surface. One molecule implicated in the P. brasiliensis-host interaction is the 14-3-3 protein. The 14-3-3 protein belongs to a family of conserved regulatory molecules that are expressed in all eukaryotic cells and are involved in diverse cellular functions. Here, we investigated the relevance of the 14-3-3 protein to the virulence of P. brasiliensis. Using antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated a 14-3-3-silenced strain (expression reduced by ˜55%). This strain allowed us to investigate the interaction between 14-3-3 and the host and to correlate the functions of P. brasiliensis 14-3-3 with cellular features, such as morphological characteristics and virulence, that are important for pathogenesis. PMID:26646480

  4. Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach*

    PubMed Central

    Jasinski-Bergner, Simon; Stehle, Franziska; Gonschorek, Evamaria; Kalich, Jana; Schulz, Kristin; Huettelmaier, Stefan; Braun, Juliane; Seliger, Barbara

    2014-01-01

    Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G+, miR-152low cells, and their miR-152-overexpressing (miRhigh) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity. PMID:25228695

  5. The pro-inflammatory cytokine 14-3-3ε is a ligand of CD13 in cartilage

    PubMed Central

    Nefla, Meriam; Sudre, Laure; Denat, Guillaume; Priam, Sabrina; Andre-Leroux, Gwenaëlle; Berenbaum, Francis; Jacques, Claire

    2015-01-01

    ABSTRACT Osteoarthritis is a whole-joint disease characterized by the progressive destruction of articular cartilage involving abnormal communication between subchondral bone and cartilage. Our team previously identified 14-3-3ε protein as a subchondral bone soluble mediator altering cartilage homeostasis. The aim of this study was to investigate the involvement of CD13 (also known as aminopeptidase N, APN) in the chondrocyte response to 14-3-3ε. After identifying CD13 in chondrocytes, we knocked down CD13 with small interfering RNA (siRNA) and blocking antibodies in articular chondrocytes. 14-3-3ε-induced MMP-3 and MMP-13 was significantly reduced with CD13 knockdown, which suggests that it has a crucial role in 14-3-3ε signal transduction. Aminopeptidase N activity was identified in chondrocytes, but the activity was unchanged after stimulation with 14-3-3ε. Direct interaction between CD13 and 14-3-3ε was then demonstrated by surface plasmon resonance. Using labeled 14-3-3ε, we also found that 14-3-3ε binds to the surface of chondrocytes in a manner that is dependent on CD13. Taken together, these results suggest that 14-3-3ε might directly bind to CD13, which transmits its signal in chondrocytes to induce a catabolic phenotype similar to that observed in osteoarthritis. The 14-3-3ε–CD13 interaction could be a new therapeutic target in osteoarthritis. PMID:26208633

  6. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells.

    PubMed

    Li, Tong; Paudel, Hemant K

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer's disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445-6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  7. 14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

    PubMed Central

    Li, Tong; Paudel, Hemant K.

    2016-01-01

    Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated in vitro with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect in vitro tau aggregation (Qureshi et al (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. In vitro, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ. PMID:27548710

  8. Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS).

    PubMed

    Volkening, Kathryn; Leystra-Lantz, Cheryl; Yang, Wenchang; Jaffee, Howard; Strong, Michael J

    2009-12-11

    Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive motor neuron degeneration in association with neurofilament (NF) aggregate formation. This process is accompanied by an alteration in the stoichiometry of NF subunit protein expression such that the steady state levels of the low molecular weight NF (NFL) mRNA levels are selectively suppressed. We have previously shown that each of TDP-43, 14-3-3 and mutant SOD1 can function as NFL mRNA 3'UTR binding proteins that directly affect the stability of NFL transcripts. In this study, we demonstrate that the interaction of TDP-43 with the NFL mRNA 3' UTR involves ribonucleotide (UG) motifs present on stem loops of the 3'UTR as well as the RRM1 and RRM2 motifs of TDP-43. Ex vivo, TDP-43, 14-3-3 and SOD1 proteins interact to modulate NFL mRNA stability, although in vivo, only TDP-43 and either mutant or wild-type SOD1 co-localize in ALS motor neurons. TDP-43 was observed to co-localize to RNA transport granules (Staufen immunoreactive) in both control and ALS spinal motor neurons. In contrast, both stress granules (TIA-1 immunoreactive) and processing bodies (P-bodies; XRN-1 immunoreactive) were more prevalent in ALS motor neurons than in controls and demonstrated strong co-localization with TDP-43. Using RNA-IP-PCR, we further demonstrate that NFL mRNA is preferentially sequestered to both stress granules and P-bodies in ALS. These data suggest that NFL mRNA processing is fundamentally altered in ALS spinal motor neurons to favour compartmentalization within both stress granules and P-bodies, and that TDP-43 plays a fundamental role in this process.

  9. Comparative analysis of the 14-3-3 gene and its expression in Echinococcus granulosus and Echinococcus multilocularis metacestodes.

    PubMed

    Siles-Lucas, M; Nunes, C P; Zaha, A

    2001-03-01

    It was suggested that the unlimited proliferative capacity of the Echinococcus multilocularis metacestode may be related to overproduction of the 14-3-3 protein. As is known, the proliferative capacities of E. granulosus and E. multilocularis metacestodes are very different. By comparing the expression levels of the 14-3-3 gene between in vitro-obtained E. granulosus and E. multilocularis metacestodes, we were able to provide experimental evidence of the potential relation between 14-3-3 over-expression and tumour-like growth in E. multilocularis metacestodes. RT-PCR and Northern blot experiments indicated that 14-3-3 expression level is about 4-fold higher in the E. multilocularis metacestode. This differential expression was confirmed both by immunoblotting and immunocytochemistry experiments, which allowed detection of the protein in the cyst wall from E. multilocularis but not in the cyst wall from E. granulosus. The alignment of the Echinococcus 14-3-3 cDNA sequence with known 14-3-3 isoforms from other organisms, grouped the parasite sequence into the tumour growth-related isoforms. The known relation between over-expression of some 14-3-3 isoforms and tumour-related processes, together with the present results, suggest that the Echinococcus 14-3-3 protein could be one of the molecules responsible for the differences between E. granulosus and E. multilocularis metacestode growth behaviour.

  10. Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress.

    PubMed

    Li, Ruihua; Jiang, Xiaotong; Jin, Donghao; Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

    14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses.

  11. Identification of 14-3-3 Family in Common Bean and Their Response to Abiotic Stress

    PubMed Central

    Dhaubhadel, Sangeeta; Bian, Shaomin; Li, Xuyan

    2015-01-01

    14-3-3s are a class of conserved regulatory proteins ubiquitously found in eukaryotes, which play important roles in a variety of cellular processes including response to diverse stresses. Although much has been learned about 14-3-3s in several plant species, it remains unknown in common bean. In this study, 9 common bean 14-3-3s (PvGF14s) were identified by exhaustive data mining against the publicly available common bean genomic database. A phylogenetic analysis revealed that each predicted PvGF14 was clustered with two GmSGF14 paralogs from soybean. Both epsilon-like and non-epsilon classes of PvGF14s were found in common bean, and the PvGF14s belonging to each class exhibited similar gene structure. Among 9 PvGF14s, only 8 are transcribed in common bean. Expression patterns of PvGF14s varied depending on tissue type, developmental stage and exposure of plants to stress. A protein-protein interaction study revealed that PvGF14a forms dimer with itself and with other PvGF14 isoforms. This study provides a first comprehensive look at common bean 14-3-3 proteins, a family of proteins with diverse functions in many cellular processes, especially in response to stresses. PMID:26599110

  12. Ustilago maydis Rho1 and 14-3-3 homologues participate in pathways controlling cell separation and cell polarity.

    PubMed

    Pham, Cau D; Yu, Zhanyang; Sandrock, Björn; Bölker, Michael; Gold, Scott E; Perlin, Michael H

    2009-07-01

    Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3epsilon and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.

  13. Histone Deacetylase 6 (HDAC6) Promotes the Pro-survival Activity of 14-3-3ζ via Deacetylation of Lysines within the 14-3-3ζ Binding Pocket*

    PubMed Central

    Mortenson, Jeffrey B.; Heppler, Lisa N.; Banks, Courtney J.; Weerasekara, Vajira K.; Whited, Matthew D.; Piccolo, Stephen R.; Johnson, William E.; Thompson, J. Will; Andersen, Joshua L.

    2015-01-01

    The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys49 and Lys120, is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys49/Lys120 deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser112 and Thr642, respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser112/Thr642 phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity. PMID:25770209

  14. Phosphorylation-related modification at the dimer interface of 14-3-3ω dramatically alters monomer interaction dynamics.

    PubMed

    Denison, Fiona C; Gökirmak, Tufan; Ferl, Robert J

    2014-01-01

    14-3-3 proteins are generally believed to function as dimers in a broad range of eukaryotic signaling pathways. The consequences of altering dimer stability are not fully understood. Phosphorylation at Ser58 in the dimer interface of mammalian 14-3-3 isoforms has been reported to destabilise dimers. An equivalent residue, Ser62, is present across most Arabidopsis isoforms but the effects of phosphorylation have not been studied in plants. Here, we assessed the effects of phosphorylation at the dimer interface of Arabidopsis 14-3-3ω. Protein kinase A phosphorylated 14-3-3ω at Ser62 and also at a previously unreported residue, Ser67, resulting in a monomer-sized band on native-PAGE. Phosphorylation at Ser62 alone, or with additional Ser67 phosphorylation, was investigated using phosphomimetic versions of 14-3-3ω. In electrophoretic and chromatographic analyses, these mutants showed mobilities intermediate between dimers and monomers. Mobility was increased by detergents, by reducing protein concentration, or by increasing pH or temperature. Urea gradient gels showed complex structural transitions associated with alterations of dimer stability, including a previously unreported 14-3-3 aggregation phenomenon. Overall, our analyses showed that dimer interface modifications such as phosphorylation reduce dimer stability, dramatically affecting the monomer-dimer equilibrium and denaturation trajectory. These findings may have dramatic implications for 14-3-3 structure and function in vivo.

  15. Sporadic Creutzfeldt-Jakob disease diagnostic accuracy is improved by a new CSF ELISA 14-3-3γ assay.

    PubMed

    Leitão, M J; Baldeiras, I; Almeida, M R; Ribeiro, M H; Santos, A C; Ribeiro, M; Tomás, J; Rocha, S; Santana, I; Oliveira, C R

    2016-05-13

    Protein 14-3-3 is a reliable marker of rapid neuronal damage, specifically increased in cerebrospinal fluid (CSF) of sporadic Creutzfeldt-Jakob disease (sCJD) patients. Its detection is usually performed by Western Blot (WB), prone to methodological issues. Our aim was to evaluate the diagnostic performance of a recently developed quantitative enzyme-linked immunosorbent (ELISA) assay for 14-3-3γ, in comparison with WB and other neurodegeneration markers. CSF samples from 145 patients with suspicion of prion disease, later classified as definite sCJD (n=72) or Non-prion diseases (Non-CJD; n=73) comprised our population. 14-3-3 protein was determined by WB and ELISA. Total Tau (t-Tau) and phosphorylated Tau (p-Tau) were also evaluated. Apolipoprotein E gene (ApoE) and prionic protein gene (PRNP) genotyping was assessed. ELISA 14-3-3γ levels were significantly increased in sCJD compared to Non-CJD patients (p<0.001), showing very good accuracy (AUC=0.982; sensitivity=97%; specificity=94%), and matching WB results in 81% of all cases. It strongly correlated with t-Tau and p-Tau (p<0.0001), showing slightly higher specificity (14-3-3 WB - 63%; Tau - 90%; p-Tau/t-Tau ratio - 88%). From WB inconclusive results (n=44), ELISA 14-3-3γ correctly classified 41 patients. Additionally, logistic regression analysis selected ELISA 14-3-3γ as the best single predictive marker for sCJD (overall accuracy=93%). ApoE and PRNP genotypes did not influence ELISA 14-3-3γ levels. Despite specificity for 14-3-3γ isoform, ELISA results not only match WB evaluation but also help discrimination of inconclusive results. Our results therefore reinforce this assay as a single screening test, allowing higher sample throughput and unequivocal results. PMID:26940479

  16. Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B

    PubMed Central

    Russo, Roberta; Zito, Francesca; Costa, Caterina; Bonaventura, Rosa

    2010-01-01

    Members of the 14-3-3 protein family are involved in many important cellular events, including stress response, survival and apoptosis. Genes of the 14-3-3 family are conserved from plants to humans, and some members are responsive to UV radiation. Here, we report the isolation of the complete cDNA encoding the 14-3-3 epsilon isoform from Paracentrotus lividus sea urchin embryos, referred to as Pl14-3-3ε, and the phylogenetic relationship with other homologues described in different phyla. Pl14-3-3ε mRNA levels were measured by QPCR during development and found to increase from the mesenchyme blastula to the prism stage. In response to UV-B (312 nm) exposure, early stage embryos collected 2 h later showed a 2.3-fold (at 400 J/m2) and a 2.7-fold (at 800 J/m2) increase in Pl14-3-3ε transcript levels compared with controls. The spatial expression of Pl14-3-3ε mRNA, detected by whole mount in situ hybridization in both control and UV-B exposed embryos, harvested at late developmental stages, showed transcripts to be located in the archenteron of gastrula stage and widely distributed in all germ layers, respectively. The Pl14-3-3ε mRNA delocalization parallels the failure in archenteron elongation observed morphologically, as well as the lack of specific endoderm markers, investigated by indirect immuno-fluorescence on whole mount embryos. Results confirm the involvement of 14-3-3ε in the stress response elicited by UV-B and demonstrate, for the first time, its contribution at the transcriptional level in the sea urchin embryo. PMID:20607471

  17. Intracellular Generation of a Diterpene-Peptide Conjugate that Inhibits 14-3-3-Mediated Interactions.

    PubMed

    Parvatkar, Prakash; Kato, Nobuo; Uesugi, Motonari; Sato, Shin-Ichi; Ohkanda, Junko

    2015-12-23

    Synthetic agents that disrupt intracellular protein-protein interactions (PPIs) are highly desirable for elucidating signaling networks and developing new therapeutics. However, designing cell-penetrating large molecules equipped with the many functional groups necessary for binding to large interfaces remains challenging. Here, we describe a rational strategy for the intracellular oxime ligation-mediated generation of an amphipathic bivalent inhibitor composed of a peptide and diterpene natural product, fusicoccin, which binds 14-3-3 protein with submicromolar affinity. Our results demonstrate that co-treatment of cells with small module molecules, the aldehyde-containing fusicoccin 1 and the aminooxy-containing peptide 2, generates the corresponding conjugate 3 in cells, resulting in significant cytotoxicity. In contrast, chemically synthesized 3 is not cytotoxic, likely due to its inability to penetrate cells. Compound 3, but not 1 or 2, disrupts endogenous 14-3-3/cRaf interactions, suggesting that cell death is caused by inhibition of 14-3-3 activity. These results suggest that intracellular generation of large-sized molecules may serve as a new approach for modulating PPIs.

  18. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability

    PubMed Central

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  19. The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

    PubMed

    Seo, Gi Won; Jo, Yong Hun; Seong, Jeong Hwan; Park, Ki Beom; Patnaik, Bharat Bhusan; Tindwa, Hamisi; Kim, Sun-Am; Lee, Yong Seok; Kim, Yu Jung; Han, Yeon Soo

    2016-01-01

    The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform. PMID:27556493

  20. Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    PubMed Central

    Singh, Lalit P.; Jiang, Yan; Cheng, Davis W.

    2007-01-01

    Recently we demonstrated that IGF-1 expression is increased in the diabetic kidney and that it may involve in renal hypertrophy and extracellular matrix protein (ECM) accumulation in mesangial cells as seen in diabetic glomerulopathy. The present study investigates the molecular mechanism(s) of IGF-1 and Akt/glycogen synthase kinase-3beta (GSK-3β) signaling pathway in the regulation of fibronectin and cyclin D1 expression and survival of renal mesangial cells. A proteomic approach is also employed to identify protein targets of IGF-1 signaling via GSK-3β inhibition in mesangial cells. We show that IGF-1 (100 ng/ml) significantly increases the protein kinase Akt/PKB activity (1.5-2-fold, p<0.05) within 1-5 minutes, which is completely blocked by the presence of 100 nM Wortmannin (phosphatidyl-inositol 3-kinase inhibitor). Akt activation is coupled with Ser9 phosphorylation and inactivation of its down-stream target GSK-3β. IGF-1 increases the cyclic AMP-responsive element (CRE) binding transcription factor CREB phosphorylation at Ser 133 and CRE-binding activity in mesangial cells, which parallels cyclin D1 and fibronectin expressions. Both proteins are known to have CRE-sequences in their promoter regions upstream of the transcription start site. Suppression of GSK-3β by SB216763 (100 nM) increases CREB phosphorylation, cyclin D1 and fibronectin levels. Two dimensional gel electrophoresis followed by MALDI-TOF mass spectrometric analysis of mesangial proteins reveals that IGF-1 treatment or an inhibition of GSK-3β increases the expression of the phosphorylated Ser/Thr binding signal adapter protein 14-3-3ζ. Immuno-precipitation of 14-3-3ζ followed by Western blotting validates the association of phosphorylated GSK-3β with 14-3-3ζ in renal mesangial cells. Stable expression of a constitutively active GSK-3β(Ser9Ala) induces cell death while overexpression of HA-tagged 14-3-3ζ increases cell viability as measured by MTT assays. These results indicate that

  1. Drosophila 14-3-3ε has a crucial role in anti-microbial peptide secretion and innate immunity.

    PubMed

    Shandala, Tetyana; Woodcock, Joanna M; Ng, Yeap; Biggs, Lisa; Skoulakis, Efthimios M C; Brooks, Doug A; Lopez, Angel F

    2011-07-01

    The secretion of anti-microbial peptides is recognised as an essential step in innate immunity, but there is limited knowledge of the molecular mechanism controlling the release of these effectors from immune response cells. Here, we report that Drosophila 14-3-3ε mutants exhibit reduced survival when infected with either Gram-positive or Gram-negative bacteria, indicating a functional role for 14-3-3ε in innate immunity. In 14-3-3ε mutants, there was a reduced release of the anti-microbial peptide Drosomycin into the haemolymph, which correlated with an accumulation of Drosomycin-containing vesicles near the plasma membrane of cells isolated from immune response tissues. Drosomycin appeared to be delivered towards the plasma membrane in Rab4- and Rab11-positive vesicles and smaller Rab11-positive vesicles. RNAi silencing of Rab11 and Rab4 significantly blocked the anterograde delivery of Drosomycin from the perinuclear region to the plasma membrane. However, in 14-3-3ε mutants there was an accumulation of small Rab11-positive vesicles near the plasma membrane. This vesicular phenotype was similar to that observed in response to the depletion of the vesicular Syntaxin protein Syx1a. In wild-type Drosophila immune tissue, 14-3-3ε was detected adjacent to Rab11, and partially overlapping with Syx1a, on vesicles near the plasma membrane. We conclude that 14-3-3ε is required for Rab11-positive vesicle function, which in turn enables antimicrobial peptide secretion during an innate immune response.

  2. Regulator of G-protein signaling 18 integrates activating and inhibitory signaling in platelets.

    PubMed

    Gegenbauer, Kristina; Elia, Giuliano; Blanco-Fernandez, Alfonso; Smolenski, Albert

    2012-04-19

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein for the G-α-q and G-α-i subunits of heterotrimeric G-proteins that turns off signaling by G-protein coupled receptors. RGS18 is highly expressed in platelets. In the present study, we show that the 14-3-3γ protein binds to phosphorylated serines 49 and 218 of RGS18. Platelet activation by thrombin, thromboxane A2, or ADP stimulates the association of 14-3-3 and RGS18, probably by increasing the phosphorylation of serine 49. In contrast, treatment of platelets with prostacyclin and nitric oxide, which trigger inhibitory cyclic nucleotide signaling involving cyclic AMP-dependent protein kinase A (PKA) and cyclic GMP-dependent protein kinase I (PKGI), induces the phosphorylation of serine 216 of RGS18 and the detachment of 14-3-3. Serine 216 phosphorylation is able to block 14-3-3 binding to RGS18 even in the presence of thrombin, thromboxane A2, or ADP. 14-3-3-deficient RGS18 is more active compared with 14-3-3-bound RGS18, leading to a more pronounced inhibition of thrombin-induced release of calcium ions from intracellular stores. Therefore, PKA- and PKGI-mediated detachment of 14-3-3 activates RGS18 to block Gq-dependent calcium signaling. These findings indicate cross-talk between platelet activation and inhibition pathways at the level of RGS18 and Gq. PMID:22234696

  3. IFNγ-induced suppression of β-catenin signaling: evidence for roles of Akt and 14.3.3ζ

    PubMed Central

    Nava, Porfirio; Kamekura, Ryuta; Quirós, Miguel; Medina-Contreras, Oscar; Hamilton, Ross W.; Kolegraff, Keli N.; Koch, Stefan; Candelario, Aurora; Romo-Parra, Hector; Laur, Oskar; Hilgarth, Roland S.; Denning, Timothy L.; Parkos, Charles A.; Nusrat, Asma

    2014-01-01

    The proinflammatory cytokine interferon γ (IFNγ ) influences intestinal epithelial cell (IEC) homeostasis in a biphasic manner by acutely stimulating proliferation that is followed by sustained inhibition of proliferation despite continued mucosal injury. β-Catenin activation has been classically associated with increased IEC proliferation. However, we observed that IFNγ inhibits IEC proliferation despite sustained activation of Akt/β-catenin signaling. Here we show that inhibition of Akt/β-catenin–mediated cell proliferation by IFNγ is associated with the formation of a protein complex containing phosphorylated β-catenin 552 (pβ-cat552) and 14.3.3ζ. Akt1 served as a bimodal switch that promotes or inhibits β-catenin transactivation in response to IFNγ stimulation. IFNγ initially promotes β-catenin transactivation through Akt-dependent C-terminal phosphorylation of β-catenin to promote its association with 14.3.3ζ. Augmented β-catenin transactivation leads to increased Akt1 protein levels, and active Akt1 accumulates in the nucleus, where it phosphorylates 14.3.3ζ to translocate 14.3.3ζ/β-catenin from the nucleus, thereby inhibiting β-catenin transactivation and IEC proliferation. These results outline a dual function of Akt1 that suppresses IEC proliferation during intestinal inflammation. PMID:25079689

  4. Cyclic Nucleotide Dependent Dephosphorylation of Regulator of G-Protein Signaling 18 in Human Platelets

    PubMed Central

    Gegenbauer, Kristina; Nagy, Zoltan; Smolenski, Albert

    2013-01-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets. PMID:24244663

  5. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    PubMed

    Gegenbauer, Kristina; Nagy, Zoltan; Smolenski, Albert

    2013-01-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets. PMID:24244663

  6. Interactions of c-Raf-1 with phosphatidylserine and 14-3-3.

    PubMed

    McPherson, R A; Harding, A; Roy, S; Lane, A; Hancock, J F

    1999-07-01

    Activation of Raf-1 occurs at the plasma membrane. We recently showed that 14-3-3 must be complexed with Raf-1 for efficient recruitment to the plasma membrane and activation by Ras, but that 14-3-3 is completely displaced from Raf-1 following plasma membrane binding. We show here that the Raf-1 zinc finger is not absolutely required for 14-3-3 binding but is required to stabilize the interaction between Raf-1 and 14-3-3. Incubation of Raf-1 with phosphatidylserine, an inner plasma membrane phospholipid, results in removal of 14-3-3 and an increase in Raf-1 kinase activity, whereas removal of 14-3-3 from Raf-1 using specific phosphopeptides substantially reduces Raf-1 basal kinase activity. Displacement of 14-3-3 from activated Raf-1 by phosphopeptides has no effect on kinase activity if Raf-1 is first removed from solution, but completely eradicates kinase activity of soluble activated Raf-1. These results suggest a mechanism for the removal of 14-3-3 from Raf-1 at the plasma membrane and show that removal of 14-3-3 from Raf-1 has markedly different effects depending on experimental conditions.

  7. Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells

    PubMed Central

    Lee, Young-Sun; Lee, Jae Kwang; Bae, Yeonju; Lee, Bok-Soon; Kim, Eunju; Cho, Chang-Hoon; Ryoo, Kanghyun; Yoo, Jiyun; Kim, Chul-Ho; Yi, Gwan-Su; Lee, Seok-Geun; Lee, C. Justin; Kang, Sang Soo; Hwang, Eun Mi; Park, Jae-Yong

    2016-01-01

    Anoctamin-1 (ANO1) acts as a Ca2+-activated Cl− channel in various normal tissues, and its expression is increased in several different types of cancer. Therefore, understanding the regulation of ANO1 surface expression is important for determining its physiological and pathophysiological functions. However, the trafficking mechanism of ANO1 remains elusive. Here, we report that segment a (N-terminal 116 amino acids) of ANO1 is crucial for its surface expression, and we identified 14-3-3γ as a binding partner for anterograde trafficking using yeast two-hybrid screening. The surface expression of ANO1 was enhanced by 14-3-3γ, and the Thr9 residue of ANO1 was critical for its interaction with 14-3-3γ. Gene silencing of 14-3-3γ and/or ANO1 demonstrated that suppression of ANO1 surface expression inhibited migration and invasion of glioblastoma cells. These findings provide novel therapeutic implications for glioblastomas, which are associated with poor prognosis. PMID:27212225

  8. 14-3-3 gene family in hybrid poplar and its involvement in tree defence against pathogens.

    PubMed

    Lapointe, G; Luckevich, M D; Cloutier, M; Séguin, A

    2001-06-01

    In ongoing investigations of the role of the signal transduction pathway in tree-pathogen interactions, four complete and two partial 14-3-3 cDNAs have been isolated which are members of a gene family. Comparisons of DNA sequences reveal a high degree of identity among the cDNAs, and, in some cases, higher than 75% sequence similarity with previously published sequences. Sequence analysis at the amino acid level uncovered potential phosphorylation sites, some of which were identical among the proteins, and some of which varied. Treatment of trees with chitosan, jasmonates or by wounding of leaves, caused increases in the levels of 14-3-3 mRNA transcripts. Since jasmonates and chitosan are signal transducers of defence reactions in plants, these results suggest a possible role for 14-3-3 proteins in the pathogen defence response of deciduous trees. Effects of elicitors on transcription of the pal gene were also monitored. Pal is a well-characterized, pathogen response-related gene.

  9. Impaired Binding of 14-3-3 to C-RAF in Noonan Syndrome Suggests New Approaches in Diseases with Increased Ras Signaling▿

    PubMed Central

    Molzan, Manuela; Schumacher, Benjamin; Ottmann, Corinna; Baljuls, Angela; Polzien, Lisa; Weyand, Michael; Thiel, Philipp; Rose, Rolf; Rose, Micheline; Kuhenne, Philipp; Kaiser, Markus; Rapp, Ulf R.; Kuhlmann, Jürgen; Ottmann, Christian

    2010-01-01

    The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser259, a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS259 crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer259/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway. PMID:20679480

  10. Overexpression of 14-3-3z promotes tau phosphorylation at Ser262 and accelerates proteosomal degradation of synaptophysin in rat primary hippocampal neurons.

    PubMed

    Qureshi, Hamid Y; Han, Dong; MacDonald, Ryen; Paudel, Hemant K

    2013-01-01

    b-Amyloid peptide accumulation, tau hyperphosphorylation, and synapse loss are characteristic neuropathological symptoms of Alzheimer's disease (AD). Tau hyperphosphorylation is suggested to inhibit the association of tau with microtubules, making microtubules unstable and causing neurodegeneration. The mechanism of tau phosphorylation in AD brain, therefore, is of considerable significance. Although PHF-tau is phosphorylated at over 40 Ser/Thr sites, Ser(262) phosphorylation was shown to mediate b-amyloid neurotoxicity and formation of toxic tau lesions in the brain. In vitro, PKA is one of the kinases that phosphorylates tau at Ser(262), but the mechanism by which it phosphorylates tau in AD brain is not very clear. 14-3-3z is associated with neurofibrillary tangles and is upregulated in AD brain. In this study, we show that 14-3-3z promotes tau phosphorylation at Ser(262) by PKA in differentiating neurons. When overexpressed in rat hippocampal primary neurons, 14-3-3z causes an increase in Ser(262) phosphorylation, a decrease in the amount of microtubule-bound tau, a reduction in the amount of polymerized microtubules, as well as microtubule instability. More importantly, the level of pre-synaptic protein synaptophysin was significantly reduced. Downregulation of synaptophysin in 14-3-3z overexpressing neurons was mitigated by inhibiting the proteosome, indicating that 14-3-3z promotes proteosomal degradation of synaptophysin. When 14-3-3z overexpressing neurons were treated with the microtubule stabilizing drug taxol, tau Ser(262) phosphorylation decreased and synaptophysin level was restored. Our data demonstrate that overexpression of 14-3-3z accelerates proteosomal turnover of synaptophysin by promoting the destabilization of microtubules. Synaptophysin is involved in synapse formation and neurotransmitter release. Our results suggest that 14-3-3z may cause synaptic pathology by reducing synaptophysin levels in the brains of patients suffering from AD. PMID

  11. Oxidative damage of 14-3-3 zeta and gamma isoforms in Alzheimer's disease and cerebral amyloid angiopathy.

    PubMed

    Santpere, G; Puig, B; Ferrer, I

    2007-06-01

    Previous studies have shown oxidative damage resulting from amyloid Abeta exposure to cultured cells and in murine models. A target of oxidation is 14-3-3 which comprises a group of proteins involved in kinase activation and chaperone activity. The present study shows glycoxidative damage, as revealed with mono and bi-dimensional gel electrophoresis and Western blotting, followed by in-gel digestion and mass spectrometry, in the frontal cortex in Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA), a neurodegenerative disease with deposition of Abeta in cerebral blood vessels and in diffuse plaques unaccompanied by intraneuronal hyper-phosphorylated tau deposition. malondialdehyde-lysine (MDA-Lys)-, but not 4-hydroxy-2-nonenal (HNE)-immunoreactive adducts, and N-carboxyethyl-lysine (CEL), but not N-carboxymethyl-lysine (CML)-products, were present in 14-3-3 involving zeta and gamma isoforms in both AD and CAA. These findings demonstrate that 14-3-3 glyco- and lipoxidation occurs in AD and CAA, probably as a direct consequence of Abeta deposition.

  12. Upregulation of lactate dehydrogenase a by 14-3-3ζ leads to increased glycolysis critical for breast cancer initiation and progression

    PubMed Central

    Chang, Chia-Chi; Zhang, Chenyu; Zhang, Qingling; Sahin, Ozgur; Wang, Hai; Xu, Jia; Xiao, Yi; Zhang, Jian; Rehman, Sumaiyah K.; Li, Ping; Hung, Mien-Chie; Behbod, Fariba; Yu, Dihua

    2016-01-01

    Metabolic reprogramming is a hallmark of cancer. Elevated glycolysis in cancer cells switches the cellular metabolic flux to produce more biological building blocks, thereby sustaining rapid proliferation. Recently, new evidence has emerged that metabolic dysregulation may occur at early-stages of neoplasia and critically contribute to cancer initiation. Here, our bioinformatics analysis of microarray data from early-stages breast neoplastic lesions revealed that 14-3-3ζ expression is strongly correlated with the expression of canonical glycolytic genes, particularly lactate dehydrogenase A (LDHA). Experimentally, increasing 14-3-3ζ expression in human mammary epithelial cells (hMECs) up-regulated LDHA expression, elevated glycolytic activity, and promoted early transformation. Knockdown of LDHA in the 14-3-3ζ-overexpressing hMECs significantly reduced glycolytic activity and inhibited transformation. Mechanistically, 14-3-3ζ overexpression activates the MEK-ERK-CREB axis, which subsequently up-regulates LDHA. In vivo, inhibiting the activated the MEK/ERK pathway in 14-3-3ζ-overexpressing hMEC-derived MCF10DCIS.COM lesions led to effective inhibition of tumor growth. Therefore, targeting the MEK/ERK pathway could be an effective strategy for intervention of 14-3-3ζ-overexpressing early breast lesions. Together, our data demonstrate that overexpression of 14-3-3ζ in early stage pre-cancerous breast epithelial cells may trigger an elevated glycolysis and transcriptionally up-regulating LDHA, thereby contributes to human breast cancer initiation. PMID:27150057

  13. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  14. Proteomic screening for Rho-kinase substrates by combining kinase and phosphatase inhibitors with 14-3-3ζ affinity chromatography.

    PubMed

    Nishioka, Tomoki; Nakayama, Masanori; Amano, Mutsuki; Kaibuchi, Kozo

    2012-01-01

    The small GTPase RhoA is a molecular switch in various extracellular signals. Rho-kinase/ROCK/ROK, a major effector of RhoA, regulates diverse cellular functions by phosphorylating cytoskeletal proteins, endocytic proteins, and polarity proteins. More than twenty Rho-kinase substrates have been reported, but the known substrates do not fully explain the Rho-kinase functions. Herein, we describe the comprehensive screening for Rho-kinase substrates by treating HeLa cells with Rho-kinase and phosphatase inhibitors. The cell lysates containing the phosphorylated substrates were then subjected to affinity chromatography using beads coated with 14-3-3 protein, which interacts with proteins containing phosphorylated serine or threonine residues, to enrich the phosphorylated proteins. The identities of the molecules and phosphorylation sites were determined by liquid chromatography tandem mass spectrometry (LC/MS/MS) after tryptic digestion and phosphopeptide enrichment. The phosphorylated proteins whose phosphopeptide ion peaks were suppressed by treatment with the Rho-kinase inhibitor were regarded as candidate substrates. We identified 121 proteins as candidate substrates. We also identified phosphorylation sites in Partitioning defective 3 homolog (Par-3) at Ser143 and Ser144. We found that Rho-kinase phosphorylated Par-3 at Ser144 both in vitro and in vivo. The method used in this study would be applicable and useful to identify novel substrates of other kinases.

  15. In-vivo administration of clozapine affects behaviour but does not reverse dendritic spine deficits in the 14-3-3ζ KO mouse model of schizophrenia-like disorders.

    PubMed

    Jaehne, Emily J; Ramshaw, Hayley; Xu, Xiangjun; Saleh, Eiman; Clark, Scott R; Schubert, Klaus Oliver; Lopez, Angel; Schwarz, Quenten; Baune, Bernhard T

    2015-11-01

    Clozapine is an atypical antipsychotic drug used in the treatment of schizophrenia, which has been shown to reverse behavioural and dendritic spine deficits in mice. It has recently been shown that deficiency of 14-3-3ζ has an association with schizophrenia, and that a mouse model lacking this protein displays several schizophrenia-like behavioural deficits. To test the effect of clozapine in this mouse model, 14-3-3ζ KO mice were administered clozapine (5mg/kg) for two weeks prior to being analysed in a test battery of cognition, anxiety, and despair (depression-like) behaviours. Following behavioural testing brain samples were collected for analysis of specific anatomical defects and dendritic spine formation. We found that clozapine reduced despair behaviour of 14-3-3ζ KO mice in the forced swim test (FST) and altered the behaviour of wild types and 14-3-3ζ KO mice in the Y-maze task. In contrast, clozapine had no effects on hippocampal laminar defects or decreased dendritic spine density observed in 14-3-3ζ KO mice. Our results suggest that clozapine may have beneficial effects on clinical behaviours associated with deficiencies in the 14-3-3ζ molecular pathway, despite having no effects on morphological defects. These findings may provide mechanistic insight to the action of this drug.

  16. p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1

    PubMed Central

    Tollenaere, Maxim A. X.; Villumsen, Bine H.; Blasius, Melanie; Nielsen, Julie C.; Wagner, Sebastian A.; Bartek, Jiri; Beli, Petra; Mailand, Niels; Bekker-Jensen, Simon

    2015-01-01

    Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress. PMID:26616734

  17. Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

    PubMed

    Carrasco, José Luis; Castelló, María José; Naumann, Kai; Lassowskat, Ines; Navarrete-Gómez, Marisa; Scheel, Dierk; Vera, Pablo

    2014-01-01

    Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

  18. IκB kinase-induced interaction of TPL-2 kinase with 14-3-3 is essential for Toll-like receptor activation of ERK-1 and -2 MAP kinases.

    PubMed

    Ben-Addi, Abduelhakem; Mambole-Dema, Agnes; Brender, Christine; Martin, Stephen R; Janzen, Julia; Kjaer, Sven; Smerdon, Stephen J; Ley, Steven C

    2014-06-10

    The MEK-1/2 kinase TPL-2 is critical for Toll-like receptor activation of the ERK-1/2 MAP kinase pathway during inflammatory responses, but it can transform cells following C-terminal truncation. IκB kinase (IKK) complex phosphorylation of the TPL-2 C terminus regulates full-length TPL-2 activation of ERK-1/2 by a mechanism that has remained obscure. Here, we show that TPL-2 Ser-400 phosphorylation by IKK and TPL-2 Ser-443 autophosphorylation cooperated to trigger TPL-2 association with 14-3-3. Recruitment of 14-3-3 to the phosphorylated C terminus stimulated TPL-2 MEK-1 kinase activity, which was essential for TPL-2 activation of ERK-1/2. The binding of 14-3-3 to TPL-2 was also indispensible for lipopolysaccharide-induced production of tumor necrosis factor by macrophages, which is regulated by TPL-2 independently of ERK-1/2 activation. Our data identify a key step in the activation of TPL-2 signaling and provide a mechanistic insight into how C-terminal deletion triggers the oncogenic potential of TPL-2 by rendering its kinase activity independent of 14-3-3 binding.

  19. Cellular regulation by protein phosphorylation.

    PubMed

    Fischer, Edmond H

    2013-01-11

    A historical account of the discovery of reversible protein phosphorylation is presented. This process was uncovered in the mid 1950s in a study undertaken with Edwin G. Krebs to elucidate the complex hormonal regulation of skeletal muscle glycogen phosphorylase. Contrary to the known activation of this enzyme by AMP which serves as an allosteric effector, its hormonal regulation results from a phosphorylation of the protein by phosphorylase kinase following the activation of the latter by Ca(2+) and ATP. The study led to the establishment of the first hormonal cascade of successive enzymatic reactions, kinases acting on kinases, initiated by cAMP discovered by Earl Sutherland. It also showed how two different physiological processes, carbohydrate metabolism and muscle contraction, could be regulated in concert.

  20. Selective 14-3-3γ induction quenches p-β-catenin Ser37/Bax-enhanced cell death in cerebral cortical neurons during ischemia.

    PubMed

    Lai, X J; Ye, S Q; Zheng, L; Li, L; Liu, Q R; Yu, S B; Pang, Y; Jin, S; Li, Q; Yu, A C H; Chen, X Q

    2014-01-01

    Ischemia-induced cell death is a major cause of disability or death after stroke. Identifying the key intrinsic protective mechanisms induced by ischemia is critical for the development of effective stroke treatment. Here, we reported that 14-3-3γ was a selective ischemia-inducible survival factor in cerebral cortical neurons reducing cell death by downregulating Bax depend direct 14-3-3γ/p-β-catenin Ser37 interactions in the nucleus. 14-3-3γ, but not other 14-3-3 isoforms, was upregulated in primary cerebral cortical neurons upon oxygen-glucose deprivation (OGD) as measured by quantitative PCR, western blot and fluorescent immunostaining. The selective induction of 14-3-3γ in cortical neurons by OGD was verified by the in vivo ischemic stroke model. Knocking down 14-3-3γ alone or inhibiting 14-3-3/client interactions was sufficient to induce cell death in normal cultured neurons and exacerbate OGD-induced neuronal death. Ectopic overexpression of 14-3-3γ significantly reduced OGD-induced cell death in cultured neurons. Co-immunoprecipitation and fluorescence resonance energy transfer demonstrated that endogenous 14-3-3γ bound directly to more p-β-catenin Ser37 but not p-Bad, p-Ask-1, p-p53 and Bax. During OGD, p-β-catenin Ser37 but not p-β-catenin Ser45 was increased prominently, which correlated with Bax elevation in cortical neurons. OGD promoted the entry of 14-3-3γ into the nuclei, in correlation with the increase of nuclear p-β-catenin Ser37 in neurons. Overexpression of 14-3-3γ significantly reduced Bax expression, whereas knockdown of 14-3-3γ increased Bax in cortical neurons. Abolishing β-catenin phosphorylation at Ser37 (S37A) significantly reduced Bax and cell death in neurons upon OGD. Finally, 14-3-3γ overexpression completely suppressed β-catenin-enhanced Bax and cell death in neurons upon OGD. Based on these data, we propose that the 14-3-3γ/p-β-catenin Ser37/Bax axis determines cell survival or death of neurons during ischemia

  1. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  2. 14-3-3ζ deficient mice in the BALB/c background display behavioural and anatomical defects associated with neurodevelopmental disorders

    PubMed Central

    Xu, Xiangjun; Jaehne, Emily J.; Greenberg, Zarina; McCarthy, Peter; Saleh, Eiman; Parish, Clare L.; Camera, Daria; Heng, Julian; Haas, Matilda; Baune, Bernhard T.; Ratnayake, Udani; Buuse, Maarten van den; Lopez, Angel F.; Ramshaw, Hayley S.; Schwarz, Quenten

    2015-01-01

    Sequencing and expression analyses implicate 14-3-3ζ as a genetic risk factor for neurodevelopmental disorders such as schizophrenia and autism. In support of this notion, we recently found that 14-3-3ζ−/− mice in the Sv/129 background display schizophrenia-like defects. As epistatic interactions play a significant role in disease pathogenesis we generated a new congenic strain in the BALB/c background to determine the impact of genetic interactions on the 14-3-3ζ−/− phenotype. In addition to replicating defects such as aberrant mossy fibre connectivity and impaired spatial memory, our analysis of 14-3-3ζ−/− BALB/c mice identified enlarged lateral ventricles, reduced synaptic density and ectopically positioned pyramidal neurons in all subfields of the hippocampus. In contrast to our previous analyses, 14-3-3ζ−/− BALB/c mice lacked locomotor hyperactivity that was underscored by normal levels of the dopamine transporter (DAT) and dopamine signalling. Taken together, our results demonstrate that dysfunction of 14-3-3ζ gives rise to many of the pathological hallmarks associated with the human condition. 14-3-3ζ-deficient BALB/c mice therefore provide a novel model to address the underlying biology of structural defects affecting the hippocampus and ventricle, and cognitive defects such as hippocampal-dependent learning and memory. PMID:26207352

  3. Epstein-Barr virus Rta-mediated transactivation of p21 and 14-3-3σ arrests cells at the G1/S transition by reducing cyclin E/CDK2 activity.

    PubMed

    Huang, Sheng-Yen; Hsieh, Min-Jie; Chen, Chu-Ying; Chen, Yen-Ju; Chen, Jen-Yang; Chen, Mei-Ru; Tsai, Ching-Hwa; Lin, Su-Fang; Hsu, Tsuey-Ying

    2012-01-01

    Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2'-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation. PMID:21918011

  4. Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

    PubMed

    Sato, Shogo; Jung, Hunmin; Nakagawa, Tsutomu; Pawlosky, Robert; Takeshima, Tomomi; Lee, Wan-Ru; Sakiyama, Haruhiko; Laxman, Sunil; Wynn, R Max; Tu, Benjamin P; MacMillan, John B; De Brabander, Jef K; Veech, Richard L; Uyeda, Kosaku

    2016-05-13

    The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis. PMID:26984404

  5. Protein kinase C-mediated phosphorylation and activation of PDE3A regulate cAMP levels in human platelets.

    PubMed

    Hunter, Roger W; Mackintosh, Carol; Hers, Ingeborg

    2009-05-01

    The elevation of [cAMP](i) is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492), in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE(1) and forskolin-induced phosphorylation of Ser(312) and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE(1)-evoked cAMP accumulation by thrombin required both G(i) and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492) leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding. PMID:19261611

  6. Regulation of protein turnover by heat shock proteins.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.

  7. Adjudin disrupts spermatogenesis via the action of some unlikely partners: Eps8, Arp2/3 complex, drebrin E, PAR6 and 14-3-3.

    PubMed

    Cheng, C Yan; Lie, Pearl Py; Wong, Elissa Wp; Mruk, Dolores D; Silvestrini, Bruno

    2011-10-01

    expression of PAR6 (partitioning defective protein 6) and 14-3-3 (also known as PAR5) considerably at the apical ES, disrupting the homeostasis of endocytic vesicle-mediated protein trafficking, which in turn leads to an increase in protein endocytosis. The net result of these changes destabilizes cell adhesion and induces degeneration of the apical ES, causing premature release of spermatids, mimicking spermiation.

  8. Role of regulator of G protein signaling proteins in bone

    PubMed Central

    Keinan, David; Yang, Shuying; Cohen, Robert E.; Yuan, Xue; Liu, Tongjun; Li, Yi-Ping

    2014-01-01

    Regulators of G protein signaling (RGS) proteins are a family with more than 30 proteins that all contain an RGS domain. In the past decade, increasing evidence has indicated that RGS proteins play crucial roles in the regulation of G protein coupling receptors (GPCR), G proteins, and calcium signaling during cell proliferation, migration, and differentiation in a variety of tissues. In bone, those proteins modulate bone development and remodeling by influencing various signaling pathways such as GPCR-G protein signaling, Wnt, calcium oscillations and PTH. This review summarizes the recent advances in the understanding of the regulation of RGS genes expression, as well as the functions and mechanisms of RGS proteins, especially in regulating GPCR-G protein signaling, Wnt signaling, calcium oscillations signaling and PTH signaling during bone development and remodeling. This review also highlights the regulation of different RGS proteins in osteoblasts, chondrocytes and osteoclasts. The knowledge from the recent advances of RGS study summarized in the review would provide the insights into new therapies for bone diseases. PMID:24389209

  9. Regulation of TET Protein Stability by Calpains

    PubMed Central

    Wang, Yu; Zhang, Yi

    2014-01-01

    SUMMARY DNA methylation at the fifth position of cytosine (5mC) is an important epigenetic modification that affects chromatin structure and gene expression. Recent studies have established a critical function of the Ten-eleven translocation (Tet) family of proteins in regulating DNA methylation dynamics. Three Tet genes have been identified in mammals, and they all encode for proteins capable of oxidizing 5mC as part of the DNA demethylation process. While regulation of Tet expression at the transcriptional level is well documented, how TET proteins are regulated at post-translational level is poorly understood. In this study, we report that all three TET proteins are direct substrates of calpains, a family of calcium-dependent proteases. Specifically, calpain1 mediates TET1 and TET2 turnover in mouse ES cells, and calpain2 regulates TET3 level during differentiation. This study provides the first evidence that TET proteins are subject to calpain-mediated degradation. PMID:24412366

  10. Induction of AID-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5′-CpG-3′-rich 14-3-3γ promoter and is sustained by E2A

    PubMed Central

    Mai, Thach; Pone, Egest J.; Li, Guideng; Lam, Tonika S.; Moehlman, J’aime; Xu, Zhenming; Casali, Paolo

    2013-01-01

    Class switch DNA recombination (CSR) crucially diversifies antibody biological effectors functions. 14-3-3γ specifically binds to the 5′-AGCT-3′ repeats in the IgH locus switch (S) regions. By directly interacting with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. Here, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by lipopolysaccharides (LPS), and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites (TSSs) for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A. PMID:23851690

  11. Induction of activation-induced cytidine deaminase-targeting adaptor 14-3-3γ is mediated by NF-κB-dependent recruitment of CFP1 to the 5'-CpG-3'-rich 14-3-3γ promoter and is sustained by E2A.

    PubMed

    Mai, Thach; Pone, Egest J; Li, Guideng; Lam, Tonika S; Moehlman, J'aime; Xu, Zhenming; Casali, Paolo

    2013-08-15

    Class switch DNA recombination (CSR) crucially diversifies Ab biologic effector functions. 14-3-3γ specifically binds to the 5'-AGCT-3' repeats in the IgH locus switch (S) regions. By interacting directly with the C-terminal region of activation-induced cytidine deaminase (AID), 14-3-3γ targets this enzyme to S regions to mediate CSR. In this study, we showed that 14-3-3γ was expressed in germinal center B cells in vivo and induced in B cells by T-dependent and T-independent primary CSR-inducing stimuli in vitro in humans and mice. Induction of 14-3-3γ was rapid, peaking within 3 h of stimulation by LPSs, and sustained over the course of AID and CSR induction. It was dependent on recruitment of NF-κB to the 14-3-3γ gene promoter. The NF-κB recruitment enhanced the occupancy of the CpG island within the 14-3-3γ promoter by CFP1, a component of the COMPASS histone methyltransferase complex, and promoter-specific enrichment of histone 3 lysine 4 trimethylation (H3K4me3), which is indicative of open chromatin state and marks transcription-competent promoters. NF-κB also potentiated the binding of B cell lineage-specific factor E2A to an E-box motif located immediately downstream of the two closely-spaced transcription start sites for sustained 14-3-3γ expression and CSR induction. Thus, 14-3-3γ induction in CSR is enabled by the CFP1-mediated H3K4me3 enrichment in the promoter, dependent on NF-κB and sustained by E2A.

  12. Identification of candidate genes for psychosis in rat models, and possible association between schizophrenia and the 14-3-3eta gene.

    PubMed

    Wong, A H C; Macciardi, F; Klempan, T; Kawczynski, W; Barr, C L; Lakatoo, S; Wong, M; Buckle, C; Trakalo, J; Boffa, E; Oak, J; Azevedo, M-H; Dourado, A; Coelho, I; Macedo, A; Vicente, A; Valente, J; Ferreira, C P; Pato, M T; Pato, C N; Kennedy, J L; Van Tol, H H M

    2003-02-01

    Although the genetic contribution to schizophrenia is substantial, positive findings in whole-genome linkage scans have not been consistently replicated. We analyzed gene expression in various rat conditions to identify novel candidate genes for schizophrenia. Suppression subtraction hybridization (SSH), with polyA mRNA from temporal and frontal cortex of rats, was used to identify differentially expressed genes. Expression of mRNA was compared between adult Lewis and Fischer 344 (F344) rats, adult and postnatal day 6 (d6) F344, and adult F344 treated with haloperidol or control vehicle. These groups were chosen because each highlights a particular aspect of schizophrenia: differences in strain vulnerability to behavioral analogs of psychosis; factors that may relate to disease onset in relation to CNS development; and improvement of symptoms by haloperidol. The 14-3-3 gene family, as represented by 14-3-3gamma and 14-3-3zeta isoforms in the SSH study, and SNAP-25 were among the candidate genes. Genetic association between schizophrenia and the 14-3-3eta gene, positioned close to a genomic locus implicated in schizophrenia, and SNAP-25 genes was analyzed in 168 schizophrenia probands and their families. These findings address three different genes in the 14-3-3 family. We find a significant association with schizophrenia for two polymorphisms in the 14-3-3eta gene: a 7 bp variable number of tandem repeats in the 5' noncoding region (P=0.036, 1 df), and a 3' untranslated region SNP (753G/A) that is an RFLP visualized with Ava II (P=0.028). There was no significant genetic association with SNAP-25. The candidate genes identified may be of functional importance in the etiology, pathophysiology or treatment response of schizophrenia or psychotic symptoms. This is to our knowledge the first report of a significant association between the 14-3-3eta-chain gene and schizophrenia in a family-based sample, strengthening prior association reports in case-control studies and

  13. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    NASA Astrophysics Data System (ADS)

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-08-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells.

  14. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells.

    PubMed

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including "p38 MAPK Signaling", "p53 Signaling", "14-3-3-mediated Signaling", "p70S6K Signaling" and "Protein Ubiquitination Pathway". This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  15. Mitochondria-Mediated Protein Regulation Mechanism of Polymorphs-Dependent Inhibition of Nanoselenium on Cancer Cells

    PubMed Central

    Wang, Ge; Guo, Yuming; Yang, Gai; Yang, Lin; Ma, Xiaoming; Wang, Kui; Zhu, Lin; Sun, Jiaojiao; Wang, Xiaobing; Zhang, Hua

    2016-01-01

    The present study was (i) to prepare two types of selenium nanoparticles, namely an amorphous form of selenium quantum dots (A-SeQDs) and a crystalline form of selenium quantum dots (C-SeQDs); and (ii) to investigate the nano-bio interactions of A-SeQDs and C-SeQDs in MCF-7, HepG2, HeLa, NIH/3T3, L929 cells and BRL-3A cells. It was found that A-SeQDs could induce the mitochondria-mediated apoptosis, necrosis and death of cells, while C-SeQDs had much weaker effects. This polymorphs-dependent anti-proliferative activity of nano-selenium was scarcely reported. Further investigation demonstrated that A-SeQDs could differentially regulate 61 proteins and several pathways related to stress response, protein synthesis, cell migration and cell cycle, including “p38 MAPK Signaling”, “p53 Signaling”, “14-3-3-mediated Signaling”, “p70S6K Signaling” and “Protein Ubiquitination Pathway”. This was the first report to demonstrate the involvement of protein synthesis and post-translational modification pathways in the anti-proliferative activity associated with NMs. Compared with previously fragmentary studies, this study use a nanomics approach combining bioinformatics and proteomics to systematically investigate the nano-bio interactions of selenium nanoparticles in cancer cells. PMID:27514819

  16. FET proteins regulate lifespan and neuronal integrity

    PubMed Central

    Therrien, Martine; Rouleau, Guy A.; Dion, Patrick A.; Parker, J. Alex

    2016-01-01

    The FET protein family includes FUS, EWS and TAF15 proteins, all of which have been linked to amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motor neurons. Here, we show that a reduction of FET proteins in the nematode Caenorhabditis elegans causes synaptic dysfunction accompanied by impaired motor phenotypes. FET proteins are also involved in the regulation of lifespan and stress resistance, acting partially through the insulin/IGF-signalling pathway. We propose that FET proteins are involved in the maintenance of lifespan, cellular stress resistance and neuronal integrity. PMID:27117089

  17. Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses.

    PubMed

    Cao, Hui; Xu, Yuxing; Yuan, Linlin; Bian, Yanwei; Wang, Lihui; Zhen, Shoumin; Hu, Yingkao; Yan, Yueming

    2016-01-01

    The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd(2+), Cr(3+), Cu(2+), and Zn(2+)) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up

  18. Molecular Characterization of the 14-3-3 Gene Family in Brachypodium distachyon L. Reveals High Evolutionary Conservation and Diverse Responses to Abiotic Stresses

    PubMed Central

    Cao, Hui; Xu, Yuxing; Yuan, Linlin; Bian, Yanwei; Wang, Lihui; Zhen, Shoumin; Hu, Yingkao; Yan, Yueming

    2016-01-01

    The 14-3-3 gene family identified in all eukaryotic organisms is involved in a wide range of biological processes, particularly in resistance to various abiotic stresses. Here, we performed the first comprehensive study on the molecular characterization, phylogenetics, and responses to various abiotic stresses of the 14-3-3 gene family in Brachypodium distachyon L. A total of seven 14-3-3 genes from B. distachyon and 120 from five main lineages among 12 species were identified, which were divided into five well-conserved subfamilies. The molecular structure analysis showed that the plant 14-3-3 gene family is highly evolutionarily conserved, although certain divergence had occurred in different subfamilies. The duplication event investigation revealed that segmental duplication seemed to be the predominant form by which the 14-3-3 gene family had expanded. Moreover, seven critical amino acids were detected, which may contribute to functional divergence. Expression profiling analysis showed that BdGF14 genes were abundantly expressed in the roots, but showed low expression in the meristems. All seven BdGF14 genes showed significant expression changes under various abiotic stresses, including heavy metal, phytohormone, osmotic, and temperature stresses, which might play important roles in responses to multiple abiotic stresses mainly through participating in ABA-dependent signaling and reactive oxygen species-mediated MAPK cascade signaling pathways. In particular, BdGF14 genes generally showed upregulated expression in response to multiple stresses of high temperature, heavy metal, abscisic acid (ABA), and salicylic acid (SA), but downregulated expression under H2O2, NaCl, and polyethylene glycol (PEG) stresses. Meanwhile, dynamic transcriptional expression analysis of BdGF14 genes under longer treatments with heavy metals (Cd2+, Cr3+, Cu2+, and Zn2+) and phytohormone (ABA) and recovery revealed two main expression trends in both roots and leaves: up-down and up

  19. Regulation of cardiac C-protein phosphorylation

    SciTech Connect

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased (/sup 32/P)phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and (/sup 32/P)phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 ..mu..M Iso and 17% in hearts exposed to Iso plus 1 ..mu..M methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed.

  20. Regulation of cellular transport by klotho protein.

    PubMed

    Sopjani, Mentor; Rinnerthaler, Mark; Almilaji, Ahmad; Ahmeti, Salih; Dermaku-Sopjani, Miribane

    2014-01-01

    The antiaging protein of Klotho is a transmembrane protein mainly expressed in the kidney, parathyroid glands and choroid plexus of the brain. The Klotho protein exists in two forms, a full-length membrane form and a soluble secreted form. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it acts as β-glucuronidase and a hormone. Soluble Klotho can be found in the blood, cerebrospinal fluid, and the urine of mammals. Klotho deficiency results in early appearance of multiple age-related disorders and premature death, whereas overexpression of Klotho exerts the opposite effect. Klotho may influence cellular transport processes across the cell membrane by inhibiting calcitriol (1,25(OH) (2)D(3)), formation or by directly affecting transporter proteins, including ion channels, carriers and pumps. Accordingly, Klotho protein is a powerful regulator of transport mechanisms across the cell membrane. Klotho regulates diverse calcium and potassium ion channels, as well as several carriers including the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, the Na(+)-coupled phosphate cotransporters, NaPi-IIa and NaPi-IIb, and a Na(+)/K(+)-ATPase. All those cellular transport regulations contribute in the aging suppressor role of Klotho. Future studies will help to determine if the Klotho protein regulates cell-surface expression of other transport proteins and is affecting underlying mechanisms.

  1. Regulation of cell proliferation by G proteins.

    PubMed

    Dhanasekaran, N; Tsim, S T; Dermott, J M; Onesime, D

    1998-09-17

    G Proteins provide signal transduction mechanisms to seven transmembrane receptors. Recent studies have indicated that the alpha-subunits as well as the betagamma-subunits of these proteins regulate several critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Of the 17 alpha-subunits that have been cloned, at least ten of them have been shown to couple mitogenic signaling in fibroblast cells. Activating mutations in G alpha(s), G alpha(i)2, and G alpha12 have been correlated with different types of tumors. In addition, the ability of the betagamma-subunits to activate mitogenic pathways in different cell-types has been defined. The present review briefly summarizes the diverse and novel signaling pathways regulated by the alpha- as well as the betagamma-subunits of G proteins in regulating cell proliferation. PMID:9779986

  2. [RGS proteins (regulators of G protein signaling) and their roles in regulation of immune response].

    PubMed

    Lewandowicz, Anna M; Kowalski, Marek L; Pawliczak, Rafał

    2004-01-01

    RGS proteins (Regulators of G-protein Signaling) comprise a protein family responsible for regulating G proteins. By enhancing the GTPase activity of the a subunit, they speed up the reconstruction of the heterotrimeric structure of G protein, thus inhibiting its signal transduction. Sst2 protein in yeast Saccharomyces cervisiae, FlbA in fungus Aspergillus nidulans, and Egl-10 in the nematode Caenorhabditis elegans are the first native G regulators with GTPase activity (GAPs:--GTPase-activating proteins). The existence of over 30 RGS human proteins has been confirmed thus far, and they have been grouped and classified into six subfamilies. In immunocompetent cells, RGS proteins are entangled in a complicate net of different interrelating signal pathways. They are connected with B- and T-cell chemokine susceptibility, efficient T cell proliferation, and the regulation of B cell maturation. They also take an essential part in inflammation. High hopes are held for drugs, which handle would be RGS proteins and which would further provide the possibility of modifying the pharmacokinetics of drugs acting through G protein- coupled receptors. The aim of this review is to discuss the new RGS protein family and explain the potential involvement of RGS proteins in the modulation of the immune response PMID:15459549

  3. The Scaffold Protein POSH Regulates Axon Outgrowth

    PubMed Central

    Taylor, Jennifer; Chung, Kwan-Ho; Figueroa, Claudia; Zurawski, Jonathan; Dickson, Heather M.; Brace, E. J.; Avery, Adam W.; Turner, David L.

    2008-01-01

    How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth. Complementation analysis reveals a requirement for the third Src homology (SH) 3 domain of POSH, and we find that the actomyosin regulatory protein Shroom3 interacts with this domain of POSH. Inhibition of Shroom3 expression by RNAi leads to increased process lengths, as observed for POSH RNAi, suggesting that POSH and Shroom function together to inhibit process outgrowth. Complementation analysis and interference of protein function by dominant-negative approaches suggest that Shroom3 recruits Rho kinase to inhibit process outgrowth. Furthermore, inhibition of myosin II function reverses the POSH or Shroom3 RNAi phenotype, indicating a role for myosin II regulation as a target of the POSH–Shroom complex. Collectively, these results suggest that the molecular scaffold protein POSH assembles an inhibitory complex that links to the actin–myosin network to regulate neuronal process outgrowth. PMID:18829867

  4. The scaffold protein POSH regulates axon outgrowth.

    PubMed

    Taylor, Jennifer; Chung, Kwan-Ho; Figueroa, Claudia; Zurawski, Jonathan; Dickson, Heather M; Brace, E J; Avery, Adam W; Turner, David L; Vojtek, Anne B

    2008-12-01

    How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth. Complementation analysis reveals a requirement for the third Src homology (SH) 3 domain of POSH, and we find that the actomyosin regulatory protein Shroom3 interacts with this domain of POSH. Inhibition of Shroom3 expression by RNAi leads to increased process lengths, as observed for POSH RNAi, suggesting that POSH and Shroom function together to inhibit process outgrowth. Complementation analysis and interference of protein function by dominant-negative approaches suggest that Shroom3 recruits Rho kinase to inhibit process outgrowth. Furthermore, inhibition of myosin II function reverses the POSH or Shroom3 RNAi phenotype, indicating a role for myosin II regulation as a target of the POSH-Shroom complex. Collectively, these results suggest that the molecular scaffold protein POSH assembles an inhibitory complex that links to the actin-myosin network to regulate neuronal process outgrowth.

  5. Acetylation regulates Jun protein turnover in Drosophila.

    PubMed

    Zhang, Daoyong; Suganuma, Tamaki; Workman, Jerry L

    2013-11-01

    C-Jun is a major transcription factor belonging to the activating protein 1 (AP-1) family. Phosphorylation has been shown to be critical for c-Jun activation and stability. Here, we report that Jra, the Drosophila Jun protein, is acetylated in vivo. We demonstrate that the acetylation of Jra leads to its rapid degradation in response to osmotic stress. Intriguingly, we also found that Jra phosphorylation antagonized its acetylation, indicating the opposite roles of acetylation and phosphorylation in Jra degradation process under osmotic stress. Our results provide new insights into how c-Jun proteins are precisely regulated by the interplay of different posttranslational modifications.

  6. Redox regulation of protein damage in plasma

    PubMed Central

    Griffiths, Helen R.; Dias, Irundika H.K.; Willetts, Rachel S.; Devitt, Andrew

    2014-01-01

    The presence and concentrations of modified proteins circulating in plasma depend on rates of protein synthesis, modification and clearance. In early studies, the proteins most frequently analysed for damage were those which were more abundant in plasma (e.g. albumin and immunoglobulins) which exist at up to 10 orders of magnitude higher concentrations than other plasma proteins e.g. cytokines. However, advances in analytical techniques using mass spectrometry and immuno-affinity purification methods, have facilitated analysis of less abundant, modified proteins and the nature of modifications at specific sites is now being characterised. The damaging reactive species that cause protein modifications in plasma principally arise from reactive oxygen species (ROS) produced by NADPH oxidases (NOX), nitric oxide synthases (NOS) and oxygenase activities; reactive nitrogen species (RNS) from myeloperoxidase (MPO) and NOS activities; and hypochlorous acid from MPO. Secondary damage to proteins may be caused by oxidized lipids and glucose autooxidation. In this review, we focus on redox regulatory control of those enzymes and processes which control protein maturation during synthesis, produce reactive species, repair and remove damaged plasma proteins. We have highlighted the potential for alterations in the extracellular redox compartment to regulate intracellular redox state and, conversely, for intracellular oxidative stress to alter the cellular secretome and composition of extracellular vesicles. Through secreted, redox-active regulatory molecules, changes in redox state may be transmitted to distant sites. PMID:24624332

  7. Regulation of Mutant p53 Protein Expression

    PubMed Central

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation. PMID:26734569

  8. SUMOylation regulates the SNF1 protein kinase.

    PubMed

    Simpson-Lavy, Kobi J; Johnston, Mark

    2013-10-22

    The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK's homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source.

  9. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  10. Distortion of homeostatic signaling proteins by simulated microgravity in rat hypothalamus: A(16) O/(18) O-labeled comparative integrated proteomic approach.

    PubMed

    Iqbal, Javed; Li, Wang; Hasan, Murtaza; Juan Li, Yu; Ullah, Kaleem; Yun, Wang; Awan, Umer; Qing, Hong; Deng, Yulin

    2014-02-01

    Microgravity generates oxidative stress in central nervous system, causing distortion of various vital signaling cascades involved in many homeostatic functions. Here, we performed comparative (16) O/(18) O labeled integrated proteomic strategy to observe the differential expression of signaling proteins involved in homeostasis. In this study, rat-tail suspension model is employed to induce simulated microgravity in CNS. By wide proteomic analysis, total of 35 and 97 significantly differentially expressed proteins were found by HPLC/ESI-TOF and HPLC-Q-TOF analysis, respectively. Among the total of 132 proteins quantified, 25 proteins were found related to various signaling cascades. Protein Thy-1, 14-3-3 gamma, 14-3-3 epsilon, 14-3-3 theta, 14-3-3 eta, and 14-3-3 beta/alpha proteins, calmodulin and calcium/calmodulin-dependent protein kinase type-II subunit beta were found upregulated under the influence of simulated microgravity. These proteins are found involved in disrupting homeostatic pathways like sleep/wake cycle, drinking behavior, hypothalamic-pituitary-adrenocortical regulation and fight and/or flee actions under stress. Furthermore, MS results for protein Thy-1 were verified by Western blot analysis showing the quantification accuracy of MS instruments. Results presented here will serve as means to understand the mechanism of action of microgravity and further reference for future detailed study of consequences of microgravity on astronauts and their possible countermeasures.

  11. Heat shock proteins: essential proteins for apoptosis regulation

    PubMed Central

    Lanneau, D; Brunet, M; Frisan, E; Solary, E; Fontenay, M; Garrido, C

    2008-01-01

    Abstract Many different external and intrinsic apoptotic stimuli induce the accumulation in the cells of a set of proteins known as stress or heat shock proteins (HSPs). HSPs are conserved proteins present in both prokaryotes and eukaryotes. These proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. HSPs have a protective function, that is they allow the cells to survive to otherwise lethal conditions. Various mechanisms have been proposed to account for the cytoprotective functions of HSPs. Several of these proteins have demonstrated to directly interact with components of the cell signalling pathways, for example those of the tightly regulated caspasedependent programmed cell death machinery, upstream, downstream and at the mitochondrial level. HSPs can also affect caspase-independent apoptosis-like process by interacting with apoptogenic factors such as apoptosis-inducing factor (AIF) or by acting at the lysosome level. This review will describe the different key apoptotic proteins interacting with HSPs and the consequences of these interactions in cell survival, proliferation and apoptotic processes. Our purpose will be illustrated by emerging strategies in targeting these protective proteins to treat haematological malignancies. PMID:18266962

  12. Regulation of Pluripotency by RNA Binding Proteins

    PubMed Central

    Ye, Julia; Blelloch, Robert

    2015-01-01

    Establishment, maintenance, and exit from pluripotency require precise coordination of a cell’s molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells. PMID:25192462

  13. Proteomic pathway analysis of the hippocampus in schizophrenia and bipolar affective disorder implicates 14-3-3 signaling, aryl hydrocarbon receptor signaling, and glucose metabolism: potential roles in GABAergic interneuron pathology.

    PubMed

    Schubert, Klaus Oliver; Föcking, Melanie; Cotter, David R

    2015-09-01

    Neuropathological changes of the hippocampus have been associated with psychotic disorders such as schizophrenia and bipolar disorder. Recent work has particularly implicated hippocampal GABAergic interneurons in the pathophysiology of these diseases. However, the molecular mechanisms underlying structural and cellular hippocampal pathology remain poorly understood. We used data from comprehensive difference-in-gel electrophoresis (2-D DIGE) investigations of postmortem human hippocampus of people with schizophrenia and bipolar disorder, covering the acidic (isoelectric point (pI) between pH4 and 7) and, separately, the basic (pI between pH6 and 11) sub-proteome, for Ingenuity Pathway Analysis (IPA) of implicated protein networks and pathways. Comparing disease and control cases, we identified 58 unique differentially expressed proteins in schizophrenia, and 70 differentially expressed proteins in bipolar disorder, using mass spectrometry. IPA implicated, most prominently, 14-3-3 and aryl hydrocarbon receptor signaling in schizophrenia, and gluconeogenesis/glycolysis in bipolar disorder. Both disorders were characterized by alterations of proteins involved in the oxidative stress response, mitochondrial function, and protein-endocytosis, -trafficking, -degradation, and -ubiquitination. These findings are interpreted with a focus on GABAergic interneuron pathology in the hippocampus.

  14. The FAD-dependent glycerol-3-phosphate dehydrogenase of Giardia duodenalis: an unconventional enzyme that interacts with the g14-3-3 and it is a target of the antitumoral compound NBDHEX

    PubMed Central

    Lalle, Marco; Camerini, Serena; Cecchetti, Serena; Finelli, Renata; Sferra, Gabriella; Müller, Joachim; Ricci, Giorgio; Pozio, Edoardo

    2015-01-01

    The flagellated protozoan Giardia duodenalis is a worldwide parasite causing giardiasis, an acute and chronic diarrheal disease. Metabolism in G. duodenalis has a limited complexity thus making metabolic enzymes ideal targets for drug development. However, only few metabolic pathways (i.e., carbohydrates) have been described so far. Recently, the parasite homolog of the mitochondrial-like glycerol-3-phosphate dehydrogenase (gG3PD) has been identified among the interactors of the g14-3-3 protein. G3PD is involved in glycolysis, electron transport, glycerophospholipids metabolism, and hyperosmotic stress response, and is emerging as promising target in tumor treatment. In this work, we demonstrate that gG3PD is a functional flavoenzyme able to convert glycerol-3-phosphate into dihydroxyacetone phosphate and that its activity and the intracellular glycerol level increase during encystation. Taking advantage of co-immunoprecipitation assays and deletion mutants, we provide evidence that gG3PD and g14-3-3 interact at the trophozoite stage, the intracellular localization of gG3PD is stage dependent and it partially co-localizes with mitosomes during cyst development. Finally, we demonstrate that the gG3PD activity is affected by the antitumoral compound 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol, that results more effective in vitro at killing G. duodenalis trophozoites than the reference drug metronidazole. Overall, our results highlight the involvement of gG3PD in processes crucial for the parasite survival thus proposing this enzyme as target for novel antigiardial interventions. PMID:26082764

  15. Regulation of gene transcription by Polycomb proteins

    PubMed Central

    Aranda, Sergi; Mas, Gloria; Di Croce, Luciano

    2015-01-01

    The Polycomb group (PcG) of proteins defines a subset of factors that physically associate and function to maintain the positional identity of cells from the embryo to adult stages. PcG has long been considered a paradigmatic model for epigenetic maintenance of gene transcription programs. Despite intensive research efforts to unveil the molecular mechanisms of action of PcG proteins, several fundamental questions remain unresolved: How many different PcG complexes exist in mammalian cells? How are PcG complexes targeted to specific loci? How does PcG regulate transcription? In this review, we discuss the diversity of PcG complexes in mammalian cells, examine newly identified modes of recruitment to chromatin, and highlight the latest insights into the molecular mechanisms underlying the function of PcGs in transcription regulation and three-dimensional chromatin conformation. PMID:26665172

  16. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  17. Abundant protein phosphorylation potentially regulates Arabidopsis anther development

    PubMed Central

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-01-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana. However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4–7 and 8–12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  18. Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

    PubMed

    Ye, Juanying; Zhang, Zaibao; You, Chenjiang; Zhang, Xumin; Lu, Jianan; Ma, Hong

    2016-09-01

    As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development. PMID:27531888

  19. Matricellular protein Cfl1 regulates cell differentiation.

    PubMed

    Tian, Xiuyun; Lin, Xiaorong

    2013-11-01

    Like higher eukaryotic cells in tissues, microbial cells in a community act in concert in response to environmental stimuli. They coordinate gene expression and their physiological and morphological states through intercellular communication mediated by matricellular signals. The adhesion protein Cfl1 was recently shown to be a matricellular signal in regulating morphogenesis and biofilm formation in the eukaryotic microbe Cryptococcus neoformans. Cfl1 is naturally highly expressed in the hyphal subpopulation during the mating colony development. Some Cfl1 proteins are cleaved and released to the ECM (extracellular matrix). The released exogenous Cfl1 activates Cryptococcus cells to express their endogenous Cfl1, to undergo filamentation, and to form structured biofilm colonies. In this study, we demonstrate that the N-terminal signal peptide and the novel conserved cysteine-rich SIGC domain at the C-terminus are critical for the adherence property and the signaling activity of this multifunctional protein. The investigation of this fungal matricellular signaling network involving Cfl1 and the master regulator of morphogenesis Znf2 provides a foundation to further elucidate intercellular communication in microbial development.

  20. YB-1 protein: functions and regulation.

    PubMed

    Lyabin, Dmitry N; Eliseeva, Irina A; Ovchinnikov, Lev P

    2014-01-01

    The Y-box binding protein 1 (YB-1, YBX1) is a member of the family of DNA- and RNA-binding proteins with an evolutionarily ancient and conserved cold shock domain. It falls into a group of intrinsically disordered proteins that do not follow the classical rule 'one protein-one function' but introduce a novel principle stating that a disordered structure suggests many functions. YB-1 participates in a wide variety of DNA/RNA-dependent events, including DNA reparation, pre-mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. At the cell level, the multiple activities of YB-1 are manifested as its involvement in cell proliferation and differentiation, stress response, and malignant cell transformation. WIREs RNA 2014, 5:95-110. doi: 10.1002/wrna.1200 CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article. For further resources related to this article, please visit the WIREs website.

  1. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  2. Regulators of G-protein-signaling proteins: negative modulators of G-protein-coupled receptor signaling.

    PubMed

    Woodard, Geoffrey E; Jardín, Isaac; Berna-Erro, A; Salido, Gines M; Rosado, Juan A

    2015-01-01

    Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.

  3. The insulator protein CTCF regulates Drosophila steroidogenesis.

    PubMed

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B; Espinàs, M Lluisa

    2015-01-01

    The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis. PMID:25979705

  4. The insulator protein CTCF regulates Drosophila steroidogenesis

    PubMed Central

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B.; Espinàs, M. Lluisa

    2015-01-01

    ABSTRACT The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis. PMID:25979705

  5. The insulator protein CTCF regulates Drosophila steroidogenesis.

    PubMed

    Fresán, Ujué; Cuartero, Sergi; O'Connor, Michael B; Espinàs, M Lluisa

    2015-05-15

    The steroid hormone ecdysone is a central regulator of insect development. In this report we show that CTCF expression in the prothoracic gland is required for full transcriptional activation of the Halloween genes spookier, shadow and noppera-bo, which encode ecdysone biosynthetic enzymes, and for proper timing of ecdysone-responsive gene expression. Loss of CTCF results in delayed and less synchronized larval development that can only be rescued by feeding larvae with both, the steroid hormone 20-hydroxyecdysone and cholesterol. Moreover, CTCF-knockdown in prothoracic gland cells leads to increased lipid accumulation. In conclusion, the insulator protein CTCF is required for Halloween gene expression and cholesterol homeostasis in ecdysone-producing cells controlling steroidogenesis.

  6. Dynamics in enzymatic protein complexes offer a novel principle for the regulation of melatonin synthesis in the human pineal gland.

    PubMed

    Maronde, Erik; Saade, Anastasia; Ackermann, Katrin; Goubran-Botros, Hany; Pagan, Cecile; Bux, Roman; Bourgeron, Thomas; Dehghani, Faramarz; Stehle, Jörg H

    2011-08-01

    Time of day is communicated to the body through rhythmic cues, including pineal gland melatonin synthesis, which is restricted to nighttime. Whereas in most rodents transcriptional regulation of the arylalkylamine N-acetyltransferase (Aanat) gene is essential for rhythmic melatonin synthesis, investigations into nonrodent mammalian species have shown post-transcriptional regulation to be of central importance, with molecular mechanisms still elusive. Therefore, human pineal tissues, taken from routine autopsies were allocated to four time-of-death groups (night/dawn/day/dusk) and analyzed for daytime-dependent changes in phosphorylated AANAT (p31T-AANAT) and in acetyl-serotonin-methyltransferase (ASMT) expression and activity. Protein content, intracellular localization, and colocalization of p31T-AANAT and ASMT were assessed, using immunoblotting, immunofluorescence, and immunoprecipitation techniques. Fresh sheep pineal gland preparations were used for comparative purposes. The amount of p31T-AANAT and ASMT proteins as well as their intracellular localization showed no diurnal variation in autoptic human and fresh sheep pineal glands. Moreover, in human and sheep pineal extracts, AANAT could not be dephosphorylated, which was at variance to data derived from rat pineal extracts. P31T-AANAT and ASMT were often found to colocalize in cellular rod-like structures that were also partly immunoreactive for the pinealocyte process-specific marker S-antigen (arrestin) in both, human and sheep pinealocytes. Protein-protein interaction studies with p31T-AANAT, ASMT, and S-antigen demonstrated a direct association and formation of robust complexes, involving also 14-3-3. This work provides evidence for a regulation principle for AANAT activity in the human pineal gland, which may not be based on a p31T-AANAT phosphorylation/dephosphorylation switch, as described for other mammalian species.

  7. An Overview of Chromatin-Regulating Proteins in Cells

    PubMed Central

    Zhang, Pingyu; Torres, Keila; Liu, Xiuping; Liu, Chang-gong; Pollock, Raphael E.

    2016-01-01

    In eukaryotic cells, gene expressions on chromosome DNA are orchestrated by a dynamic chromosome structure state that is largely controlled by chromatin-regulating proteins, which regulate chromatin structures, release DNA from the nucleosome, and activate or suppress gene expression by modifying nucleosome histones or mobilizing DNA-histone structure. The two classes of chromatin- regulating proteins are 1) enzymes that modify histones through methylation, acetylation, phosphorylation, adenosine diphosphate–ribosylation, glycosylation, sumoylation, or ubiquitylation and 2) enzymes that remodel DNA-histone structure with energy from ATP hydrolysis. Chromatin-regulating proteins, which modulate DNA-histone interaction, change chromatin conformation, and increase or decrease the binding of functional DNA-regulating protein complexes, have major functions in nuclear processes, including gene transcription and DNA replication, repair, and recombination. This review provides a general overview of chromatin-regulating proteins, including their classification, molecular functions, and interactions with the nucleosome in eukaryotic cells. PMID:26796306

  8. Id proteins: small molecules, mighty regulators.

    PubMed

    Ling, Flora; Kang, Bin; Sun, Xiao-Hong

    2014-01-01

    The family of inhibitor of differentiation (Id) proteins is a group of evolutionarily conserved molecules, which play important regulatory roles in organisms ranging from Drosophila to humans. Id proteins are small polypeptides harboring a helix-loop-helix (HLH) motif, which are best known to mediate dimerization with other basic HLH proteins, primarily E proteins. Because Id proteins do not possess the basic amino acids adjacent to the HLH motif necessary for DNA binding, Id proteins inhibit the function of E protein homodimers, as well as heterodimers between E proteins and tissue-specific bHLH proteins. However, Id proteins have also been shown to have E protein-independent functions. The Id genes are broadly but differentially expressed in a variety of cell types. Transcription of the Id genes is controlled by transcription factors such as C/EBPβ and Egr as well as by signaling pathways triggered by different stimuli, which include bone morphogenic proteins, cytokines, and ligands of T cell receptors. In general, Id proteins are capable of inhibiting the differentiation of progenitors of different cell types, promoting cell-cycle progression, delaying cellular senescence, and facilitating cell migration. These properties of Id proteins enable them to play significant roles in stem cell maintenance, vasculogenesis, tumorigenesis and metastasis, the development of the immune system, and energy metabolism. In this review, we intend to highlight the current understanding of the function of Id proteins and discuss gaps in our knowledge about the mechanisms whereby Id proteins exert their diverse effects in multiple cellular processes.

  9. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators. PMID:27515397

  10. Regulation, Signaling, and Physiological Functions of G-Proteins.

    PubMed

    Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

    2016-09-25

    Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators.

  11. Regulation of intestinal protein metabolism by amino acids.

    PubMed

    Bertrand, Julien; Goichon, Alexis; Déchelotte, Pierre; Coëffier, Moïse

    2013-09-01

    Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50% per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.

  12. HnRNP-like proteins as post-transcriptional regulators.

    PubMed

    Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

    2014-10-01

    Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses.

  13. HnRNP-like proteins as post-transcriptional regulators.

    PubMed

    Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

    2014-10-01

    Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. PMID:25219311

  14. Roles for Regulator of G Protein Signaling Proteins in Synaptic Signaling and Plasticity.

    PubMed

    Gerber, Kyle J; Squires, Katherine E; Hepler, John R

    2016-02-01

    The regulator of G protein signaling (RGS) family of proteins serves critical roles in G protein-coupled receptor (GPCR) and heterotrimeric G protein signal transduction. RGS proteins are best understood as negative regulators of GPCR/G protein signaling. They achieve this by acting as GTPase activating proteins (GAPs) for Gα subunits and accelerating the turnoff of G protein signaling. Many RGS proteins also bind additional signaling partners that either regulate their functions or enable them to regulate other important signaling events. At neuronal synapses, GPCRs, G proteins, and RGS proteins work in coordination to regulate key aspects of neurotransmitter release, synaptic transmission, and synaptic plasticity, which are necessary for central nervous system physiology and behavior. Accumulating evidence has revealed key roles for specific RGS proteins in multiple signaling pathways at neuronal synapses, regulating both pre- and postsynaptic signaling events and synaptic plasticity. Here, we review and highlight the current knowledge of specific RGS proteins (RGS2, RGS4, RGS7, RGS9-2, and RGS14) that have been clearly demonstrated to serve critical roles in modulating synaptic signaling and plasticity throughout the brain, and we consider their potential as future therapeutic targets.

  15. Bcl-2 family proteins: master regulators of cell survival.

    PubMed

    Hatok, Jozef; Racay, Peter

    2016-08-01

    The most prominent function of proteins of the Bcl-2 family is regulation of the initiation of intrinsic (mitochondrial) pathways of apoptosis. However, recent research has revealed that in addition to regulation of mitochondrial apoptosis, proteins of the Bcl-2 family play important roles in regulating other cellular pathways with a strong impact on cell survival like autophagy, endoplasmic reticulum (ER) stress response, intracellular calcium dynamics, cell cycle progression, mitochondrial dynamics and energy metabolism. This review summarizes the recent knowledge about functions of Bcl-2 family proteins that are related to cell survival. PMID:27505095

  16. Transcriptional Regulation by Trithorax-Group Proteins

    PubMed Central

    Kingston, Robert E.; Tamkun, John W.

    2014-01-01

    The trithorax group of genes (trxG) was identified in mutational screens that examined developmental phenotypes and suppression of Polycomb mutant phenotypes. The protein products of these genes are primarily involved in gene activation, although some can also have repressive effects. There is no central function for these proteins. Some move nucleosomes about on the genome in an ATP-dependent manner, some covalently modify histones such as methylating lysine 4 of histone H3, and some directly interact with the transcription machinery or are a part of that machinery. It is interesting to consider why these specific members of large families of functionally related proteins have strong developmental phenotypes. PMID:25274705

  17. MTBreg: The Database of Conditionally Regulated Proteins in Mycobacterium Tuberculosis

    DOE Data Explorer

    Kaufman, Markus; Pal, Debnath; Eisenberg, David

    Proteins up- and down- regulated in Mycobacterium tuberculosis grown under conditions mimicking infection are included in this database. It also includes information on proteins that are regulated by selected transcription factors or other regulatory proteins. The literature data provided here is complimentary to the databases provided by Michael Strong that include recent TB computational functional linkages and the Prolinks Database by Peter Bowers. The experimental condition, the experimental dataset and a literature reference will be displayed, including links to the computationally linked proteins in the Prolinks Database and the entry in the Mycobacterium tuberculosis Structural Genomics Database.[Copied from information at http://www.doe-mbi.ucla.edu/Services/MTBreg/

  18. Copper Delivery to Chloroplast Proteins and its Regulation

    PubMed Central

    Aguirre, Guadalupe; Pilon, Marinus

    2016-01-01

    Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field. PMID:26793223

  19. Regulating Rap small G-proteins in time and space.

    PubMed

    Gloerich, Martijn; Bos, Johannes L

    2011-10-01

    Signaling by the small G-protein Rap is under tight regulation by its GEFs and GAPs. These are multi-domain proteins that are themselves controlled by distinct upstream pathways, and thus couple different extra- and intracellular cues to Rap. The individual RapGEFs and RapGAPs are, in addition, targeted to specific cellular locations by numerous anchoring mechanisms and, consequently, may control different pools of Rap. Here, we review the various activating signals and targeting mechanisms of these proteins and discuss their contribution to the spatiotemporal regulation and biological functions of the Rap proteins.

  20. Trafficking regulation of proteins in Alzheimer’s disease

    PubMed Central

    2014-01-01

    The β-amyloid (Aβ) peptide has been postulated to be a key determinant in the pathogenesis of Alzheimer’s disease (AD). Aβ is produced through sequential cleavage of the β-amyloid precursor protein (APP) by β- and γ-secretases. APP and relevant secretases are transmembrane proteins and traffic through the secretory pathway in a highly regulated fashion. Perturbation of their intracellular trafficking may affect dynamic interactions among these proteins, thus altering Aβ generation and accelerating disease pathogenesis. Herein, we review recent progress elucidating the regulation of intracellular trafficking of these essential protein components in AD. PMID:24410826

  1. Regulation of tomato Prf by Pto-like protein kinases.

    PubMed

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  2. Minimalist Design of Allosterically Regulated Protein Catalysts.

    PubMed

    Makhlynets, O V; Korendovych, I V

    2016-01-01

    Nature facilitates chemical transformations with exceptional selectivity and efficiency. Despite a tremendous progress in understanding and predicting protein function, the overall problem of designing a protein catalyst for a given chemical transformation is far from solved. Over the years, many design techniques with various degrees of complexity and rational input have been developed. Minimalist approach to protein design that focuses on the bare minimum requirements to achieve activity presents several important advantages. By focusing on basic physicochemical properties and strategic placing of only few highly active residues one can feasibly evaluate in silico a very large variety of possible catalysts. In more general terms minimalist approach looks for the mere possibility of catalysis, rather than trying to identify the most active catalyst possible. Even very basic designs that utilize a single residue introduced into nonenzymatic proteins or peptide bundles are surprisingly active. Because of the inherent simplicity of the minimalist approach computational tools greatly enhance its efficiency. No complex calculations need to be set up and even a beginner can master this technique in a very short time. Here, we present a step-by-step protocol for minimalist design of functional proteins using basic, easily available, and free computational tools. PMID:27586334

  3. Tor1 regulates protein solubility in Saccharomyces cerevisiae

    PubMed Central

    Peters, Theodore W.; Rardin, Matthew J.; Czerwieniec, Gregg; Evani, Uday S.; Reis-Rodrigues, Pedro; Lithgow, Gordon J.; Mooney, Sean D.; Gibson, Bradford W.; Hughes, Robert E.

    2012-01-01

    Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase. PMID:23097491

  4. Coevolution of RAC Small GTPases and their Regulators GEF Proteins

    PubMed Central

    Jiménez-Sánchez, Alejandro

    2016-01-01

    RAC proteins are small GTPases involved in important cellular processes in eukaryotes, and their deregulation may contribute to cancer. Activation of RAC proteins is regulated by DOCK and DBL protein families of guanine nucleotide exchange factors (GEFs). Although DOCK and DBL proteins act as GEFs on RAC proteins, DOCK and DBL family members are evolutionarily unrelated. To understand how DBL and DOCK families perform the same function on RAC proteins despite their unrelated primary structure, phylogenetic analyses of the RAC, DBL, and DOCK families were implemented, and interaction patterns that may suggest a coevolutionary process were searched. Interestingly, while RAC and DOCK proteins are very well conserved in humans and among eukaryotes, DBL proteins are highly divergent. Moreover, correlation analyses of the phylogenetic distances of RAC and GEF proteins and covariation analyses between residues in the interacting domains showed significant coevolution rates for both RAC–DOCK and RAC–DBL interactions. PMID:27226705

  5. UCP2, a mitochondrial protein regulated at multiple levels.

    PubMed

    Donadelli, Massimo; Dando, Ilaria; Fiorini, Claudia; Palmieri, Marta

    2014-04-01

    An ever-increasing number of studies highlight the role of uncoupling protein 2 (UCP2) in a broad range of physiological and pathological processes. The knowledge of the molecular mechanisms of UCP2 regulation is becoming fundamental in both the comprehension of UCP2-related physiological events and the identification of novel therapeutic strategies based on UCP2 modulation. The study of UCP2 regulation is a fast-moving field. Recently, several research groups have made a great effort to thoroughly understand the various molecular mechanisms at the basis of UCP2 regulation. In this review, we describe novel findings concerning events that can occur in a concerted manner at various levels: Ucp2 gene mutation (single nucleotide polymorphisms), UCP2 mRNA and protein expression (transcriptional, translational, and protein turn-over regulation), UCP2 proton conductance (ligands and post-transcriptional modifications), and nutritional and pharmacological regulation of UCP2.

  6. Regulation of Wnt/β-Catenin Signaling by Protein Kinases

    PubMed Central

    Verheyen, Esther M.; Gottardi, Cara J.

    2011-01-01

    The Wnt/β-catenin signaling pathway plays essential roles during development and adult tissue homeostasis. Inappropriate activation of the pathway can result in a variety of malignancies. Protein kinases have emerged as key regulators at multiple steps of the Wnt pathway. In this review, we present a synthesis covering the latest information on how Wnt signaling is regulated by diverse protein kinases. PMID:19623618

  7. Whey proteins in the regulation of food intake and satiety.

    PubMed

    Luhovyy, Bohdan L; Akhavan, Tina; Anderson, G Harvey

    2007-12-01

    Whey protein has potential as a functional food component to contribute to the regulation of body weight by providing satiety signals that affect both short-term and long-term food intake regulation. Because whey is an inexpensive source of high nutritional quality protein, the utilization of whey as a physiologically functional food ingredient for weight management is of current interest. At present, the role of individual whey proteins and peptides in contributing to food intake regulation has not been fully defined. However, Whey protein reduces short-term food intake relative to placebo, carbohydrate and other proteins. Whey protein affects satiation and satiety by the actions of: (1) whey protein fractions per se; (2) bioactive peptides; (3) amino-acids released after digestion; (4) combined action of whey protein and/or peptides and/or amino acids with other milk constituents. Whey ingestion activates many components of the food intake regulatory system. Whey protein is insulinotropic, and whey-born peptides affect the renin-angiotensin system. Therefore whey protein has potential as physiologically functional food component for persons with obesity and its co-morbidities (hypertension, type II diabetes, hyper- and dislipidemia). It remains unclear, however, if the favourable effects of whey on food intake, subjective satiety and intake regulatory mechanisms in humans are obtained from usual serving sizes of dairy products. The effects described have been observed in short-term experiments and when whey is consumed in much higher amounts.

  8. Regulation of receptor protein-tyrosine phosphatase dimerization.

    PubMed

    van der Wijk, Thea; Blanchetot, Christophe; den Hertog, Jeroen

    2005-01-01

    Receptor protein-tyrosine phosphatases (RPTPs) are single membrane spanning proteins belonging to the family of PTPs that, together with the antagonistically acting protein-tyrosine kinases (PTKs), regulate the protein phosphotyrosine levels in cells. Protein-tyrosine phosphorylation is an important post-translational modification that has a major role in cell signaling by affecting protein-protein interactions and enzymatic activities. Increasing evidence indicates that RPTPs, like RPTKs, are regulated by dimerization. For RPTPalpha, we have shown that rotational coupling of the constitutive dimers in the cell membrane determines enzyme activity. Furthermore, oxidative stress, identified as an important second messenger during the past decade, is a regulator of rotational coupling of RPTPalpha dimers. In this review, we discuss the biochemical and cell biological techniques that we use to study the regulation of RPTPs by dimerization. These techniques include (co-) immunoprecipitation, RPTP activity assays, chemical and genetic cross-linking, detection of cell surface proteins by biotinylation, and analysis of RPTPalpha dimers, using conformation-sensitive antibody binding.

  9. Autocrine regulation of milk secretion by a protein in milk.

    PubMed Central

    Wilde, C J; Addey, C V; Boddy, L M; Peaker, M

    1995-01-01

    Frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. Screening of goat's milk proteins in rabbit mammary explant cultures identified a single whey protein of M(r) 7600 able to inhibit synthesis of milk constituents. The active whey protein, which we term FIL (Feedback inhibitor of Lactation), also decreased milk secretion temporarily when introduced into a mammary gland of lactating goats. FIL was synthesized by primary cultures of goat mammary epithelial cells, and was secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by this milk protein. Images Figure 1 Figure 2 Figure 5 PMID:7826353

  10. Developmental Regulation of the Plastid Protein Import Apparatus.

    PubMed Central

    Dahlin, C; Cline, K

    1991-01-01

    Plastid development involves the programmed accumulation of proteins. Most plastid proteins are synthesized in the cytosol and imported into the organelle by an envelope-based protein import apparatus. Previous studies have shown that developmental rates of protein accumulation correspond to mRNA levels. Here, we examined the relationship between plastid development and the activity of the protein import apparatus. Developing plastids, primarily from wheat leaves, were analyzed for their protein import capability in vitro. Import capability, initially high in proplastids, declined as much as 20-fold as plastid development approached either the mature etioplast or the mature chloroplast. The observed decline was not due to senescence, nonspecific inhibitors, or protein turnover. Furthermore, the import capability of mature etioplasts, initially very low, was transiently reactivated during light-mediated redifferentiation into chloroplasts. These results suggest that plant cells regulate the import apparatus in concert with the protein demands of the developing plastids. PMID:12324584

  11. Regulation of the gibberellin pathway by auxin and DELLA proteins.

    PubMed

    O'Neill, Damian P; Davidson, Sandra E; Clarke, Victoria C; Yamauchi, Yukika; Yamaguchi, Shinjiro; Kamiya, Yuji; Reid, James B; Ross, John J

    2010-10-01

    The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel's LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA(20) showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA. PMID:20706734

  12. Structural Basis for Protein Phosphatase 1 Regulation and Specificity

    PubMed Central

    Peti, Wolfgang; Nairn, Angus C.; Page, Rebecca

    2012-01-01

    The ubiquitous Ser/Thr Protein Phosphatase 1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. However, the free catalytic subunit of PP1, while an effective enzyme, lacks substrate specificity. Instead, it depends on a diverse set of regulatory proteins (≥200) to confer specificity towards distinct substrates. Here, we discuss recent advances in structural studies of PP1 holoenzyme complexes and summarize the new insights these studies have provided into the molecular basis of PP1 regulation and specificity. PMID:22284538

  13. Piezo proteins: regulators of mechanosensation and other cellular processes.

    PubMed

    Bagriantsev, Sviatoslav N; Gracheva, Elena O; Gallagher, Patrick G

    2014-11-14

    Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature.

  14. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  15. Computerized video time lapse study of cell cycle delay and arrest, mitotic catastrophe, apoptosis and clonogenic survival in irradiated 14-3-3sigma and CDKN1A (p21) knockout cell lines.

    PubMed

    Chu, Kenneth; Teele, Noella; Dewey, Michael W; Albright, Norman; Dewey, William C

    2004-09-01

    Computerized video time lapse (CVTL) microscopy was used to observe cellular events induced by ionizing radiation (10-12 Gy) in nonclonogenic cells of the wild-type HCT116 colorectal carcinoma cell line and its three isogenic derivative lines in which p21 (CDKN1A), 14-3-3sigma or both checkpoint genes (double-knockout) had been knocked out. Cells that fused after mitosis or failed to complete mitosis were classified together as cells that underwent mitotic catastrophe. Seventeen percent of the wild-type cells and 34-47% of the knockout cells underwent mitotic catastrophe to enter generation 1 with a 4N content of DNA, i.e., the same DNA content as irradiated cells arrested in G(2) at the end of generation 0. Radiation caused a transient division delay in generation 0 before the cells divided or underwent mitotic catastrophe. Compared with the division delay for wild-type cells that express CDKN1A and 14-3-3sigma, knocking out CDKN1A reduced the delay the most for cells irradiated in G(1) (from approximately 15 h to approximately 3- 5 h), while knocking out 14-3-3sigma reduced the delay the most for cells irradiated in late S and G(2) (from approximately 18 h to approximately 3-4 h). However, 27% of wild-type cells and 17% of 14-3-3sigma(-/-) cells were arrested at 96 h in generation 0 compared with less than 1% for CDKN1A(-/-) and double-knockout cells. Thus expression of CDKN1A is necessary for the prolonged delay or arrest in generation 0. Furthermore, CDKN1A plays a crucial role in generation 1, greatly inhibiting progression into subsequent generations of both diploid cells and polyploid cells produced by mitotic catastrophe. Thus, in CDKN1A-deficient cell lines, a series of mitotic catastrophe events occurred to produce highly polyploid progeny during generations 3 and 4. Most importantly, the polyploid progeny produced by mitotic catastrophe events did not die sooner than the progeny of dividing cells. Death was identified as loss of cell movement, i

  16. Light-Inducible Gene Regulation with Engineered Zinc Finger Proteins

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2014-01-01

    The coupling of light-inducible protein-protein interactions with gene regulation systems has enabled the control of gene expression with light. In particular, heterodimer protein pairs from plants can be used to engineer a gene regulation system in mammalian cells that is reversible, repeatable, tunable, controllable in a spatiotemporal manner, and targetable to any DNA sequence. This system, Light-Inducible Transcription using Engineered Zinc finger proteins (LITEZ), is based on the blue light-induced interaction of GIGANTEA and the LOV domain of FKF1 that drives the localization of a transcriptional activator to the DNA-binding site of a highly customizable engineered zinc finger protein. This chapter provides methods for modifying LITEZ to target new DNA sequences, engineering a programmable LED array to illuminate cell cultures, and using the modified LITEZ system to achieve spatiotemporal control of transgene expression in mammalian cells. PMID:24718797

  17. Regulation of cilia assembly, disassembly, and length by protein phosphorylation.

    PubMed

    Cao, Muqing; Li, Guihua; Pan, Junmin

    2009-01-01

    The exact mechanism by which cells are able to assemble, regulate, and disassemble cilia or flagella is not yet completely understood. Recent studies in several model systems, including Chlamydomonas, Tetrahymena, Leishmania, Caenorhabditis elegans, and mammals, provide increasing biochemical and genetic evidence that phosphorylation of multiple protein kinases plays a key role in cilia assembly, disassembly, and length regulation. Members of several protein kinase families--including aurora kinases, never in mitosis A (NIMA)-related protein kinases, mitogen-activated protein (MAP) kinases, and a novel cyclin-dependent protein kinase--are involved in the ciliary regulation process. Among the newly identified protein kinase substrates are Chlamydomonas kinesin-13 (CrKinesin13), a microtubule depolymerizer, and histone deacetylase 6 (HDAC6), a microtubule deacetylase. Chlamydomonas aurora/Ipl1p-like protein kinase (CALK) and CrKinesin13 are two proteins that undergo phosphorylation changes correlated with flagellar assembly or disassembly. CALK becomes phosphorylated when flagella are lost, whereas CrKinesin13 is phosphorylated when new flagella are assembled. Conversely, suppressing CrKinesin13 expression results in cells with shorter flagella. PMID:20362099

  18. Small G proteins and their regulators in cellular signalling.

    PubMed

    Csépányi-Kömi, Roland; Lévay, Magdolna; Ligeti, Erzsébet

    2012-04-28

    Small molecular weight GTPases (small G proteins) are essential in the transduction of signals from different plasma membrane receptors. Due to their endogenous GTP-hydrolyzing activity, these proteins function as time-dependent biological switches controlling diverse cellular functions including cell shape and migration, cell proliferation, gene transcription, vesicular transport and membrane-trafficking. This review focuses on endocrine diseases linked to small G proteins. We provide examples for the regulation of the activity of small G proteins by various mechanisms such as posttranslational modifications, guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) or guanine nucleotide dissociation inhibitors (GDIs). Finally we summarize endocrine diseases where small G proteins or their regulatory proteins have been revealed as the cause.

  19. Regulation of bacterial RecA protein function.

    PubMed

    Cox, Michael M

    2007-01-01

    The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes. PMID:17364684

  20. Regulation of Ras proteins by reactive nitrogen species.

    PubMed

    Davis, Michael F; Vigil, Dom; Campbell, Sharon L

    2011-08-01

    Ras GTPases have been a subject of intense investigation since the early 1980s, when single point mutations in Ras were shown to cause deregulated cell growth control. Subsequently, Ras was identified as the most prevalent oncogene found in human cancer. Ras proteins regulate a host of pathways involved in cell growth, differentiation, and apoptosis by cycling between inactive GDP-bound and active GTP-bound states. Regulation of Ras activity is controlled by cellular factors that alter guanine nucleotide cycling. Oncogenic mutations prevent protein regulatory factors from down-regulating Ras activity, thereby maintaining Ras in a chronically activated state. The central dogma in the field is that protein modulatory factors are the primary regulators of Ras activity. Since the mid-1990s, however, evidence has accumulated that small molecule reactive nitrogen species (RNS) can also influence Ras guanine nucleotide cycling. Herein, we review the basic chemistry behind RNS formation and discuss the mechanism through which various RNS enhance nucleotide exchange in Ras proteins. In addition, we present studies that demonstrate the physiological relevance of RNS-mediated Ras activation within the context of immune system function, brain function, and cancer development. We also highlight future directions and experimental methods that may enhance our ability to detect RNS-mediated activation in cell cultures and in vivo. The development of such methods may ultimately pave new directions for detecting and elucidating how Ras proteins are regulated by redox species, as well as for targeting redox-activated Ras in cancer and other disease states.

  1. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    PubMed

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  2. New Insights into the Protein Turnover Regulation in Ethylene Biosynthesis.

    PubMed

    Yoon, Gyeong Mee

    2015-07-01

    Biosynthesis of the phytohormone ethylene is under tight regulation to satisfy the need for appropriate levels of ethylene in plants in response to exogenous and endogenous stimuli. The enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which catalyzes the rate-limiting step of ethylene biosynthesis, plays a central role to regulate ethylene production through changes in ACS gene expression levels and the activity of the enzyme. Together with molecular genetic studies suggesting the roles of post-translational modification of the ACS, newly emerging evidence strongly suggests that the regulation of ACS protein stability is an alternative mechanism that controls ethylene production, in addition to the transcriptional regulation of ACS genes. In this review, recent new insight into the regulation of ACS protein turnover is highlighted, with a special focus on the roles of phosphorylation, ubiquitination, and novel components that regulate the turnover of ACS proteins. The prospect of cross-talk between ethylene biosynthesis and other signaling pathways to control turnover of the ACS protein is also considered.

  3. Regulation of Mitochondrial Processes by Protein S-Nitrosylation

    PubMed Central

    Piantadosi, Claude A.

    2011-01-01

    Background Nitric oxide (NO) exerts powerful physiological effects through guanylate cyclase (GC), a non-mitochondrial enzyme, and through the generation of protein cysteinyl-NO (SNO) adducts— a post-translational modification relevant to mitochondrial biology. A small number of SNO proteins, generated by various mechanisms, are characteristically found in mammalian mitochondria and influence the regulation of oxidative phosphorylation and other aspects of mitochondrial function. Scope of Review The principles by which mitochondrial SNO proteins are formed and their actions, independently or collectively with NO binding to heme, iron-sulfur centers, or to glutathione (GSH) are reviewed on a molecular background of SNO-based signal transduction. Major Conclusions Mitochondrial SNO-proteins have been demonstrated to inhibit Complex I of the electron transport chain, to modulate mitochondrial reactive oxygen species (ROS) production, influence calcium-dependent opening of the mitochondrial permeability transition pore (MPTP), promote selective importation of mitochondrial protein, and stimulate mitochondrial fission. The ease of reversibility and the affirmation of regulated S-nitros(yl)ating and denitros(yl)ating enzymatic reactions supports hypotheses that SNO regulates the mitochondrion through redox mechanisms. SNO modification of mitochondrial proteins, whether homeostatic or adaptive (physiological), or pathogenic, is an area of active investigation. General Significance Mitochondrial SNO proteins are associated with mainly protective, bur soem pathological effects; the former mainly in inflammatory and ischemia/reperfusion syndromes and the latter in neurodegenerative diseases. Experimentally, mitochondrial SNO delivery is also emerging as a potential new area of therapeutics. PMID:21397666

  4. The regulation and function of the Id proteins in lymphocyte development.

    PubMed

    Rivera, R; Murre, C

    2001-12-20

    Helix-loop-helix (HLH) proteins are essential factors for lymphocyte development and function. One class of HLH proteins, the E-proteins, regulate many aspects of lymphocyte maturation, survival, proliferation, and differentiation. E-proteins are negatively regulated by another class of HLH proteins known as the Id proteins. The Id proteins function as dominant negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here we discuss the function and regulation of the Id proteins in lymphocyte development.

  5. Role of Regulators of G Protein Signaling Proteins in Bone Physiology and Pathophysiology

    PubMed Central

    Jules, Joel; Yang, Shuying; Chen, Wei; Li, Yi-Ping

    2016-01-01

    Regulators of G protein signaling (RGS) proteins enhance the intrinsic GTPase activity of α subunits of the heterotrimeric G protein complex of G protein-coupled receptors (GPCRs) and thereby inactivate signal transduction initiated by GPCRs. The RGS family consists of nearly 37 members with a conserved RGS homology domain which is critical for their GTPase accelerating activity. RGS proteins are expressed in most tissues, including heart, lung, brain, kidney, and bone and play essential roles in many physiological and pathological processes. In skeletal development and bone homeostasis as well as in many bone disorders, RGS proteins control the functions of various GPCRs, including the parathyroid hormone receptor type 1 and calcium-sensing receptor and also regulate various critical signaling pathways, such as Wnt and calcium oscillations. This chapter will discuss the current findings on the roles of RGS proteins in regulating signaling of key GPCRs in skeletal development and bone homeostasis. We also will examine the current updates of RGS proteins’ regulation of calcium oscillations in bone physiology and highlight the roles of RGS proteins in selected bone pathological disorders. Despite the recent advances in bone and mineral research, RGS proteins remain understudied in the skeletal system. Further understanding of the roles of RGS proteins in bone should not only provide great insights into the molecular basis of various bone diseases but also generate great therapeutic drug targets for many bone diseases. PMID:26123302

  6. G protein-coupled receptors and the regulation of autophagy

    PubMed Central

    Wauson, Eric M.; Dbouk, Hashem A.; Ghosh, Anwesha B.; Cobb, Melanie H.

    2014-01-01

    Autophagy is an important catabolic cellular process that eliminates damaged and unnecessary cytoplasmic proteins and organelles. Basal autophagy occurs during normal physiological conditions, but the activity of this process can be significantly altered in human diseases. Thus, defining the regulatory inputs and signals that control autophagy is essential. Nutrients are key modulators of autophagy. While autophagy is generally accepted to be regulated in a cell autonomous fashion, recent studies suggest nutrients can modulate autophagy in a systemic manner by inducing the secretion of hormones and neurotransmitters that regulate G protein-coupled receptors (GPCRs). Emerging studies show that GPCRs also regulate autophagy by directly detecting extracellular nutrients. We review the role of GPCRs in autophagy regulation, highlighting their potential as therapeutic drug targets. PMID:24751357

  7. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses

    PubMed Central

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-01-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events. PMID:27669825

  8. Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

    PubMed

    Kim, Hyun-Soo; Fernandes, Gary; Lee, Chang-Woo

    2016-09-01

    Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events. PMID:27669825

  9. COMPARTMENTALIZED PHOSPHORYLATION OF IAP BY PROTEIN KINASE A REGULATES CYTOPROTECTION

    PubMed Central

    Dohi, Takehiko; Xia, Fang; Altieri, Dario C.

    2007-01-01

    SUMMARY Cell death pathways are likely regulated in specialized subcellular microdomains, but how this occurs is not understood. Here, we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates the Inhibitor of Apoptosis (IAP) protein survivin on Ser20 in the cytosol, but not in mitochondria. This phosphorylation event disrupts the binding interface between survivin and its antiapoptotic cofactor, XIAP. Conversely, mitochondrial survivin or a non-PKA phosphorylatable survivin mutant binds XIAP avidly, enhances XIAP stability, synergistically inhibits apoptosis, and accelerates tumor growth, in vivo. Therefore, differential phosphorylation of survivin by PKA in subcellular microdomains regulates tumor cell apoptosis via its interaction with XIAP. PMID:17612487

  10. Network motifs in integrated cellular networks of transcription-regulation and protein-protein interaction

    NASA Astrophysics Data System (ADS)

    Yeger-Lotem, Esti; Sattath, Shmuel; Kashtan, Nadav; Itzkovitz, Shalev; Milo, Ron; Pinter, Ron Y.; Alon, Uri; Margalit, Hanah

    2004-04-01

    Genes and proteins generate molecular circuitry that enables the cell to process information and respond to stimuli. A major challenge is to identify characteristic patterns in this network of interactions that may shed light on basic cellular mechanisms. Previous studies have analyzed aspects of this network, concentrating on either transcription-regulation or protein-protein interactions. Here we search for composite network motifs: characteristic network patterns consisting of both transcription-regulation and protein-protein interactions that recur significantly more often than in random networks. To this end we developed algorithms for detecting motifs in networks with two or more types of interactions and applied them to an integrated data set of protein-protein interactions and transcription regulation in Saccharomyces cerevisiae. We found a two-protein mixed-feedback loop motif, five types of three-protein motifs exhibiting coregulation and complex formation, and many motifs involving four proteins. Virtually all four-protein motifs consisted of combinations of smaller motifs. This study presents a basic framework for detecting the building blocks of networks with multiple types of interactions.

  11. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

    PubMed Central

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  12. Transcriptional regulation of protein complexes within and across species.

    PubMed

    Tan, Kai; Shlomi, Tomer; Feizi, Hoda; Ideker, Trey; Sharan, Roded

    2007-01-23

    Yeast two-hybrid and coimmunoprecipitation experiments have defined large-scale protein-protein interaction networks for many model species. Separately, systematic chromatin immunoprecipitation experiments have enabled the assembly of large networks of transcriptional regulatory interactions. To investigate the functional interplay between these two interaction types, we combined both within a probabilistic framework that models the cell as a network of transcription factors regulating protein complexes. This framework identified 72 putative coregulated complexes in yeast and allowed the prediction of 120 previously uncharacterized transcriptional interactions. Several predictions were tested by new microarray profiles, yielding a confirmation rate (58%) comparable with that of direct immunoprecipitation experiments. Furthermore, we extended our framework to a cross-species setting, identifying 24 coregulated complexes that were conserved between yeast and fly. Analyses of these conserved complexes revealed different conservation levels of their regulators and provided suggestive evidence that protein-protein interaction networks may evolve more slowly than transcriptional interaction networks. Our results demonstrate how multiple molecular interaction types can be integrated toward a global wiring diagram of the cell, and they provide insights into the evolutionary dynamics of protein complex regulation.

  13. Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; Camacho-Arroyo, Ignacio

    2015-01-01

    In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone. PMID:26635640

  14. Eosinophil granule cationic proteins regulate the classical pathway of complement.

    PubMed Central

    Weiler, J M; Edens, R E; Bell, C S; Gleich, G J

    1995-01-01

    Major basic protein, the primary constituent of eosinophil granules, regulates the alternative and classical pathways of complement. Major basic protein and other eosinophil granule cationic proteins, which are important in mediating tissue damage in allergic disease, regulate the alternative pathway by interfering with C3b interaction with factor B to assemble an alternative pathway C3 convertase. In the present study, eosinophil peroxidase, eosinophil cationic protein and eosinophil-derived neurotoxin, as well as major basic protein, were examined for capacity to regulate the classical pathway. Eosinophil peroxidase, eosinophil cationic protein and major basic protein inhibited formation of cell-bound classical pathway C3 convertase (EAC1,4b,2a), causing 50% inhibition of complement-mediated lysis at about 0.19, 0.75 and 0.5 micrograms/10(7) cellular intermediates, respectively. Eosinophil-derived neurotoxin had no activity on this pathway of complement. The eosinophil granule proteins were examined for activity on the formation of the membrane attack complex. Major basic protein and eosinophil cationic protein had no activity on terminal lysis. In contrast, eosinophil peroxidase inhibited lysis of EAC1,4b,2a,3b,5b, but had only minimal activity on later events in complement lysis. These polycations were then examined to determine the site(s) at which they regulated the early classical pathway. Eosinophil granule polycationic proteins: (1) reduced the Zmax at all time points but had only minimal effect on the Tmax during the formation of the classical pathway C3 convertase (EAC1,4b,2a); (2) inhibited formation of EAC1,4b,2a proportional to C4 but independent of C2 concentration; (3) inhibited fluid phase formation of C1,4b,2a, as reflected by a decrease in C1-induced consumption of C2 over time; and (4) inhibited C1 activity over time without a direct effect on either C4 or C2. These observations suggest that polycations regulate the early classical pathway by

  15. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley.

    PubMed

    Mork-Jansson, Astrid; Bue, Ann Kristin; Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).

  16. Binding-regulated click ligation for selective detection of proteins.

    PubMed

    Cao, Ya; Han, Peng; Wang, Zhuxin; Chen, Weiwei; Shu, Yongqian; Xiang, Yang

    2016-04-15

    Herein, a binding-regulated click ligation (BRCL) strategy for endowing selective detection of proteins is developed with the incorporation of small-molecule ligand and clickable DNA probes. The fundamental principle underlying the strategy is the regulating capability of specific protein-ligand binding against the ligation between clickable DNA probes, which could efficiently combine the detection of particular protein with enormous DNA-based sensing technologies. In this work, the feasibly of the BRCL strategy is first verified through agarose gel electrophoresis and electrochemical impedance spectroscopy measurements, and then confirmed by transferring it to a nanomaterial-assisted fluorescence assay. Significantly, the BRCL strategy-based assay is able to respond to target protein with desirable selectivity, attributing to the specific recognition between small-molecule ligand and its target. Further experiments validate the general applicability of the sensing method by tailoring the ligand toward different proteins (i.e., avidin and folate receptor), and demonstrate its usability in complex biological samples. To our knowledge, this work pioneers the practice of click chemistry in probing specific small-molecule ligand-protein binding, and therefore may pave a new way for selective detection of proteins.

  17. HypoxiaDB: a database of hypoxia-regulated proteins.

    PubMed

    Khurana, Pankaj; Sugadev, Ragumani; Jain, Jaspreet; Singh, Shashi Bala

    2013-01-01

    There has been intense interest in the cellular response to hypoxia, and a large number of differentially expressed proteins have been identified through various high-throughput experiments. These valuable data are scattered, and there have been no systematic attempts to document the various proteins regulated by hypoxia. Compilation, curation and annotation of these data are important in deciphering their role in hypoxia and hypoxia-related disorders. Therefore, we have compiled HypoxiaDB, a database of hypoxia-regulated proteins. It is a comprehensive, manually-curated, non-redundant catalog of proteins whose expressions are shown experimentally to be altered at different levels and durations of hypoxia. The database currently contains 72 000 manually curated entries taken on 3500 proteins extracted from 73 peer-reviewed publications selected from PubMed. HypoxiaDB is distinctive from other generalized databases: (i) it compiles tissue-specific protein expression changes under different levels and duration of hypoxia. Also, it provides manually curated literature references to support the inclusion of the protein in the database and establish its association with hypoxia. (ii) For each protein, HypoxiaDB integrates data on gene ontology, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway, protein-protein interactions, protein family (Pfam), OMIM (Online Mendelian Inheritance in Man), PDB (Protein Data Bank) structures and homology to other sequenced genomes. (iii) It also provides pre-compiled information on hypoxia-proteins, which otherwise requires tedious computational analysis. This includes information like chromosomal location, identifiers like Entrez, HGNC, Unigene, Uniprot, Ensembl, Vega, GI numbers and Genbank accession numbers associated with the protein. These are further cross-linked to respective public databases augmenting HypoxiaDB to the external repositories. (iv) In addition, HypoxiaDB provides an online sequence-similarity search tool for

  18. Mutual Regulation of FOXM1, NPM and ARF Proteins.

    PubMed

    Pandit, Bulbul; Gartel, Andrei L

    2015-01-01

    ARF, NPM and FOXM1 proteins interact with each other in mammalian cells. We showed previously that proteasome inhibitors suppress not only FOXM1 expression, but also the expression of ARF and NPM proteins. Using RNA interference we found that the depletion of each of these proteins by RNAi in human cancer HeLa cells leads to down-regulation of the two other partners, suggesting that these proteins stabilize each other in human cancer cells. Since the suppression of FOXM1 is one of hallmarks of proteasome inhibition, suppression of ARF and NPM by proteasome inhibitors may be explained in part as a secondary effect of downregulation of FOXM1 that modulate stability of ARF and NPM1 proteins.

  19. Mutual Regulation of FOXM1, NPM and ARF Proteins

    PubMed Central

    Pandit, Bulbul; Gartel, Andrei L.

    2015-01-01

    ARF, NPM and FOXM1 proteins interact with each other in mammalian cells. We showed previously that proteasome inhibitors suppress not only FOXM1 expression, but also the expression of ARF and NPM proteins. Using RNA interference we found that the depletion of each of these proteins by RNAi in human cancer HeLa cells leads to down-regulation of the two other partners, suggesting that these proteins stabilize each other in human cancer cells. Since the suppression of FOXM1 is one of hallmarks of proteasome inhibition, suppression of ARF and NPM by proteasome inhibitors may be explained in part as a secondary effect of downregulation of FOXM1 that modulate stability of ARF and NPM1 proteins. PMID:26000045

  20. Novel regulators of cardiac inflammation: Matricellular proteins expand their repertoire.

    PubMed

    Rienks, Marieke; Papageorgiou, Anna-Pia

    2016-02-01

    More than 20years ago, Paul Bornstein coined the term matricellular protein to describe a group of secreted extracellular matrix proteins with de-adhesive properties. Though this is still true today, this family of proteins is vastly expanding with new emerging functions pushing the boundaries of this classic definition. In the heart, matricellular proteins have been extensively investigated in models of myocardial infarction, pressure overload, viral myocarditis and age-related cardiomyopathy with clear implications during cardiac fibrosis yet their involvement in regulating cardiac inflammation is less established. In this review, we describe our current understanding of the immune activation by damage- or pathogen-associated molecular pattern molecules during cardiac injury making a distinction between sterile versus non-sterile cardiac inflammation, and explain how matricellular proteins influence this crucial pathophysiological response in the heart.

  1. Rho regulation: DLC proteins in space and time.

    PubMed

    Braun, Anja C; Olayioye, Monilola A

    2015-08-01

    Rho GTPases function as molecular switches that connect changes of the external environment to intracellular signaling pathways. They are active at various subcellular sites and require fast and tight regulation to fulfill their role as transducers of extracellular stimuli. New imaging technologies visualizing the active states of Rho proteins in living cells elucidated the necessity of precise spatiotemporal activation of the GTPases. The local regulation of Rho proteins is coordinated by the interaction with different guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that turn on and off GTPase signaling to downstream effectors. GEFs and GAPs thus serve as critical signaling nodes that specify the amplitude and duration of a particular Rho signaling pathway. Despite their importance in Rho regulation, the molecular aspects underlying the spatiotemporal control of the regulators themselves are still largely elusive. In this review we will focus on the Deleted in Liver Cancer (DLC) family of RhoGAP proteins and summarize the evidence gathered over the past years revealing their different subcellular localizations that might account for isoform-specific functions. We will also highlight the importance of their tightly controlled expression in the context of neoplastic transformation.

  2. Lipid droplet meets a mitochondrial protein to regulate adipocyte lipolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In response to adrenergic stimulation, adipocytes undergo protein kinase A (PKA)-stimulated lipolysis. A key PKA target in this context is perilipin 1, a major regulator of lipolysis on lipid droplets (LDs). A study published in this issue of The EMBO Journal (Pidoux et al, 2011) identifies optic at...

  3. Emerging roles of zinc finger proteins in regulating adipogenesis

    PubMed Central

    Wei, Shengjuan; Zhang, Lifan; Zhou, Xiang; Du, Min; Jiang, Zhihua; Hausman, Gary J.; Bergen, Werner G.; Zan, Linsen; Dodson, Michael V.

    2014-01-01

    Proteins containing the zinc finger domain(s) are named zinc finger proteins (ZFPs), which are one of the largest classes of transcription factors in eukaryotic genomes. A large number of ZFPs have been studied and many of them were found to be involved regulating normal growth and development of cells and tissues through diverse signal transduction pathways. Recent studies revealed that a small but increasing number of ZFPs could function as key transcriptional regulators involved in adipogenesis. As the prevalence of obesity and metabolic disorders, the investigation of molecular regulatory mechanisms of adipocyte development must be more completely understood to develop novel and long term impact strategies for ameliorating obesity. In this review, we discuss recent work which has documented that ZFPs are important functional contributors to the regulation of adipogenesis. Taken altogether these data lead to the conclusion that ZFPs may become promising targets to combat human obesity. PMID:23760207

  4. Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

    PubMed Central

    Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

    2016-01-01

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

  5. Regulated eukaryotic DNA replication origin firing with purified proteins.

    PubMed

    Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

    2015-03-26

    Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

  6. BCL-2 family proteins as regulators of mitochondria metabolism.

    PubMed

    Gross, Atan

    2016-08-01

    The BCL-2 family proteins are major regulators of apoptosis, and one of their major sites of action are the mitochondria. Mitochondria are the cellular hubs for metabolism and indeed selected BCL-2 family proteins also possess roles related to mitochondria metabolism and dynamics. Here we discuss the link between mitochondrial metabolism/dynamics and the fate of stem cells, with an emphasis on the role of the BID-MTCH2 pair in regulating this link. We also discuss the possibility that BCL-2 family proteins act as metabolic sensors/messengers coming on and off of mitochondria to "sample" the cytosol and provide the mitochondria with up-to-date metabolic information. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  7. Protein Phosphorylation: A Major Switch Mechanism for Metabolic Regulation.

    PubMed

    Humphrey, Sean J; James, David E; Mann, Matthias

    2015-12-01

    Metabolism research is undergoing a renaissance because many diseases are increasingly recognized as being characterized by perturbations in intracellular metabolic regulation. Metabolic changes can be conferred through changes to the expression of metabolic enzymes, the concentrations of substrates or products that govern reaction kinetics, or post-translational modification (PTM) of the proteins that facilitate these reactions. On the 60th anniversary since its discovery, reversible protein phosphorylation is widely appreciated as an essential PTM regulating metabolism. With the ability to quantitatively measure dynamic changes in protein phosphorylation on a global scale - hereafter referred to as phosphoproteomics - we are now entering a new era in metabolism research, with mass spectrometry (MS)-based proteomics at the helm. PMID:26498855

  8. The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

    PubMed Central

    Sun, Jun; Li, Chen; Wang, Suwen

    2015-01-01

    Background Pairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation. Methodology/Principal Findings To investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females. Conclusions/Significance Our findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile. PMID:26070205

  9. Regulation of Ras Proteins by Reactive Nitrogen Species†

    PubMed Central

    Davis, Michael F.; Vigil, Dom; Campbell, Sharon L.

    2012-01-01

    Ras GTPases have been a subject of intense investigation since the early-80’s, when single point mutations in Ras were shown to cause deregulated cell growth control. Subsequently, Ras was identified as the most prevalent oncogene found in human cancer. Ras proteins regulate a host of pathways involved in cell growth, differentiation, and apoptosis by cycling between inactive GDP-bound and active GTP-bound states. Regulation of Ras activity is controlled by cellular factors that alter guanine nucleotide cycling. Oncogenic mutations prevent protein regulatory factors from down-regulating Ras activity, thereby maintaining Ras in a chronically activated state. The central dogma in the field is that protein modulatory factors are the primary regulators of Ras activity. Since the mid-90’s, however, evidence has accumulated that small molecule reactive nitrogen species (RNS) can also influence Ras guanine nucleotide cycling. Herein, we review the basic chemistry behind RNS formation and discuss the mechanism through which various RNS enhance nucleotide exchange in Ras proteins. In addition, we present studies that demonstrate the physiological relevance of RNS-mediated Ras activation within the context of immune system function, brain function, and cancer development. We also highlight future directions and experimental methods that may enhance our ability to detect RNS-mediated activation in cell cultures and in vivo. The development of such methods may ultimately pave new directions for detecting and elucidating how Ras proteins are regulated by redox species, as well as for targeting redox-activated Ras in cancer and other disease states. PMID:21616138

  10. Regulation of bone morphogenetic proteins in early embryonic development

    NASA Astrophysics Data System (ADS)

    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  11. Regulation of thrombosis and vascular function by protein methionine oxidation.

    PubMed

    Gu, Sean X; Stevens, Jeff W; Lentz, Steven R

    2015-06-18

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis.

  12. [PPR proteins--modular factors regulating expression of organellar genomes].

    PubMed

    Zapisek, Bartosz; Piątkowski, Jakub

    2015-01-01

    PPR proteins make up the most numerous family of RNA-binding proteins identified to date. They localize almost exclusively to plastids and mitochondria of eukaryotic organisms. The most striking feature of this family is the expansion of PPR protein-encoding genes in vascular plants, which likely coincided with plants colonizing land. PPR proteins participate in stabilizing, editing, splicing, degradation and processing of policistronic transcripts, as well as translation activation in mitochondria and plastids. Although the number of PPR proteins in non-plant organisms is significantly smaller than in plants, they still play a crucial role in regulating the expression of mtDNA. Disruptions of PPR protein-encoding genes usually result in severe phenotypic consequences. Plant PPR proteins bind RNA in a sequence-specific manner, where a single PPR motif recognizes an individual nucleotide in a given sequence. This opens up possibilities for engineering de novo synthetic protein sequences that would interact with precisely determined organellar sequences, thus enabling modulation of mtDNA and ctDNA expression.

  13. Protein Kinases of the Hippo Pathway: Regulation and Substrates

    PubMed Central

    Avruch, Joseph; Zhou, Dawang; Fitamant, Julien; Bardeesy, Nabeel; Mou, Fan; Barrufet, Laura Regué

    2012-01-01

    The “Hippo” signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in S. cerevisiae e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologues Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP

  14. Wrecked regulation of intrinsically disordered proteins in diseases: pathogenicity of deregulated regulators

    PubMed Central

    Uversky, Vladimir N.

    2014-01-01

    Biologically active proteins without stable tertiary structure are common in all known proteomes. Functions of these intrinsically disordered proteins (IDPs) are typically related to regulation, signaling, and control. Cellular levels of these important regulators are tightly regulated by a variety mechanisms ranging from firmly controlled expression to precisely targeted degradation. Functions of IDPs are controlled by binding to specific partners, alternative splicing, and posttranslational modifications among other means. In the norm, right amounts of precisely activated IDPs have to be present in right time at right places. Wrecked regulation brings havoc to the ordered world of disordered proteins, leading to protein misfolding, misidentification, and missignaling that give rise to numerous human diseases, such as cancer, cardiovascular disease, neurodegenerative diseases, and diabetes. Among factors inducing pathogenic transformations of IDPs are various cellular mechanisms, such as chromosomal translocations, damaged splicing, altered expression, frustrated posttranslational modifications, aberrant proteolytic degradation, and defective trafficking. This review presents some of the aspects of deregulated regulation of IDPs leading to human diseases. PMID:25988147

  15. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation*

    PubMed Central

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O.; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  16. ORMDL proteins regulate ceramide levels during sterile inflammation.

    PubMed

    Cai, Lin; Oyeniran, Clement; Biswas, Debolina D; Allegood, Jeremy; Milstien, Sheldon; Kordula, Tomasz; Maceyka, Michael; Spiegel, Sarah

    2016-08-01

    The bioactive sphingolipid metabolite, ceramide, regulates physiological processes important for inflammation and elevated levels of ceramide have been implicated in IL-1-mediated events. Although much has been learned about ceramide generation by activation of sphingomyelinases in response to IL-1, the contribution of the de novo pathway is not completely understood. Because yeast ORM1 and ORM2 proteins negatively regulate ceramide levels through inhibition of serine palmitoyltransferase, the first committed step in ceramide biosynthesis, we examined the functions of individual mammalian ORM orthologs, ORM (yeast)-like (ORMDL)1-3, in regulation of ceramide levels. In HepG2 liver cells, downregulation of ORMDL3 markedly increased the ceramide precursors, dihydrosphingosine and dihydroceramide, primarily from de novo biosynthesis based on [U-(13)C]palmitate incorporation into base-labeled and dual-labeled dihydroceramides, whereas downregulation of each isoform increased dihydroceramides [(13)C]labeled in only the amide-linked fatty acid. IL-1 and the IL-6 family cytokine, oncostatin M, increased dihydroceramide and ceramide levels in HepG2 cells and concomitantly decreased ORMDL proteins. Moreover, during irritant-induced sterile inflammation in mice leading to induction of the acute-phase response, which is dependent on IL-1, expression of ORMDL proteins in the liver was strongly downregulated and accompanied by increased ceramide levels in the liver and accumulation in the blood. Together, our results suggest that ORMDLs may be involved in regulation of ceramides during IL-1-mediated sterile inflammation. PMID:27313060

  17. Regulation of the MAPK pathway by raf kinase inhibitory protein.

    PubMed

    Vandamme, Drieke; Herrero, Ana; Al-Mulla, Fahd; Kolch, Walter

    2014-01-01

    The Raf kinase inhibitor protein 1 (RKIP-1) was the first reported endogenous inhibitor of Raf-1-MEK-ERK/MAPK cascade, by interfering with the phosphorylation of MEK by Raf-1. However, RKIP's functions related to the MAPK signaling are far more complex. Newer data indicate that by modulating different protein-protein interactions, RKIP is involved in fine-tuning cell signaling, modulating ERK dynamics, and regulating cross talk between different pathways. Here, we describe the molecular mechanisms by which RKIP controls MAPK signaling at different levels and vice versa and its regulation via feedback phosphorylation. We also focus on several discrepancies and questions that remain, such as the RKIP binding regulation by Raf-1 N-region phosphorylation, the possible B-Raf inhibition, and the effects of RKIP-lipid binding. We also describe how RKIP's role as key signaling modulator of many cell fate decisions leads to the fact that fine control of RKIP activity and regulation is crucial to avoid pathological processes, such as metastasis, pulmonary arterial hypertension, and heart failure.

  18. Differential regulation of oligodendrocyte markers by glucocorticoids: Post-transcriptional regulation of both proteolipid protein and myelin basic protein and transcriptional regulation of glycerol phosphate dehydrogenase

    SciTech Connect

    Kumar, S.; Cole, R.; Chiappelli, F.; De Vellis, J. )

    1989-09-01

    During neonatal development glucocorticoids potentiate oligodendrocyte differentiation and myelinogenesis by regulating the expression of myelin basic protein, proteolipid protein, and glycerol phosphate dehydrogenase. The actual locus at which hydrocortisone exerts its developmental influence on glial physiology is, however, not well understood. Gycerol phosphate dehydrogenase is glucocorticoid-inducible in oligodendrocytes at all stages of development both in vivo and in vitro. In newborn rat cerebral cultures, between 9 and 15 days in vitro, a 2- to 3-fold increase in myelin basic protein and proteolipid protein mRNA levels occurs in oligodendrocytes within 12 hr of hydrocortisone treatment. Immunostaining demonstrates that this increase in mRNAs is followed by a 2- to 3-fold increase in the protein levels within 24 hr. In vitro transcription assays performed with oligodendrocyte nuclei show an 11-fold increase in the transcriptional activity of glycerol phosphate dehydrogenase in response to hydrocortisone but no increase in transcription of myelin basic protein or proteolipid protein. These results indicate that during early myelinogeneis, glucocorticoids influence the expression of key oligodendroglial markers by different processes: The expression of glycerol phosphate dehydrogenase is regulated at the transcriptional level, whereas the expression of myelin basic protein and proteolipid protein is modulated via a different, yet uncharacterized, mechanism involving post-transcriptional regulation.

  19. Reactive Oxygen Species Regulate Nucleostemin Oligomerization and Protein Degradation*

    PubMed Central

    Huang, Min; Whang, Patrick; Chodaparambil, Jayanth V.; Pollyea, Daniel A.; Kusler, Brenda; Xu, Liwen; Felsher, Dean W.; Mitchell, Beverly S.

    2011-01-01

    Nucleostemin (NS) is a nucleolar-nucleoplasmic shuttle protein that regulates cell proliferation, binds p53 and Mdm2, and is highly expressed in tumor cells. We have identified NS as a target of oxidative regulation in transformed hematopoietic cells. NS oligomerization occurs in HL-60 leukemic cells and Raji B lymphoblasts that express high levels of c-Myc and have high intrinsic levels of reactive oxygen species (ROS); reducing agents dissociate NS into monomers and dimers. Exposure of U2OS osteosarcoma cells with low levels of intrinsic ROS to hydrogen peroxide (H2O2) induces thiol-reversible disulfide bond-mediated oligomerization of NS. Increased exposure to H2O2 impairs NS degradation, immobilizes the protein within the nucleolus, and results in detergent-insoluble NS. The regulation of NS by ROS was validated in a murine lymphoma tumor model in which c-Myc is overexpressed and in CD34+ cells from patients with chronic myelogenous leukemia in blast crisis. In both instances, increased ROS levels were associated with markedly increased expression of NS protein and thiol-reversible oligomerization. Site-directed mutagenesis of critical cysteine-containing regions of nucleostemin altered both its intracellular localization and its stability. MG132, a potent proteasome inhibitor and activator of ROS, markedly decreased degradation and increased nucleolar retention of NS mutants, whereas N-acetyl-l-cysteine largely prevented the effects of MG132. These results indicate that NS is a highly redox-sensitive protein. Increased intracellular ROS levels, such as those that result from oncogenic transformation in hematopoietic malignancies, regulate the ability of NS to oligomerize, prevent its degradation, and may alter its ability to regulate cell proliferation. PMID:21242306

  20. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    PubMed

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation. PMID:24646224

  1. Regulation of heartbeat by G protein-coupled ion channels.

    PubMed

    Brown, A M

    1990-12-01

    The coupling of ion channels to receptors by G proteins is the subject of this American Physiological Society Walter B. Cannon Memorial "Physiology in Perspective" Lecture. This subject is particularly appropriate because it includes a molecular explanation of a homeostatic mechanism involving the autonomic nervous system and the latter subject preoccupied Dr. Cannon during most of his career. With the use of reconstitution methods, we and others have shown that heterotrimeric guanine nucleotide-binding (G) proteins couple receptors to ion channels by both membrane-delimited, direct pathways and cytoplasmic second messenger pathways. Furthermore, one set of receptors may be coupled to as many as three different sets of ion channels to form networks. Dual G protein pathways lead to the prediction of biphasic ion current responses in cell signaling, and this prediction was confirmed. In sinoatrial pacemaker cells, the pacemaking hyperpolarization-activated inward current (If) is directly regulated by the G proteins Gs and Go, and the two can act simultaneously. This could explain the classical observation that vagal inhibition of heart rate is greater during sympathetic stimulation. Because deactivation of the muscarinic response occurs much faster than the G protein alpha-subunit hydrolyzes guanosine 5'-triphosphate, we looked for accessory cellular factors. A surprising result was that the small monomeric ras G protein blocked the muscarinic pathway. The significance of this observation is unknown, but it appears that small and large G proteins may interact in ion channel signaling pathways.

  2. Regulation of heartbeat by G protein-coupled ion channels.

    PubMed

    Brown, A M

    1990-12-01

    The coupling of ion channels to receptors by G proteins is the subject of this American Physiological Society Walter B. Cannon Memorial "Physiology in Perspective" Lecture. This subject is particularly appropriate because it includes a molecular explanation of a homeostatic mechanism involving the autonomic nervous system and the latter subject preoccupied Dr. Cannon during most of his career. With the use of reconstitution methods, we and others have shown that heterotrimeric guanine nucleotide-binding (G) proteins couple receptors to ion channels by both membrane-delimited, direct pathways and cytoplasmic second messenger pathways. Furthermore, one set of receptors may be coupled to as many as three different sets of ion channels to form networks. Dual G protein pathways lead to the prediction of biphasic ion current responses in cell signaling, and this prediction was confirmed. In sinoatrial pacemaker cells, the pacemaking hyperpolarization-activated inward current (If) is directly regulated by the G proteins Gs and Go, and the two can act simultaneously. This could explain the classical observation that vagal inhibition of heart rate is greater during sympathetic stimulation. Because deactivation of the muscarinic response occurs much faster than the G protein alpha-subunit hydrolyzes guanosine 5'-triphosphate, we looked for accessory cellular factors. A surprising result was that the small monomeric ras G protein blocked the muscarinic pathway. The significance of this observation is unknown, but it appears that small and large G proteins may interact in ion channel signaling pathways. PMID:1701981

  3. HOX13 proteins: the molecular switcher in Hoxd bimodal regulation.

    PubMed

    Ros, Marian A

    2016-05-15

    The striking correlation between the genomic arrangement of Hox genes and their temporal and spatial pattern of expression during embryonic development has been a source of fascination since its discovery. This correspondence has been used as a privileged example in the investigation of the connection between genomic architecture and function. In this issue of Genes & Development, Beccari and colleagues (pp. 1172-1186) make a big step forward in understanding Hox gene regulation during limb development by showing the pivotal role of HOXA13 and HOXD13 proteins in the transition from a proximal to a distal type of Hoxd transcriptional regulation. PMID:27222515

  4. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  5. Bone morphogenetic protein (BMP) signaling regulates mitotic checkpoint protein levels in human breast cancer cells.

    PubMed

    Yan, Hualong; Zhu, Songcheng; Song, Chenlin; Liu, Naifa; Kang, Jiuhong

    2012-04-01

    Aberrant expression of mitotic checkpoint genes compromises mitotic checkpoint, leads to chromosome instability and tumorigenesis. However, the cell signals that control mitotic checkpoint gene expression have not been reported so far. In the present study we show that, in human breast cancer cells, chemical inhibition of Bone morphogenetic proteins (BMPs), but not Transforming Growth Factor-β (TGF-β), abrogates the mitotic arrest induced by nocodazole. Protein expression analysis reveals that inhibition of BMP signaling dramatically down regulates protein levels of mitotic checkpoint components BUB3, Hec1, TTK and MAD2, but inhibition of TGF-β has relatively minor effect on the expression of these proteins. Activation of BMP signaling specifically up regulates BUB3, and activation of Activin A signaling globally down regulates these proteins level. Furthermore, overexpressing MAD2, TTK, BUB3 or Hec1 significantly rescues the mitotic arrest defect caused by BMP inhibition. Our results demonstrated for the first time that TGF-β family cytokines are cellular signals regulating mitotic checkpoint and perturbations in intrinsic BMP signaling could lead to suppression of mitotic checkpoint signaling by downregulating key checkpoint proteins. The results suggest a possible mechanism by which dysregulation of TGF-β signaling causes mitotic checkpoint defects and drives tumorigenesis. The finding also provides a potential and more specific strategy for cancer prevention by targeting BMP and mitotic checkpoint connection. PMID:22234345

  6. Regulation of protein degradation in muscle by calcium

    NASA Technical Reports Server (NTRS)

    Zeman, Richard J.; Kameyama, Tsuneo; Matsumoto, Kazue; Bernstein, Paul; Etlinger, Joseph D.

    1985-01-01

    Calcium-dependent regulation of intracellular protein degradation was studied in isolated rat skeletal muscles incubated in vitro in the presence of a large variety of agents known to affect calcium movement and distribution. The effect of different classes of protease inhibitors was tested to determine the responsible proteolytic systems involved in calcium-dependent degradation. The results suggest that nonlysosomal leupetin- and E-64-c-sensitive proteases are resposible for calcium-dependent proteolysis in muscle.

  7. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  8. Redox regulation by reversible protein S-thiolation in bacteria

    PubMed Central

    Loi, Vu Van; Rossius, Martina; Antelmann, Haike

    2015-01-01

    Low molecular weight (LMW) thiols function as thiol-redox buffers to maintain the reduced state of the cytoplasm. The best studied LMW thiol is the tripeptide glutathione (GSH) present in all eukaryotes and Gram-negative bacteria. Firmicutes bacteria, including Bacillus and Staphylococcus species utilize the redox buffer bacillithiol (BSH) while Actinomycetes produce the related redox buffer mycothiol (MSH). In eukaryotes, proteins are post-translationally modified to S-glutathionylated proteins under conditions of oxidative stress. S-glutathionylation has emerged as major redox-regulatory mechanism in eukaryotes and protects active site cysteine residues against overoxidation to sulfonic acids. First studies identified S-glutathionylated proteins also in Gram-negative bacteria. Advances in mass spectrometry have further facilitated the identification of protein S-bacillithiolations and S-mycothiolation as BSH- and MSH-mixed protein disulfides formed under oxidative stress in Firmicutes and Actinomycetes, respectively. In Bacillus subtilis, protein S-bacillithiolation controls the activities of the redox-sensing OhrR repressor and the methionine synthase MetE in vivo. In Corynebacterium glutamicum, protein S-mycothiolation was more widespread and affected the functions of the maltodextrin phosphorylase MalP and thiol peroxidase (Tpx). In addition, novel bacilliredoxins (Brx) and mycoredoxins (Mrx1) were shown to function similar to glutaredoxins in the reduction of BSH- and MSH-mixed protein disulfides. Here we review the current knowledge about the functions of the bacterial thiol-redox buffers glutathione, bacillithiol, and mycothiol and the role of protein S-thiolation in redox regulation and thiol protection in model and pathogenic bacteria. PMID:25852656

  9. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis.

    PubMed

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S; Cuéllar Pérez, Amparo; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-08-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  10. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

    Gonzalez, Nathalie; Pauwels, Laurens; Baekelandt, Alexandra; De Milde, Liesbeth; Van Leene, Jelle; Besbrugge, Nienke; Heyndrickx, Ken S.; Pérez, Amparo Cuéllar; Durand, Astrid Nagels; De Clercq, Rebecca; Van De Slijke, Eveline; Vanden Bossche, Robin; Eeckhout, Dominique; Gevaert, Kris; Vandepoele, Klaas; De Jaeger, Geert; Goossens, Alain; Inzé, Dirk

    2015-01-01

    Cell number is an important determinant of final organ size. In the leaf, a large proportion of cells are derived from the stomatal lineage. Meristemoids, which are stem cell-like precursor cells, undergo asymmetric divisions, generating several pavement cells adjacent to the two guard cells. However, the mechanism controlling the asymmetric divisions of these stem cells prior to differentiation is not well understood. Here, we characterized PEAPOD (PPD) proteins, the only transcriptional regulators known to negatively regulate meristemoid division. PPD proteins interact with KIX8 and KIX9, which act as adaptor proteins for the corepressor TOPLESS. D3-type cyclin encoding genes were identified among direct targets of PPD2, being negatively regulated by PPDs and KIX8/9. Accordingly, kix8 kix9 mutants phenocopied PPD loss-of-function producing larger leaves resulting from increased meristemoid amplifying divisions. The identified conserved complex might be specific for leaf growth in the second dimension, since it is not present in Poaceae (grasses), which also lack the developmental program it controls. PMID:26232487

  11. Chromatin-regulating proteins as targets for cancer therapy

    PubMed Central

    Oike, Takahiro; Ogiwara, Hideaki; Amornwichet, Napapat; Nakano, Takashi; Kohno, Takashi

    2014-01-01

    Chromatin-regulating proteins represent a large class of novel targets for cancer therapy. In the context of radiotherapy, acetylation and deacetylation of histones by histone acetyltransferases (HATs) and histone deacetylases (HDACs) play important roles in the repair of DNA double-strand breaks generated by ionizing irradiation, and are therefore attractive targets for radiosensitization. Small-molecule inhibitors of HATs (garcinol, anacardic acid and curcumin) and HDACs (vorinostat, sodium butyrate and valproic acid) have been shown to sensitize cancer cells to ionizing irradiation in preclinical models, and some of these molecules are being tested in clinical trials, either alone or in combination with radiotherapy. Meanwhile, recent large-scale genome analyses have identified frequent mutations in genes encoding chromatin-regulating proteins, especially in those encoding subunits of the SWI/SNF chromatin-remodeling complex, in various human cancers. These observations have driven researchers toward development of targeted therapies against cancers carrying these mutations. DOT1L inhibition in MLL-rearranged leukemia, EZH2 inhibition in EZH2-mutant or MLL-rearranged hematologic malignancies and SNF5-deficient tumors, BRD4 inhibition in various hematologic malignancies, and BRM inhibition in BRG1-deficient tumors have demonstrated promising anti-tumor effects in preclinical models, and these strategies are currently awaiting clinical application. Overall, the data collected so far suggest that targeting chromatin-regulating proteins is a promising strategy for tomorrow's cancer therapy, including radiotherapy and molecularly targeted chemotherapy. PMID:24522270

  12. Function and Regulation of Heterotrimeric G Proteins during Chemotaxis.

    PubMed

    Kamp, Marjon E; Liu, Youtao; Kortholt, Arjan

    2016-01-14

    Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis.

  13. Function and Regulation of Heterotrimeric G Proteins during Chemotaxis

    PubMed Central

    Kamp, Marjon E.; Liu, Youtao; Kortholt, Arjan

    2016-01-01

    Chemotaxis, or directional movement towards an extracellular gradient of chemicals, is necessary for processes as diverse as finding nutrients, the immune response, metastasis and wound healing. Activation of G-protein coupled receptors (GPCRs) is at the very base of the chemotactic signaling pathway. Chemotaxis starts with binding of the chemoattractant to GPCRs at the cell-surface, which finally leads to major changes in the cytoskeleton and directional cell movement towards the chemoattractant. Many chemotaxis pathways that are directly regulated by Gβγ have been identified and studied extensively; however, whether Gα is just a handle that regulates the release of Gβγ or whether Gα has its own set of distinct chemotactic effectors, is only beginning to be understood. In this review, we will discuss the different levels of regulation in GPCR signaling and the downstream pathways that are essential for proper chemotaxis. PMID:26784171

  14. Protein-Tyrosine Phosphatase 1B Substrates and Metabolic Regulation

    PubMed Central

    Bakke, Jesse; Haj, Fawaz G.

    2014-01-01

    Metabolic homeostasis requires integration of complex signaling networks which, when deregulated, contribute to metabolic syndrome and related disorders. Protein-tyrosine phosphatase 1B (PTP1B) has emerged as a key regulator of signaling networks that are implicated in metabolic diseases such as obesity and type 2 diabetes. In this review, we examine mechanisms that regulate PTP1B-substrate interaction, enzymatic activity and experimental approaches to identify PTP1B substrates. We then highlight findings that implicate PTP1B in metabolic regulation. In particular, insulin and leptin signaling are discussed as well as recently identified PTP1B substrates that are involved in endoplasmic reticulum stress response, cell-cell communication, energy balance and vesicle trafficking. In summary, PTP1B exhibits exquisite substrate specificity and is an outstanding pharmaceutical target for obesity and type 2 diabetes. PMID:25263014

  15. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  16. Regulation of cholesterol esterification by protein kinase C

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-03-05

    They have recently identified acyl-CoA cholesterol acyltransferase as the key enzyme for cholesterol esterification in the central nervous system. They found that the activity of glial acyl-CoA cholesterol acyltransferase could be controlled by a phosphorylation-dephosphorylation mechanism. However, repeated attempts to identify cyclic AMP as the bioregulator for this reaction failed. Recently, they have studied the possible involvement of protein kinase C in the regulation of glial cholesterol esterification. Phorbol-12-myristate 13-acetate (PMA) can activate cellular cholesterol esterification in a complex, time-dependent manner. Phorbol analogues inactive toward protein kinase C are also ineffective in this assay. Furthermore, oleoyl-acetyl-glycerol mimics the effect of PMA, confirming the proposal that protein kinase C mediates the effect of these compounds and that the natural bioregulator is probably diacylglycerol. Receptor-mediated polyphosphatidyl-inositol cleavage often produces diacylglycerol and inositol triphosphate. The synergic effects of these two compounds are known to be necessary to elicit other biological responses. Their preliminary studies using calcium ionophore A23187 indicates that Ca/sup + +/ is not required for cellular cholesterol esterification. In sum, glial cholesterol esterification is probably regulated by a calcium-independent and protein kinase C-dependent reaction.

  17. RNA structures regulating ribosomal protein biosynthesis in bacilli.

    PubMed

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M

    2013-07-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  18. RNA structures regulating ribosomal protein biosynthesis in bacilli

    PubMed Central

    Deiorio-Haggar, Kaila; Anthony, Jon; Meyer, Michelle M.

    2013-01-01

    In Bacilli, there are three experimentally validated ribosomal-protein autogenous regulatory RNAs that are not shared with E. coli. Each of these RNAs forms a unique secondary structure that interacts with a ribosomal protein encoded by a downstream gene, namely S4, S15, and L20. Only one of these RNAs that interacts with L20 is currently found in the RNA Families Database. We created, or modified, existing structural alignments for these three RNAs and used them to perform homology searches. We have determined that each structure exhibits a narrow phylogenetic distribution, mostly relegated to the Firmicute class Bacilli. This work, in conjunction with other similar work, demonstrates that there are most likely many non-homologous RNA regulatory elements regulating ribosomal protein biosynthesis that still await discovery and characterization in other bacterial species. PMID:23611891

  19. REGULATION OF THE UNFOLDED PROTEIN RESPONSE BY MICRORNAS

    PubMed Central

    Madanecki, Piotr; Piotrowski, Arkadiusz; Ochocka, Renata; Collawn, James F.; Bartoszewski, Rafal

    2013-01-01

    The unfolded protein response (UPR) is an adaptive response to stress that is caused by an accumulation of misfolded proteins in the lumen of endoplasmic reticulum (ER) and is therefore an important component of cellular homeostasis. During ER stress, the UPR increases the protein folding capacity of the endoplasmic reticulum to relieve the stress, and failure to recover leads to apoptosis. Specific cellular mechanisms, therefore, are required for the cellular recovery phase after UPR activation. In this review, we discuss the potential role of microRNAs as key regulators of this pathway. Using bioinformatics, we identified a number of microRNAs that are predicted to decrease the mRNA expression levels for a number of critical components of the UPR and discuss how microRNAs may play an essential role in turning off the UPR after the stress has subsided. PMID:24092331

  20. Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

    NASA Technical Reports Server (NTRS)

    Howard, G.; Steffen, J. M.; Geoghegan, T. E.

    1989-01-01

    The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

  1. CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice.

    PubMed Central

    Timchenko, N A; Harris, T E; Wilde, M; Bilyeu, T A; Burgess-Beusse, B L; Finegold, M J; Darlington, G J

    1997-01-01

    CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels. PMID:9372966

  2. Protein tyrosine phosphatase regulation of endothelial cell apoptosis and differentiation.

    PubMed

    Yang, C; Chang, J; Gorospe, M; Passaniti, A

    1996-02-01

    Apoptosis, or programmed cell death, occurs during development and may also be an important factor in many diseases. However, little is known about the signal transduction pathways regulating apoptosis. In these studies, loss of endothelial cell-substrate attachment and apoptosis after removal of growth factors was associated with dephosphorylation of tyrosine residues at the cell periphery. Dephosphorylation of total cellular proteins accompanied apoptosis and was reduced by orthovanadate, an inhibitor of protein tyrosine phosphatases. Orthovanadate blocked the fragmentation of nuclear DNA, inhibited DNA laddering, and suppressed the expression of TRPM-2, an apoptosis-associated gene. The tyrosine phosphorylation levels of FAK125, erk1 (mitogen-activated kinase kinase), and cdc-2 were reduced during apoptosis. FAK125 dephosphorylation was inhibited by orthovanadate, but premature activation (tyrosine dephosphorylation) of cdc-2 was not. Orthovanadate was as effective as basic fibroblast growth factor in activating erk1 without increasing cell proliferation and in preventing the apoptosis of endothelial cells after treatment with tumor necrosis factor alpha. Endothelial cell differentiation on extracellular matrix (Matrigel) was also stimulated by orthovanadate in the absence of basic fibroblast growth factor without affecting growth arrest and inhibition of DNA synthesis. Expression of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1) was down-regulated during the early stages of differentiation, remained low for at least 6 hours as differentiation proceeded, and increased upon completion of differentiation. Cells that failed to down-regulate p21 mRNA on Matrigel in the absence of angiogenic factors underwent apoptosis. These results suggest that protein tyrosine phosphatases are actively involved in signal transduction during apoptosis and may regulate p21 expression to inhibit endothelial cell differentiation.

  3. Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins*

    PubMed Central

    Chen, Buxin; Siderovski, David P.; Neubig, Richard R.; Lawson, Mark A.; Trejo, JoAnn

    2014-01-01

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 420AKKAA424 mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins. PMID:24297163

  4. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    PubMed

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins. PMID:24297163

  5. Regulation of Sp1 by cell cycle related proteins

    PubMed Central

    Tapias, Alicia; Ciudad, Carlos J.; Roninson, Igor B.; Noé, Véronique

    2009-01-01

    Sp1 transcription factor regulates the expression of multiple genes, including the Sp1 gene itself. We analyzed the ability of different cell cycle regulatory proteins to interact with Sp1 and to affect Sp1 promoter activity. Using an antibody array, we observed that CDK4, SKP2, Rad51, BRCA2 and p21 could interact with Sp1 and we confirmed these interactions by co-immunoprecipitation. CDK4, SKP2, Rad51, BRCA2 and p21 also activated the Sp1 promoter. Among the known Sp1-interacting proteins, E2F-DP1, Cyclin D1, Stat3 and Rb activated the Sp1 promoter, whereas p53 and NFκB inhibited it. The proteins that regulated Sp1 gene expression were shown by positive chromatin immunoprecipitation to be bound to the Sp1 promoter. Moreover, SKP2, BRCA2, p21, E2F-DP1, Stat3, Rb, p53 and NFκB had similar effects on an artificial promoter containing only Sp1 binding sites. Transient transfections of CDK4, Rad51, E2F-DP1, p21 and Stat3 increased mRNA expression from the endogenous Sp1 gene in HeLa cells whereas overexpression of NFκB, and p53 decreased Sp1 mRNA levels. p21 expression from a stably integrated inducible promoter in HT1080 cells activated Sp1 expression at the promoter and mRNA levels, but at the same time it decreased Sp1 protein levels due to the activation of Sp1 degradation. The observed multiple effects of cell cycle regulators on Sp1 suggest that Sp1 may be a key mediator of cell cycle associated changes in gene expression. PMID:18769160

  6. Cytoplasmic polyadenylation element binding protein-dependent protein synthesis is regulated by calcium/calmodulin-dependent protein kinase II.

    PubMed

    Atkins, Coleen M; Nozaki, Naohito; Shigeri, Yasushi; Soderling, Thomas R

    2004-06-01

    Phosphorylation of cytoplasmic polyadenylation element binding protein (CPEB) regulates protein synthesis in hippocampal dendrites. CPEB binds the 3' untranslated region (UTR) of cytoplasmic mRNAs and, when phosphorylated, initiates mRNA polyadenylation and translation. We report that, of the protein kinases activated in the hippocampus during synaptic plasticity, calcium/calmodulin-dependent protein kinase II (CaMKII) robustly phosphorylated the regulatory site (threonine 171) in CPEB in vitro. In postsynaptic density fractions or hippocampal neurons, CPEB phosphorylation increased when CaMKII was activated. These increases in CPEB phosphorylation were attenuated by a specific peptide inhibitor of CaMKII and by the general CaM-kinase inhibitor KN-93. Inhibitors of protein phosphatase 1 increased basal CPEB phosphorylation in neurons; this was also attenuated by a CaM-kinase inhibitor. To determine whether CaM-kinase activity regulates CPEB-dependent mRNA translation, hippocampal neurons were transfected with luciferase fused to a 3' UTR containing CPE-binding elements. Depolarization of neurons stimulated synthesis of luciferase; this was abrogated by inhibitors of protein synthesis, mRNA polyadenylation, and CaMKII. These results demonstrate that CPEB phosphorylation and translation are regulated by CaMKII activity and provide a possible mechanism for how dendritic protein synthesis in the hippocampus may be stimulated during synaptic plasticity.

  7. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  8. Chapter Two - Heterotrimeric G Protein Ubiquitination as a Regulator of G Protein Signaling.

    PubMed

    Torres, M

    2016-01-01

    Ubiquitin-mediated regulation of G proteins has been known for over 20 years as a result of discoveries made independently in yeast and vertebrate model systems for pheromone and photoreception, respectively. Since that time, several details underlying the cause and effect of G protein ubiquitination have been determined-including the initiating signals, responsible enzymes, trafficking pathways, and their effects on protein structure, function, interactions, and cell signaling. The collective body of evidence suggests that Gα subunits are the primary targets of ubiquitination. However, longstanding and recent results suggest that Gβ and Gγ subunits are also ubiquitinated, in some cases impacting cell polarization-a process essential for chemotaxis and polarized cell growth. More recently, evidence from mass spectrometry (MS)-based proteomics coupled with advances in PTM bioinformatics have revealed that protein families representing G protein subunits contain several structural hotspots for ubiquitination-most of which have not been investigated for a functional role in signal transduction. Taken together, our knowledge and understanding of heterotrimeric G protein ubiquitination as a regulator of G protein signaling-despite 20 years of research-is still emerging. PMID:27378755

  9. Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR-Cas9 library.

    PubMed

    Wu, Yuanzhong; Kang, Tiebang

    2016-06-29

    The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A," recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.

  10. Autopalmitoylation of TEAD Proteins Regulates Transcriptional Output of Hippo Pathway

    PubMed Central

    Chan, PuiYee; Han, Xiao; Zheng, Baohui; DeRan, Michael; Yu, Jianzhong; Jarugumilli, Gopala K.; Deng, Hua; Pan, Duojia; Luo, Xuelian; Wu, Xu

    2016-01-01

    TEA domain (TEAD) transcription factors bind to the co-activator YAP/TAZ, and regulate the transcriptional output of Hippo pathway, playing critical roles in organ size control and tumorigenesis. Protein S-palmitoylation attaches fatty acid (palmitate) to cysteine residues, and regulates protein trafficking, membrane localization and signaling activities. Using activity-based chemical probes, we discovered that human TEADs possess intrinsic palmitoylating enzyme-like activities, and undergo autopalmitoylation at evolutionarily conserved cysteine residues under physiological conditions. We determined the crystal structures of lipid-bound TEADs, and found that the lipid chain of palmitate inserts into a conserved deep hydrophobic pocket. Strikingly, palmitoylation is required for TEAD’s binding to YAP/TAZ, but dispensable for the binding to Vgll4 tumor suppressor. In addition, palmitoylation does not alter TEAD’s localization. Moreover, TEAD palmitoylation-deficient mutants impaired TAZ-mediated muscle differentiation in vitro, and Yorkie-mediated tissue overgrowth in Drosophila in vivo. Our study directly linked autopalmitoylation to the transcriptional regulation of Hippo pathway. PMID:26900866

  11. Regulation of RAG-2 protein expression in avian thymocytes.

    PubMed Central

    Ferguson, S E; Accavitti, M A; Wang, D D; Chen, C L; Thompson, C B

    1994-01-01

    The recombinase-activating genes, RAG-1 and RAG-2, have been shown to be necessary to initiate the process of V(D)J recombination during the ontogeny of lymphocytes. While much is known about the end products of this rearrangement process, little is known about the function or regulation of the components of the recombinase system. To this end, we have generated a monoclonal antibody to the chicken RAG-2 protein. Chicken thymocytes were found to express high levels of RAG-2, part of which is phosphorylated. Within thymocytes, RAG-2 is expressed primarily within the nucleus. RAG-2 protein levels are high in the CD4- CD8- and CD4+ CD8+ immature thymocytes but absent at the single-positive CD4+ CD8- or CD4- CD8+ stage of thymocyte development. Mitogenic stimulation of thymocytes with phorbol myristate acetate and ionomycin results in down-regulation of RAG-2 expression. Consistent with these data, in vivo levels of RAG-2 are markedly lower in proliferating thymocytes than in smaller, G0/G1 cells. Down-regulation of RAG-2 expression appears to occur before cells enter S phase, suggesting that RAG-2 function may be limited to noncycling cells. Images PMID:7935443

  12. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

    PubMed

    Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

    2016-09-01

    The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  13. Negative regulation of lymphocyte activation by the adaptor protein LAX.

    PubMed

    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

  14. Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

    PubMed Central

    Zhao, Yang; Xie, Shaojun; Batelli, Giorgia; Wang, Bangshing; Duan, Cheng-Guo; Wang, Xingang; Xing, Lu; Lei, Mingguang; Yan, Jun; Zhu, Xiaohong; Zhu, Jian-Kang

    2016-01-01

    The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. PMID:26943172

  15. The RHOX Homeodomain Proteins Regulate the Expression of Insulin and Other Metabolic Regulators in the Testis*

    PubMed Central

    MacLean, James A.; Hu, Zhiying; Welborn, Joshua P.; Song, Hye-Won; Rao, Manjeet K.; Wayne, Chad M.; Wilkinson, Miles F.

    2013-01-01

    Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism. PMID:24121513

  16. The RHOX homeodomain proteins regulate the expression of insulin and other metabolic regulators in the testis.

    PubMed

    MacLean, James A; Hu, Zhiying; Welborn, Joshua P; Song, Hye-Won; Rao, Manjeet K; Wayne, Chad M; Wilkinson, Miles F

    2013-11-29

    Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism.

  17. Lysine methylation regulates the pRb tumour suppressor protein.

    PubMed

    Munro, S; Khaire, N; Inche, A; Carr, S; La Thangue, N B

    2010-04-22

    The pRb tumour suppressor protein has a central role in coordinating early cell cycle progression. An important level of control imposed on pRb occurs through post-translational modification, for example, phosphorylation. We describe here a new level of regulation on pRb, mediated through the targeted methylation of lysine residues, by the methyltransferase Set7/9. Set7/9 methylates the C-terminal region of pRb, both in vitro and in cells, and methylated pRb interacts with heterochromatin protein HP1. pRb methylation is required for pRb-dependent cell cycle arrest and transcriptional repression, as well as pRb-dependent differentiation. Our results indicate that methylation can influence the properties of pRb, and raise the interesting possibility that methylation modulates pRb tumour suppressor activity.

  18. Ribozymes, riboswitches and beyond: regulation of gene expression without proteins

    PubMed Central

    Serganov, Alexander; Patel, Dinshaw J.

    2015-01-01

    Although various functions of RNA are carried out in conjunction with proteins, some catalytic RNAs, or ribozymes, which contribute to a range of cellular processes, require little or no assistance from proteins. Furthermore, the discovery of metabolite-sensing riboswitches and other types of RNA sensors has revealed RNA-based mechanisms that cells use to regulate gene expression in response to internal and external changes. Structural studies have shown how these RNAs can carry out a range of functions. In addition, the contribution of ribozymes and riboswitches to gene expression is being revealed as far more widespread than was previously appreciated. These findings have implications for understanding how cellular functions might have evolved from RNA-based origins. PMID:17846637

  19. G protein-coupled receptors in regulation of body weight.

    PubMed

    Schiöth, Helgi B

    2006-06-01

    In this issue of CNS & Neurological Disorders-Drug Targets, we focus on G protein-coupled receptors (GPCRs) that are involved in regulating body weight. In six reviews, the melanocortins system (including MC4 and MC3 receptors, Agrp, MSH), the NPY receptors (including NPY-Y1, NPY-Y2, and NPY-Y5, PYY3-36), the cannabinoid system (including the development of rimonabant), the ghrelin (GHS, growth hormone secretagogue) system, the monoamine GPCRs (including serotonin, adrenergic and histamine receptors), orexin (hypocretin) system and the galanin receptors are covered. In this overview, an introduction to the GPCRs and the field of central regulation of food intake is provided together with brief mentioning of some other GPCRs that are also implicated in regulation of body weight, such as the melanin-concentrating hormone (MCH), neuromedin U, prolactin-releasing peptide (PrRP), bombesin, cholecystokinin (CCK), Glucagon-like peptide-1 (GLP-1) (and oxyntomodulin), neuropeptide B (NPB) and neuropeptide W (NPW), opioids peptides, free fatty acid (FFA) receptors (GPR40, GPR41). In total over 40 GPCRs are listed that have been implicated to affect regulation of body weight.

  20. Iron-Regulated Surface Determinant (Isd) Proteins of Staphylococcus lugdunensis

    PubMed Central

    Zapotoczna, Marta; Heilbronner, Simon; Speziale, Pietro

    2012-01-01

    Staphylococcus lugdunensis is the only coagulase-negative Staphylococcus species with a locus encoding iron-regulated surface determinant (Isd) proteins. In Staphylococcus aureus, the Isd proteins capture heme from hemoglobin and transfer it across the wall to a membrane-bound transporter, which delivers it into the cytoplasm, where heme oxygenases release iron. The Isd proteins of S. lugdunensis are expressed under iron-restricted conditions. We propose that S. lugdunensis IsdB and IsdC proteins perform the same functions as those of S. aureus. S. lugdunensis IsdB is the only hemoglobin receptor within the isd locus. It specifically binds human hemoglobin with a dissociation constant (Kd) of 23 nM and transfers heme on IsdC. IsdB expression promotes bacterial growth in an iron-limited medium containing human hemoglobin but not mouse hemoglobin. This correlates with weak binding of IsdB to mouse hemoglobin in vitro. Unlike IsdB and IsdC, the proteins IsdJ and IsdK are not sorted to the cell wall in S. lugdunensis. In contrast, IsdJ expressed in S. aureus and Lactococcus lactis is anchored to peptidoglycan, suggesting that S. lugdunensis sortases may differ in signal recognition or could be defective. IsdJ and IsdK are present in the culture supernatant, suggesting that they could acquire heme from the external milieu. The IsdA protein of S. aureus protects bacteria from bactericidal lipids due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected S. aureus and L. lactis as efficiently as IsdA but, possibly due to its location, was less effective in its natural host. PMID:23002220

  1. Regulation of intestinal microbiota by the NLR protein family

    PubMed Central

    2013-01-01

    The human intestine harbors a diverse microbial community consisting of a large number of bacteria and other micro-organisms that have co-evolved with the host intestinal immune system. During this process, microbiota and the host immune system shape one another by various mechanisms to achieve a successful symbiotic relationship. An increasing amount of evidence suggests that dysbiosis—the breakdown of such harmonized colonization—may result in infectious and inflammatory disorders, and recent advances in our studies indicate that receptors such as Toll-like receptors and NLR (nucleotide-binding oligomerization domain-like receptor; or nucleotide-binding domain- and leucine-rich repeat-containing receptor) proteins that detect micro-organisms and their products play a critical role in maintaining intestinal homeostasis. In this review, we summarize the role of NLR proteins in the regulation of intestinal microbiota. NLR proteins belong to a diverse family of cytoplasmic microbial sensors, mutations of which are involved in various disorders, including inflammatory bowel diseases. Understanding of the different roles of NLR family proteins in the intestine is, therefore, an important step towards the development of therapeutics against digestive diseases. PMID:23325116

  2. Redox regulation of protein folding in the mitochondrial intermembrane space

    PubMed Central

    Koehler, Carla M.; Tienson, Heather L.

    2014-01-01

    Protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. However, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. The mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. Specifically, the intermembrane space proteins with disulfide bonds are imported via the mitochondrial intermembrane space assembly (MIA) pathway. Substrates are imported via a disulfide exchange relay with two components Mia40 and Erv1. This new breakthrough has resulted in novel concepts for assembly of proteins in the intermembrane space, suggesting that this compartment may be similar to that of the endoplasmic reticulum and the prokaryotic periplasm. As a better understanding of this pathway emerges, new paradigms for thiol-disulfide exchange mechanisms may be developed. Given that the intermembrane space is important for disease processes including apoptosis and neurodegeneration, new roles in regulation by oxidation–reduction chemistry seem likely to be relevant. PMID:18761382

  3. Matricellular proteins as regulators of cancer metastasis to bone.

    PubMed

    Trotter, Timothy N; Yang, Yang

    2016-01-01

    Metastasis is the major cause of death in cancer patients, and a frequent site of metastasis for many cancers is the bone marrow. Therefore, understanding the mechanisms underlying the metastatic process is necessary for future prevention and treatment. The tumor microenvironment is now known to play a role in the metastatic cascade, both at the primary tumor and in metastatic sites, and includes both cellular and non-cellular components. The extracellular matrix (ECM) provides structural support and signaling cues to cells. One particular group of molecules associated with the ECM, known as matricellular proteins, modulate multiple aspects of tumor biology, including growth, migration, invasion, angiogenesis and metastasis. These proteins are also important for normal function in the bone by regulating bone formation and bone resorption. Recent studies have described a link between some of these proteins and metastasis of various tumors to the bone. The aim of this review is to summarize what is currently known about matricellular protein influence on bone metastasis. Particular attention to the contribution of both tumor cells and non-malignant cells in the bone has been given.

  4. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

    PubMed Central

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-01-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  5. Phospholipase D Controls Dictyostelium Development By Regulating G Protein Signaling

    PubMed Central

    Ray, Sibnath; Chen, Yi; Ayoung, Joanna; Hanna, Rachel; Brazill, Derrick

    2010-01-01

    Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling. PMID:20950684

  6. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores

    PubMed Central

    Bickel, Perry E.; Tansey, John T.; Welte, Michael A.

    2009-01-01

    Summary The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  7. Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet.

    PubMed

    Dudley, M A; Schoknecht, P A; Dudley, A W; Jiang, L; Ferraris, R P; Rosenberger, J N; Henry, J F; Reeds, P J

    2001-04-01

    The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.

  8. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

    PubMed

    Bickel, Perry E; Tansey, John T; Welte, Michael A

    2009-06-01

    The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  9. PREFACE: Physics approaches to protein interactions and gene regulation Physics approaches to protein interactions and gene regulation

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Panchenko, Anna R.; Przytycka, Teresa

    2011-06-01

    networks have been identified, including scale free distribution of the vertex degree, network motifs, and modularity, to name a few. These studies of network organization require the network to be as complete as possible, which given the limitations of experimental techniques is not currently the case. Therefore, experimental procedures for detecting biomolecular interactions should be complemented by computational approaches. The paper by Lees et al provides a review of computational methods, integrating multiple independent sources of data to infer physical and functional protein-protein interaction networks. One of the important aspects of protein interactions that should be accounted for in the prediction of protein interaction networks is that many proteins are composed of distinct domains. Protein domains may mediate protein interactions while proteins and their interaction networks may gain complexity through gene duplication and expansion of existing domain architectures via domain rearrangements. The latter mechanisms have been explored in detail in the paper by Cohen-Gihon et al. Protein-protein interactions are not the only component of the cell's interactome. Regulation of cell activity can be achieved at the level of transcription and involve a transcription factor—DNA binding which typically requires recognition of a specific DNA sequence motif. Chip-Chip and the more recent Chip-Seq technologies allow in vivo identification of DNA binding sites and, together with novel in vitro approaches, provide data necessary for deciphering the corresponding binding motifs. Such information, complemented by structures of protein-DNA complexes and knowledge of the differences in binding sites among homologs, opens the door to constructing predictive binding models. The paper by Persikov and Singh provides an example of such a model in the Cys2His2 zinc finger family. Recent studies have indicated that the presence of such binding motifs is, however, neither necessary

  10. Photoreactive synthetic regulator of protein function and methods of use thereof

    DOEpatents

    Trauner, Dirk; Isacoff, Ehud Y; Kramer, Richard H; Banghart, Matthew R; Fortin, Doris L; Mourot, Alexandre

    2015-03-31

    The present disclosure provides a photoreactive synthetic regulator of protein function. The present disclosure further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present disclosure further provides methods of modulating protein function, involving use of light.

  11. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  12. Regulation of polar auxin transport by protein and lipid kinases.

    PubMed

    Armengot, Laia; Marquès-Bueno, Maria Mar; Jaillais, Yvon

    2016-07-01

    The directional transport of auxin, known as polar auxin transport (PAT), allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima, and gradients that are instrumental in both organ initiation and shape determination. As such, PAT is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell to cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the 'non-genomic' regulation of auxin transport, placing an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK, and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some receptor-like kinases (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition, as well as root gravitropism and shoot phototropism. PMID:27242371

  13. Protein-protein interactions among ion channels regulate ion transport in the kidney.

    PubMed

    Boulpaep, E

    2009-01-01

    Epithelial ion transport in various organs has long been known to be controlled by extracellular agonists acting via membrane receptors or by intracellular messengers. Evidence is mounting for regulation of transport by direct interaction among membrane proteins or between a membrane transport protein and membrane-attached proteins. The membrane protein CFTR (Cystic Fibrosis Transmembrane Regulator) is widely expressed along the length of the nephron, but its role as a chloride channel does not appear to be critical for renal handling of salt and water. It is well established that the inward rectifying K channels (ROMK = Kir 1.1) in the thick ascending limb of Henle and in principal cells of the collecting duct are inhibited by millimolar concentrations of cytosolic Mg-ATP. However, the mechanism of this inhibition has been an enigma. We propose that the ATP-Binding Cassette (ABC) protein CFTR is a cofactor for Kir 1.1 regulation. Indeed, Mg-ATP sensitivity of Kir 1.1 is completely absent in two different mouse models of cystic fibrosis. In addition, the open-closed state of CFTR appears to provide a molecular gating switch that prevents or facilitates the ATP sensing of Kir 1.1. Does Mg-ATP sensing by the CFTR- Kir 1.1 complex play a role in coupling metabolism to ion transport? Physiological intracellular ATP concentrations in tubule cells are in the millimolar range, a saturating concentration for the gating of Kir 1.1 by Mg-ATP. Therefore, Kir 1.1 channels would be closed and unable to contribute to regulation of potassium secretion unless some other process modulated the CFTR-dependent ATP-sensitivity of Kir 1.1. The third component of the metabolic sensor-effector complex for Kir 1.1 regulation is most likely the AMP-regulated serine-threonine kinase, AMP kinase (AMPK). Changing levels in AMP rather than in ATP constitute the metabolic signal "sensed" by tubule cells. Because AMPK inhibits CFTR by modulating CFTR channel gating, we propose that renal K

  14. A molecular clock regulates angiopoietin-like protein 2 expression.

    PubMed

    Kadomatsu, Tsuyoshi; Uragami, Shota; Akashi, Makoto; Tsuchiya, Yoshiki; Nakajima, Hiroo; Nakashima, Yukiko; Endo, Motoyoshi; Miyata, Keishi; Terada, Kazutoyo; Todo, Takeshi; Node, Koichi; Oike, Yuichi

    2013-01-01

    Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.

  15. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  16. Perilipin-related protein regulates lipid metabolism in C. elegans

    PubMed Central

    Chughtai, Ahmed Ali; Kaššák, Filip; Kostrouchová, Markéta; Novotný, Jan Philipp; Krause, Michael W.; Kostrouch, Zdenek

    2015-01-01

    Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism. PMID:26357594

  17. Transcriptional regulation of muscle fatty acid-binding protein.

    PubMed Central

    Carey, J O; Neufer, P D; Farrar, R P; Veerkamp, J H; Dohm, G L

    1994-01-01

    Heart fatty acid-binding protein (H-FABP) is present in a wide variety of tissues but is found in the highest concentration in cardiac and red skeletal muscle. It has been proposed that the expression of H-FABP correlates directly with the fatty acid-oxidative capacity of the tissue. In the present study, the expression of H-FABP was measured in red and white skeletal muscle under two conditions in which fatty acid utilization is known to be increased: streptozotocin-induced diabetes and fasting. Protein concentration, mRNA concentration and transcription rate were measured under both conditions. The level of both protein and mRNA increased approximately 2-fold under each condition. The transcription rate was higher in red skeletal muscle than in white muscle, was increased 2-fold during fasting, but was unchanged by streptozotocin-induced diabetes. In addition to supporting the hypothesis that H-FABP is induced during conditions of increased fatty acid utilization, these findings demonstrate that the regulation of H-FABP expression may or may not be at the level of transcription depending on the stimulus. Images Figure 2 Figure 3 PMID:8141774

  18. Regulation of Nuclear Localization of Signaling Proteins by Cytokinin

    SciTech Connect

    Kieber, J.J.

    2010-05-01

    Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

  19. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    DOE PAGES

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; et al

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected themore » splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.« less

  20. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    SciTech Connect

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-08-20

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. In conclusion, this large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.

  1. Regulation of alternative splicing in Drosophila by 56 RNA binding proteins

    PubMed Central

    Brooks, Angela N.; Duff, Michael O.; May, Gemma; Yang, Li; Bolisetty, Mohan; Landolin, Jane; Wan, Ken; Sandler, Jeremy; Booth, Benjamin W.; Celniker, Susan E.; Graveley, Brenton R.; Brenner, Steven E.

    2015-01-01

    Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs. PMID:26294686

  2. Auxin acts independently of DELLA proteins in regulating gibberellin levels.

    PubMed

    Reid, James B; Davidson, Sandra E; Ross, John J

    2011-03-01

    Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA1, play critical roles in the control of elongation, along with environmental and endogenous factors, including other hormones such as the brassinosteroids. The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism, since auxin up-regulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and down regulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA 1. In our recent paper, we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action, since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA 1 synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level. However, our recent results, in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined. PMID:21358281

  3. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  4. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein

    PubMed Central

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-01-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation. PMID:27233051

  5. Keap1-Independent Regulation of Nrf2 Activity by Protein Acetylation and a BET Bromodomain Protein.

    PubMed

    Chatterjee, Nirmalya; Tian, Min; Spirohn, Kerstin; Boutros, Michael; Bohmann, Dirk

    2016-05-01

    Mammalian BET proteins comprise a family of bromodomain-containing epigenetic regulators with complex functions in chromatin organization and gene regulation. We identified the sole member of the BET protein family in Drosophila, Fs(1)h, as an inhibitor of the stress responsive transcription factor CncC, the fly ortholog of Nrf2. Fs(1)h physically interacts with CncC in a manner that requires the function of its bromodomains and the acetylation of CncC. Treatment of cultured Drosophila cells or adult flies with fs(1)h RNAi or with the BET protein inhibitor JQ1 de-represses CncC transcriptional activity and engages protective gene expression programs. The mechanism by which Fs(1)h inhibits CncC function is distinct from the canonical mechanism that stimulates Nrf2 function by abrogating Keap1-dependent proteasomal degradation. Consistent with the independent modes of CncC regulation by Keap1 and Fs(1)h, combinations of drugs that can specifically target these pathways cause a strong synergistic and specific activation of protective CncC- dependent gene expression and boosts oxidative stress resistance. This synergism might be exploitable for the design of combinatorial therapies to target diseases associated with oxidative stress or inflammation. PMID:27233051

  6. Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

    The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma×rat glioma hybrid cells. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5′-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A2 adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA). In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells. PMID:11959806

  7. Urea may regulate urea transporter protein abundance during osmotic diuresis.

    PubMed

    Kim, Dongun; Klein, Janet D; Racine, Sandy; Murrell, Brian P; Sands, Jeff M

    2005-01-01

    Rats with diabetes mellitus have an increase in UT-A1 urea transporter protein abundance and absolute urea excretion, but the relative amount (percentage) of urea in total urinary solute is actually decreased due to the marked glucosuria. Urea-specific signaling pathways have been identified in mIMCD3 cells and renal medulla, suggesting the possibility that changes in the percentage or concentration of urea could be a factor that regulates UT-A1 abundance. In this study, we tested the hypothesis that an increase in a urinary solute other than urea would increase UT-A1 abundance, similar to diabetes mellitus, whereas an increase in urine urea would not. In both inner medullary base and tip, UT-A1 protein abundance increased during NaCl- or glucose-induced osmotic diuresis but not during urea-induced osmotic diuresis. Next, rats undergoing NaCl or glucose diuresis were given supplemental urea to increase the percentage of urine urea to control values. UT-A1 abundance did not increase in these urea-supplemented rats compared with control rats. Additionally, both UT-A2 and UT-B protein abundances in the outer medulla increased during urea-induced osmotic diuresis but not in NaCl or glucose diuresis. We conclude that during osmotic diuresis, UT-A1 abundance increases when the percentage of urea in total urinary solute is low and UT-A2 and UT-B abundances increase when the urea concentration in the medullary interstitium is high. These findings suggest that a reduction in urine or interstitial urea results in an increase in UT-A1 protein abundance in an attempt to restore inner medullary interstitial urea and preserve urine-concentrating ability.

  8. Saccharomyces cerevisiae Tti2 Regulates PIKK Proteins and Stress Response

    PubMed Central

    Hoffman, Kyle S.; Duennwald, Martin L.; Karagiannis, Jim; Genereaux, Julie; McCarton, Alexander S.; Brandl, Christopher J.

    2016-01-01

    The TTT complex is composed of the three essential proteins Tel2, Tti1, and Tti2. The complex is required to maintain steady state levels of phosphatidylinositol 3-kinase-related kinase (PIKK) proteins, including mTOR, ATM/Tel1, ATR/Mec1, and TRRAP/Tra1, all of which serve as regulators of critical cell signaling pathways. Due to their association with heat shock proteins, and with newly synthesized PIKK peptides, components of the TTT complex may act as cochaperones. Here, we analyze the consequences of depleting the cellular level of Tti2 in Saccharomyces cerevisiae. We show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but the cells are sensitive to a number of stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of Tra1, Mec1, and Tor1, affected their localization and inhibited the stress responses in which these molecules are involved. Tti2 expression was not increased during heat shock, implying that it does not play a general role in the heat shock response. However, steady state levels of Hsp42 increase when Tti2 is depleted, and tti2L187P has a synthetic interaction with exon 1 of the human Huntingtin gene containing a 103 residue polyQ sequence, suggesting a general role in protein quality control. We also find that overexpressing Hsp90 or its cochaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. PMID:27172216

  9. Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium.

    PubMed

    Cohn, J A

    1990-02-01

    T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.

  10. Regulation of RE1 protein silencing transcription factor (REST) expression by HIP1 protein interactor (HIPPI).

    PubMed

    Datta, Moumita; Bhattacharyya, Nitai P

    2011-09-30

    Earlier we have shown that the proapoptotic protein HIPPI (huntingtin interacting protein 1 (HIP1) protein interactor) along with its molecular partner HIP1 could regulate transcription of the caspase-1 gene. Here we report that RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a new transcriptional target of HIPPI. HIPPI could bind to the promoter of REST and increased its expression in neuronal as well as non-neuronal cells. Such activation of REST down-regulated expression of REST target genes, such as brain-derived neurotrophic factor (BDNF) or proenkephalin (PENK). The ability of HIPPI to activate REST gene transcription was dependent on HIP1, the nuclear transporter of HIPPI. Using a Huntington disease cell model, we have demonstrated that feeble interaction of HIP1 with mutant huntingtin protein resulted in increased nuclear accumulation of HIPPI and HIP1, leading to higher occupancy of HIPPI at the REST promoter, triggering its transcriptional activation and consequent repression of REST target genes. This novel transcription regulatory mechanism of REST by HIPPI may contribute to the deregulation of transcription observed in the cell model of Huntington disease. PMID:21832040

  11. A versatile synthetic dimerizer for the regulation of protein-protein interactions.

    PubMed

    Amara, J F; Clackson, T; Rivera, V M; Guo, T; Keenan, T; Natesan, S; Pollock, R; Yang, W; Courage, N L; Holt, D A; Gilman, M

    1997-09-30

    The use of low molecular weight organic compounds to induce dimerization or oligomerization of engineered proteins has wide-ranging utility in biological research as well as in gene and cell therapies. Chemically induced dimerization can be used to activate intracellular signal transduction pathways or to control the activity of a bipartite transcription factor. Dimerizer systems based on the natural products cyclosporin, FK506, rapamycin, and coumermycin have been described. However, owing to the complexity of these compounds, adjusting their binding or pharmacological properties by chemical modification is difficult. We have investigated several families of readily prepared, totally synthetic, cell-permeable dimerizers composed of ligands for human FKBP12. These molecules have significantly reduced complexity and greater adaptability than natural product dimers. We report here the efficacies of several of these new synthetic compounds in regulating two types of protein dimerization events inside engineered cells--induction of apoptosis through dimerization of engineered Fas proteins and regulation of transcription through dimerization of transcription factor fusion proteins. One dimerizer in particular, AP1510, proved to be exceptionally potent and versatile in all experimental contexts tested. PMID:9380684

  12. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  13. Pbx Homeodomain Proteins: TALEnted regulators of Limb Patterning and Outgrowth

    PubMed Central

    Capellini, Terence D.; Zappavigna, Vincenzo; Selleri, Licia

    2011-01-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. PMID:21416555

  14. Regulation of G Protein-Coupled Receptors by Allosteric Ligands

    PubMed Central

    2013-01-01

    Topographically distinct, druggable, allosteric sites may be present on all G protein-coupled receptors (GPCRs). As such, targeting these sites with synthetic small molecules offers an attractive approach to develop receptor-subtype selective chemical leads for the development of novel therapies. A crucial part of drug development is to understand the acute and chronic effects of such allosteric modulators at their corresponding GPCR target. Key regulatory processes including cell-surface delivery, endocytosis, recycling, and down-regulation tightly control the number of receptors at the surface of the cell. As many GPCR therapeutics will be administered chronically, understanding how such ligands modulate these regulatory pathways forms an essential part of the characterization of novel GPCR ligands. This is true for both orthosteric and allosteric ligands. In this Review, we summarize our current understanding of GPCR regulatory processes with a particular focus on the effects and implications of allosteric targeting of GPCRs. PMID:23398684

  15. The Polarity Protein Pals1 Regulates Radial Sorting of Axons.

    PubMed

    Zollinger, Daniel R; Chang, Kae-Jiun; Baalman, Kelli; Kim, Seonhee; Rasband, Matthew N

    2015-07-22

    Myelin is essential for rapid and efficient action potential propagation in vertebrates. However, the molecular mechanisms regulating myelination remain incompletely characterized. For example, even before myelination begins in the PNS, Schwann cells must radially sort axons to form 1:1 associations. Schwann cells then ensheathe and wrap axons, and establish polarized, subcellular domains, including apical and basolateral domains, paranodes, and Schmidt-Lanterman incisures. Intriguingly, polarity proteins, such as Pals1/Mpp5, are highly enriched in some of these domains, suggesting that they may regulate the polarity of Schwann cells and myelination. To test this, we generated mice with Schwann cells and oligodendrocytes that lack Pals1. During early development of the PNS, Pals1-deficient mice had impaired radial sorting of axons, delayed myelination, and reduced nerve conduction velocities. Although myelination and conduction velocities eventually recovered, polyaxonal myelination remained a prominent feature of adult Pals1-deficient nerves. Despite the enrichment of Pals1 at paranodes and incisures of control mice, nodes of Ranvier and paranodes were unaffected in Pals1-deficient mice, although we measured a significant increase in the number of incisures. As in other polarized cells, we found that Pals1 interacts with Par3 and loss of Pals1 reduced levels of Par3 in Schwann cells. In the CNS, loss of Pals1 affected neither myelination nor the establishment of polarized membrane domains. These results demonstrate that Schwann cells and oligodendrocytes use distinct mechanisms to control their polarity, and that radial sorting in the PNS is a key polarization event that requires Pals1. Significance statement: This paper reveals the role of the canonical polarity protein Pals1 in radial sorting of axons by Schwann cells. Radial sorting is essential for efficient and proper myelination and is disrupted in some types of congenital muscular dystrophy.

  16. Integrin β4 regulates SPARC protein to promote invasion.

    PubMed

    Gerson, Kristin D; Shearstone, Jeffrey R; Maddula, V S R Krishna; Seligmann, Bruce E; Mercurio, Arthur M

    2012-03-23

    The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.

  17. Cyclin D1 expression is regulated by the retinoblastoma protein.

    PubMed Central

    Müller, H; Lukas, J; Schneider, A; Warthoe, P; Bartek, J; Eilers, M; Strauss, M

    1994-01-01

    The product of the retinoblastoma susceptibility gene, pRb, acts as a tumor suppressor and loss of its function is involved in the development of various types of cancer. DNA tumor viruses are supposed to disturb the normal regulation of the cell cycle by inactivating pRb. However, a direct function of pRb in regulation of the cell cycle has hitherto not been shown. We demonstrate here that the cell cycle-dependent expression of one of the G1-phase cyclins, cyclin D1, is dependent on the presence of a functional Rb protein. Rb-deficient tumor cell lines as well as cells expressing viral oncoproteins (large tumor antigen of simian virus 40, early region 1A of adenovirus, early region 7 of papillomavirus) have low or barely detectable levels of cyclin D1. Expression of cyclin D1, but not of cyclins A and E, is induced by transfection of the Rb gene into Rb-deficient tumor cells. Cotransfection of a reporter gene under the control of the D1 promoter, together with the Rb gene, into Rb-deficient cell lines demonstrates stimulation of the D1 promoter by Rb, which parallels the stimulation of endogenous cyclin D1 gene expression. Our finding that pRb stimulates expression of a key component of cell cycle control, cyclin D1, suggests the existence of a regulatory loop between pRb and cyclin D1 and extends existing models of tumor suppressor function. Images PMID:8159685

  18. Diabetes regulates fructose absorption through thioredoxin-interacting protein

    PubMed Central

    Dotimas, James R; Lee, Austin W; Schmider, Angela B; Carroll, Shannon H; Shah, Anu; Bilen, Julide; Elliott, Kayla R; Myers, Ronald B; Soberman, Roy J; Yoshioka, Jun; Lee, Richard T

    2016-01-01

    Metabolic studies suggest that the absorptive capacity of the small intestine for fructose is limited, though the molecular mechanisms controlling this process remain unknown. Here we demonstrate that thioredoxin-interacting protein (Txnip), which regulates glucose homeostasis in mammals, binds to fructose transporters and promotes fructose absorption by the small intestine. Deletion of Txnip in mice reduced fructose transport into the peripheral bloodstream and liver, as well as the severity of adverse metabolic outcomes resulting from long-term fructose consumption. We also demonstrate that fructose consumption induces expression of Txnip in the small intestine. Diabetic mice had increased expression of Txnip in the small intestine as well as enhanced fructose uptake and transport into the hepatic portal circulation. The deletion of Txnip in mice abolished the diabetes-induced increase in fructose absorption. Our results indicate that Txnip is a critical regulator of fructose metabolism and suggest that a diabetic state can promote fructose uptake. DOI: http://dx.doi.org/10.7554/eLife.18313.001 PMID:27725089

  19. Regulation of peptidoglycan synthesis by outer membrane proteins

    PubMed Central

    Typas, Athanasios; Banzhaf, Manuel; van den Berg van Saparoea, Bart; Verheul, Jolanda; Biboy, Jacob; Nichols, Robert J.; Zietek, Matylda; Beilharz, Katrin; Kannenberg, Kai; von Rechenberg, Moritz; Breukink, Eefjan; den Blaauwen, Tanneke; Gross, Carol A.; Vollmer, Waldemar

    2011-01-01

    Summary Growth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth. PMID:21183073

  20. Regulation of Embryonic Cell Adhesion by the Prion Protein

    PubMed Central

    Schrock, Yvonne; Geiss, Corinna; Luncz, Lydia; Thomanetz, Venus; Stuermer, Claudia A. O

    2009-01-01

    Prion proteins (PrPs) are key players in fatal neurodegenerative disorders, yet their physiological functions remain unclear, as PrP knockout mice develop rather normally. We report a strong PrP loss-of-function phenotype in zebrafish embryos, characterized by the loss of embryonic cell adhesion and arrested gastrulation. Zebrafish and mouse PrP mRNAs can partially rescue this knockdown phenotype, indicating conserved PrP functions. Using zebrafish, mouse, and Drosophila cells, we show that PrP: (1) mediates Ca+2-independent homophilic cell adhesion and signaling; and (2) modulates Ca+2-dependent cell adhesion by regulating the delivery of E-cadherin to the plasma membrane. In vivo time-lapse analyses reveal that the arrested gastrulation in PrP knockdown embryos is due to deficient morphogenetic cell movements, which rely on E-cadherin–based adhesion. Cell-transplantation experiments indicate that the regulation of embryonic cell adhesion by PrP is cell-autonomous. Moreover, we find that the local accumulation of PrP at cell contact sites is concomitant with the activation of Src-related kinases, the recruitment of reggie/flotillin microdomains, and the reorganization of the actin cytoskeleton, consistent with a role of PrP in the modulation of cell adhesion via signaling. Altogether, our data uncover evolutionarily conserved roles of PrP in cell communication, which ultimately impinge on the stability of adherens cell junctions during embryonic development. PMID:19278297

  1. Differential regulation of dentin matrix protein 1 expression during odontogenesis.

    PubMed

    Lu, Yongbo; Zhang, Shubin; Xie, Yixia; Pi, Yuli; Feng, Jian Q

    2005-01-01

    Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone. Both in vitro and in vivo data show that DMP1 is critical for mineralization and tooth morphogenesis (growth and development). In this study, we studied Dmp1 gene regulation. The in vitro transient transfection assay identified two important DNA fragments, the 2.4- and 9.6-kb promoter regions. We next generated and analyzed transgenic mice bearing the beta-galactosidase (lacZ) reporter gene driven by the 2.4- or 9.6-kb promoter with the complete 4-kb intron 1. The 9.6-kb Dmp1-lacZ mice conferred a DMP1 expression pattern in odontoblasts identical to that in the endogenous Dmp1 gene. This is reflected by lacZ expression in Dmp1-lacZ knock-in mice during all stages of odontogenesis. In contrast, the 2.4-kb Dmp1-lacZ mice display activity in odontoblast cells only at the early stage of odontogenesis. Thus, we propose that different transcription factors regulate early or later cis-regulatory domains of the Dmp1 promoter, which gives rise to the unique spatial and temporal expression pattern of Dmp1 gene at different stages of tooth development.

  2. Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean[OPEN

    PubMed Central

    2015-01-01

    Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants. PMID:26498905

  3. Abscisic acid (ABA) regulation of Arabidopsis SR protein gene expression.

    PubMed

    Cruz, Tiago M D; Carvalho, Raquel F; Richardson, Dale N; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  4. Pleiotrophin regulates bone morphogenetic protein (BMP)-induced ectopic osteogenesis.

    PubMed

    Sato, Yasuko; Takita, Hiroko; Ohata, Noboru; Tamura, Masato; Kuboki, Yoshinori

    2002-06-01

    We previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2 microg) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100 microg) or rhPTN (5 and 10 microg) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10 microg of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100 microg of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100 microg of PTN to 1.2 microg of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.

  5. Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling

    PubMed Central

    Roberson, Elle C.; Garcia, Galo; Abedin, Monika; Schurmans, Stéphane; Inoue, Takanari; Reiter, Jeremy F.

    2015-01-01

    SUMMARY Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling. PMID:26305592

  6. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis

    PubMed Central

    Floss, Daniela S.; Levy, Julien G.; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J.

    2013-01-01

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis. PMID:24297892

  7. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

    PubMed

    Floss, Daniela S; Levy, Julien G; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J

    2013-12-17

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

  8. Abscisic Acid (ABA) Regulation of Arabidopsis SR Protein Gene Expression

    PubMed Central

    Cruz, Tiago M. D.; Carvalho, Raquel F.; Richardson, Dale N.; Duque, Paula

    2014-01-01

    Serine/arginine-rich (SR) proteins are major modulators of alternative splicing, a key generator of proteomic diversity and flexible means of regulating gene expression likely to be crucial in plant environmental responses. Indeed, mounting evidence implicates splicing factors in signal transduction of the abscisic acid (ABA) phytohormone, which plays pivotal roles in the response to various abiotic stresses. Using real-time RT-qPCR, we analyzed total steady-state transcript levels of the 18 SR and two SR-like genes from Arabidopsis thaliana in seedlings treated with ABA and in genetic backgrounds with altered expression of the ABA-biosynthesis ABA2 and the ABA-signaling ABI1 and ABI4 genes. We also searched for ABA-responsive cis elements in the upstream regions of the 20 genes. We found that members of the plant-specific SC35-Like (SCL) Arabidopsis SR protein subfamily are distinctively responsive to exogenous ABA, while the expression of seven SR and SR-related genes is affected by alterations in key components of the ABA pathway. Finally, despite pervasiveness of established ABA-responsive promoter elements in Arabidopsis SR and SR-like genes, their expression is likely governed by additional, yet unidentified cis-acting elements. Overall, this study pinpoints SR34, SR34b, SCL30a, SCL28, SCL33, RS40, SR45 and SR45a as promising candidates for involvement in ABA-mediated stress responses. PMID:25268622

  9. Arf-like Protein 3 (ARL3) Regulates Protein Trafficking and Ciliogenesis in Mouse Photoreceptors.

    PubMed

    Hanke-Gogokhia, Christin; Wu, Zhijian; Gerstner, Cecilia D; Frederick, Jeanne M; Zhang, Houbin; Baehr, Wolfgang

    2016-03-25

    Arf-like protein 3 (ARL3) is a ubiquitous small GTPase expressed in ciliated cells of plants and animals. Germline deletion ofArl3in mice causes multiorgan ciliopathy reminiscent of Bardet-Biedl or Joubert syndromes. As photoreceptors are elegantly compartmentalized and have cilia, we probed the function of ARL3 (ADP-ribosylation factor (Arf)-like 3 protein) by generating rod photoreceptor-specific (prefix(rod)) and retina-specific (prefix(ret))Arl3deletions. In predegenerate(rod)Arl3(-/-)mice, lipidated phototransduction proteins showed trafficking deficiencies, consistent with the role of ARL3 as a cargo displacement factor for lipid-binding proteins. By contrast,(ret)Arl3(-/-)rods and cones expressing Cre recombinase during embryonic development formed neither connecting cilia nor outer segments and degenerated rapidly. Absence of cilia infers participation of ARL3 in ciliogenesis and axoneme formation. Ciliogenesis was rescued, and degeneration was reversed in part by subretinal injection of adeno-associated virus particles expressing ARL3-EGFP. The conditional knock-out phenotypes permitted identification of two ARL3 functions, both in the GTP-bound form as follows: one as a regulator of intraflagellar transport participating in photoreceptor ciliogenesis and the other as a cargo displacement factor transporting lipidated protein to the outer segment. Surprisingly, a farnesylated inositol polyphosphate phosphatase only trafficked from the endoplasmic reticulum to the Golgi, thereby excluding it from a role in photoreceptor cilia physiology. PMID:26814127

  10. Methylation of ribosomal protein S10 by protein-arginine methyltransferase 5 regulates ribosome biogenesis.

    PubMed

    Ren, Jinqi; Wang, Yaqing; Liang, Yuheng; Zhang, Yongqing; Bao, Shilai; Xu, Zhiheng

    2010-04-23

    Modulation of ribosomal assembly is a fine tuning mechanism for cell number and organ size control. Many ribosomal proteins undergo post-translational modification, but their exact roles remain elusive. Here, we report that ribosomal protein s10 (RPS10) is a novel substrate of an oncoprotein, protein-arginine methyltransferase 5 (PRMT5). We show that PRMT5 interacts with RPS10 and catalyzes its methylation at the Arg(158) and Arg(160) residues. The methylation of RPS10 at Arg(158) and Arg(160) plays a role in the proper assembly of ribosomes, protein synthesis, and optimal cell proliferation. The RPS10-R158K/R160K mutant is not efficiently assembled into ribosomes and is unstable and prone to degradation by the proteasomal pathway. In nucleoli, RPS10 interacts with nucleophosmin/B23 and is predominantly concentrated in the granular component region, which is required for ribosome assembly. The RPS10 methylation mutant interacts weakly with nucleophosmin/B23 and fails to concentrate in the granular component region. Our results suggest that PRMT5 is likely to regulate cell proliferation through the methylation of ribosome proteins, and thus reveal a novel mechanism for PRMT5 in tumorigenesis.

  11. A role for homologous recombination proteins in cell cycle regulation

    PubMed Central

    Kostyrko, Kaja; Bosshard, Sandra; Urban, Zuzanna; Mermod, Nicolas

    2015-01-01

    Eukaryotic cells respond to DNA breaks, especially double-stranded breaks (DSBs), by activating the DNA damage response (DDR), which encompasses DNA repair and cell cycle checkpoint signaling. The DNA damage signal is transmitted to the checkpoint machinery by a network of specialized DNA damage-recognizing and signal-transducing molecules. However, recent evidence suggests that DNA repair proteins themselves may also directly contribute to the checkpoint control. Here, we investigated the role of homologous recombination (HR) proteins in normal cell cycle regulation in the absence of exogenous DNA damage. For this purpose, we used Chinese Hamster Ovary (CHO) cells expressing the Fluorescent ubiquitination-based cell cycle indicators (Fucci). Systematic siRNA-mediated knockdown of HR genes in these cells demonstrated that the lack of several of these factors alters cell cycle distribution, albeit differentially. The knock-down of MDC1, Rad51 and Brca1 caused the cells to arrest in the G2 phase, suggesting that they may be required for the G2/M transition. In contrast, inhibition of the other HR factors, including several Rad51 paralogs and Rad50, led to the arrest in the G1/G0 phase. Moreover, reduced expression of Rad51B, Rad51C, CtIP and Rad50 induced entry into a quiescent G0-like phase. In conclusion, the lack of many HR factors may lead to cell cycle checkpoint activation, even in the absence of exogenous DNA damage, indicating that these proteins may play an essential role both in DNA repair and checkpoint signaling. PMID:26125600

  12. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  13. Protein kinase D negatively regulates hepatitis C virus secretion through phosphorylation of oxysterol-binding protein and ceramide transfer protein.

    PubMed

    Amako, Yutaka; Syed, Gulam H; Siddiqui, Aleem

    2011-04-01

    Hepatitis C virus (HCV) RNA replicates its genome on specialized endoplasmic reticulum modified membranes termed membranous web and utilizes lipid droplets for initiating the viral nucleocapsid assembly. HCV maturation and/or the egress pathway requires host sphingolipid synthesis, which occur in the Golgi. Ceramide transfer protein (CERT) and oxysterol-binding protein (OSBP) play a crucial role in sphingolipid biosynthesis. Protein kinase D (PKD), a serine/threonine kinase, is recruited to the trans-Golgi network where it influences vesicular trafficking to the plasma membrane by regulation of several important mediators via phosphorylation. PKD attenuates the function of both CERT and OSBP by phosphorylation at their respective Ser(132) and Ser(240) residues (phosphorylation inhibition). Here, we investigated the functional role of PKD in HCV secretion. Our studies show that HCV gene expression down-regulated PKD activation. PKD depletion by shRNA or inhibition by pharmacological inhibitor Gö6976 enhanced HCV secretion. Overexpression of a constitutively active form of PKD suppressed HCV secretion. The suppression by PKD was subverted by the ectopic expression of nonphosphorylatable serine mutant CERT S132A or OSBP S240A. These observations imply that PKD negatively regulates HCV secretion/release by attenuating OSBP and CERT functions by phosphorylation inhibition. This study identifies the key role of the Golgi components in the HCV maturation process. PMID:21285358

  14. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein.

    PubMed

    Choi, Il-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  15. Regulation of Airway Inflammation by G-protein Regulatory Motif Peptides of AGS3 protein

    PubMed Central

    Choi, IL-Whan; Ahn, Do Whan; Choi, Jang-Kyu; Cha, Hee-Jae; Ock, Mee Sun; You, EunAe; Rhee, SangMyung; Kim, Kwang Chul; Choi, Yung Hyun; Song, Kyoung Seob

    2016-01-01

    Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment. PMID:27270970

  16. Protein kinase CK2 regulates metal toxicity in neuronal cells.

    PubMed

    Zaman, Mohammad S; Johnson, Adam J; Bobek, Gabriele; Kueh, Sindy; Kersaitis, Cindy; Bailey, Trevor D; Buskila, Yossi; Wu, Ming J

    2016-01-01

    Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 μM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and β) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

  17. Polyglutamylation: a fine-regulator of protein function? 'Protein Modifications: beyond the usual suspects' review series.

    PubMed

    Janke, Carsten; Rogowski, Krzysztof; van Dijk, Juliette

    2008-07-01

    Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved from protists to mammals and its most prominent substrate is tubulin, the microtubule (MT) building block. Various polyglutamylation states of MTs can be distinguished within a single cell and they are also characteristic of specific cell types or organelles. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The discovery of a new family of enzymes that catalyse this modification has brought new insight into the mechanism of polyglutamylation and now allows for direct functional studies of the role of tubulin polyglutamylation. Moreover, the recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes.

  18. Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells.

    PubMed

    Wang, J; Gilchrist, A; Stern, P H

    2011-02-01

    Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα(s), Gα(q) or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G(s), 6 genes were solely mediated through G(q), and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G(s) and G(q), 3 genes were regulated by both G(s) and G₁₂, and 3 genes were controlled by G(s), G(q) and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.

  19. Prostate Androgen-Regulated Mucin-Like protein 1: A Novel Regulator of Progesterone Metabolism

    PubMed Central

    Park, Ji Yeon; Jang, Hyein; Curry, Thomas E.; Sakamoto, Aiko

    2013-01-01

    The LH surge reprograms preovulatory follicular cells to become terminally differentiated luteal cells which produce high levels of progesterone and become resistant to apoptosis. PARM1 (prostate androgen regulated mucin-like protein 1) has been implicated in cell differentiation and cell survival in nonovarian cells, but little is known about PARM1 in the ovary. This study demonstrated that the LH surge induced a dramatic increase in Parm1 expression in periovulatory follicles and newly forming CL in both cycling and immature rat models. We further demonstrated that hCG increases Parm1 expression in granulosa cell cultures. The in vitro up-regulation of Parm1 expression was mediated by hCG-activated multiple signaling pathways and transcriptional activation of this gene. Parm1 knockdown increased the viability of cultured granulosa cells but resulted in a decrease in progesterone levels. The inhibitory effect of Parm1 silencing on progesterone was reversed by adenoviral mediated add-back expression of Parm1. Parm1 silencing had little effect on the expression of genes involved in progesterone biosynthesis and metabolism such as Scarb1, Ldlr, Vldlr, Scp2, Star, Cyp11a1, Hsd3b, and Srd5a1, while decreasing the expression of Akr1c3. Analyses of culture media steroid levels revealed that Parm1 knockdown had no effect on pregnenolone levels, while resulting in time-dependent decreases in progesterone and 20α-dihydroprogesterone and accelerated accumulation of 5α-pregnanediol. This study revealed that the up-regulation of Parm1 expression promotes progesterone and 20α-dihydroprogesterone accumulation in luteinizing granulosa cells by inhibiting progesterone catabolism to 5α-pregnanediol. PARM1 contributes to ovulation and/or luteal function by acting as a novel regulator of progesterone metabolism. PMID:24085821

  20. The VQ Motif-Containing Protein Family of Plant-Specific Transcriptional Regulators1

    PubMed Central

    Jing, Yanjun; Lin, Rongcheng

    2015-01-01

    The VQ motif-containing proteins (designated as VQ proteins) are a class of plant-specific proteins with a conserved and single short FxxhVQxhTG amino acid sequence motif. VQ proteins regulate diverse developmental processes, including responses to biotic and abiotic stresses, seed development, and photomorphogenesis. In this Update, we summarize and discuss recent advances in our understanding of the regulation and function of VQ proteins and the role of the VQ motif in mediating transcriptional regulation and protein-protein interactions in signaling pathways. Based on the accumulated evidence, we propose a general mechanism of action for the VQ protein family, which likely defines a novel class of transcriptional regulators specific to plants. PMID:26220951

  1. ERM proteins regulate growth cone responses to Sema3A

    PubMed Central

    Mintz, C. David; Carcea, Ioana; McNickle, Daniel G.; Dickson, Tracey C.; Ge, Yongchao; Salton, Stephen R.J.; Benson, Deanna L.

    2008-01-01

    Axonal growth cones initiate and sustain directed growth in response to cues in their environment. A variety of events such as receptor internalization, kinase activation, and actin rearrangement can be stimulated by guidance cues and are essential for mediating targeted growth cone behavior. Surprisingly little is known about how such disparate actions are coordinated. Our data suggest that ezrin, radixin, and moesin (ERMs), a family of highly homologous, multifunctional proteins may be able to coordinate growth cone responses to the guidance cue, Sema3A. We show that active ERMs concentrate asymmetrically in neocortical growth cones, are rapidly and transiently inactivated by Sema3A, and are required for Sema3A-mediated growth cone collapse and guidance. The FERM domain of active ERMs regulates internalization of the Sema3A receptor, Npn1 and its co-receptor, L1CAM, while the ERM C-terminal domain binds and caps F-actin. Our data support a model in which ERMs can coordinate membrane and actin dynamics in response to Sema3A. PMID:18651636

  2. Interleukin 2 activates extracellular signal-regulated protein kinase 2

    PubMed Central

    1993-01-01

    Interleukin 2 (IL-2) stimulated activation of the 42-kD extracellular signal-regulated kinase 2 (Erk2) in murine IL-3-dependent cells, expressing either high or intermediate affinity IL-2 receptors. Activation was both rapid, occurring within 5 min of IL-2 addition, and prolonged, remaining elevated for 30 min. Activation of Erk2 appeared to be necessary for IL-2 stimulation of proliferation, as deletion of a region of the cytoplasmic domain of the IL-2 receptor beta chain, essential for IL-2 stimulation of proliferation, abolished Erk2 activation by IL-2. Furthermore, cells that had been deprived of cytokine for 24 h were then refractory to IL-2 stimulation of both Erk2 activity and proliferation. However, elevation of Erk2 activity was not sufficient to stimulate proliferation, as protein kinase C activation stimulated Erk2 activity but not DNA synthesis. Also, cells exposed to IL-2 in the presence of rapamycin showed full Erk2 activation but not DNA synthesis. These data suggest that IL-2 must stimulate both Erk2 activity and a further pathway(s) to trigger cell proliferation. PMID:8376945

  3. Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

    PubMed Central

    Song, Gyun Jee; Kim, Jaehong; Kim, Jong-Heon; Song, Seungeun; Park, Hana; Zhang, Zhong-Yin

    2016-01-01

    Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases. PMID:27790059

  4. Regulation of mitochondrial functions by protein phosphorylation and dephosphorylation.

    PubMed

    Lim, Sangbin; Smith, Kelly R; Lim, Ssang-Taek Steve; Tian, Rong; Lu, Jianrong; Tan, Ming

    2016-01-01

    The mitochondria are double membrane-bound organelles found in most eukaryotic cells. They generate most of the cell's energy supply of adenosine triphosphate (ATP). Protein phosphorylation and dephosphorylation are critical mechanisms in the regulation of cell signaling networks and are essential for almost all the cellular functions. For many decades, mitochondria were considered autonomous organelles merely functioning to generate energy for cells to survive and proliferate, and were thought to be independent of the cellular signaling networks. Consequently, phosphorylation and dephosphorylation processes of mitochondrial kinases and phosphatases were largely neglected. However, evidence accumulated in recent years on mitochondria-localized kinases/phosphatases has changed this longstanding view. Mitochondria are increasingly recognized as a hub for cell signaling, and many kinases and phosphatases have been reported to localize in mitochondria and play important functions. However, the strength of the evidence on mitochondrial localization and the activities of the reported kinases and phosphatases vary greatly, and the detailed mechanisms on how these kinases/phosphatases translocate to mitochondria, their subsequent function, and the physiological and pathological implications of their localization are still poorly understood. Here, we provide an updated perspective on the recent advancement in this area, with an emphasis on the implications of mitochondrial kinases/phosphatases in cancer and several other diseases.

  5. OsGF14b Positively Regulates Panicle Blast Resistance but Negatively Regulates Leaf Blast Resistance in Rice.

    PubMed

    Liu, Qing; Yang, Jianyuan; Zhang, Shaohong; Zhao, Junliang; Feng, Aiqing; Yang, Tifeng; Wang, Xiaofei; Mao, Xinxue; Dong, Jingfang; Zhu, Xiaoyuan; Leung, Hei; Leach, Jan E; Liu, Bin

    2016-01-01

    Although 14-3-3 proteins have been reported to be involved in responses to biotic stresses in plants, their functions in rice blast, the most destructive disease in rice, are largely unknown. Only GF14e has been confirmed to negatively regulate leaf blast. We report that GF14b is highly expressed in seedlings and panicles during blast infection. Rice plants overexpressing GF14b show enhanced resistance to panicle blast but are susceptible to leaf blast. In contrast, GF14b-silenced plants show increased susceptibility to panicle blast but enhanced resistance to leaf blast. Yeast one-hybrid assays demonstrate that WRKY71 binds to the promoter of GF14b and modulates its expression. Overexpression of GF14b induces expression of jasmonic acid (JA) synthesis-related genes but suppresses expression of salicylic acid (SA) synthesis-related genes. In contrast, suppressed GF14b expression causes decreased expression of JA synthesis-related genes but activation of SA synthesis-related genes. These results suggest that GF14b positively regulates<